Page 2 Fluorolog-3 v. 3.2 (2 May 2008)
Page 3 Operation Manual Rev. 3.2 http://www.jobinyvon.com USA: HORIBA Jobin Yvon Inc., 3880 Park Avenue, Edison, NJ 08820-3012, Toll-Free: +1-866-jobinyvon Tel: +1-732-494-8660, Fax: +1-732-549-5125, E-mail: info@jobinyvon.com, www.jobinyvon.com France: HORIBA Jobin Yvon S.A.S., 16-18, rue du Canal, 91165 Longjumeau Cedex, Tel: +33 (0) 1 64 54 13 00, Fax: +33 (0) 1 69 09 93 19, www.jobinyvon.fr Japan: HORIBA Ltd., JY Optical Sales Dept, Higashi-Kanda, Daiji Building, 1-7-8 Higashi-Kanda...
Page 4 Fluorolog-3 v. 3.2 (2 May 2008) Copyright © 2002, 2005–2006, 2008 by HORIBA Jobin Yvon Inc. All rights reserved. No part of this work may be reproduced, stored, in a retrieval system, or transmitted in any form by any means, including...
Page 5: Table Of Contents
Fluorolog-3 v. 3.2 (2 May 2008) Table of Contents 0: Introduction ....................0-1 ® About the Fluorolog -3..........................0-1 Chapter overview ............................ 0-2 Disclaimer ............................... 0-4 Safety summary ............................0-6 Risks of ultraviolet exposure........................0-9 Additional risks of xenon lamps ......................0-11 1: Requirements &...
Page 6 Fluorolog-3 v. 3.2 (2 May 2008) 7: Troubleshooting................... 7-1 Chart ............................... 7-1 Using diagnostic spectra ........................7-3 Further assistance…..........................7-8 8: Producing Correction Factors ................8-1 Introduction ............................. 8-1 Types of Correction-Factor Kits......................8-2 Generating emission correction factors via 1908 accessory..............8-3 Calculating emission correction factors via 1908 accessory..............
Page 7 Fluorolog-3 v. 3.2 (2 May 2008) 14: Introduction to lifetime measurements ............14-1 Introduction ............................14-1 Lifetime measurements ........................14-2 Types of lifetime scans ......................... 14-4 15: Technical Specifications ................15-1 Spectrofluorometer system........................15-2 Minimum computer requirements ......................15-4 Software..............................15-4 16: Components &...
Page 8 Fluorolog-3 v. 3.2 (2 May 2008) 19: Bibliography ....................19-1 20: Index .....................20-1...
Page 9: 0: Introduction
Fluorolog-3 v. 3.2 (2 May 2008) Introduction 0: Introduction ® About the Fluorolog ® The main parts of the Fluorolog -3 spectrofluorometer system are: • State-of-the-art optical components • A personal computer • FluorEssence™ for Windows ® , the driving software.
Page 10: Chapter Overview
Fluorolog-3 v. 3.2 (2 May 2008) Introduction Chapter overview 1: Requirements & Power and environmental requirements; select the best spot for Installation the instrument. 2: System Description ® Various Fluorolog -3 configurations; their features and benefits. 3: System Operation Operation of the spectrofluorometer system, and calibration instructions.
Page 11 Fluorolog-3 v. 3.2 (2 May 2008) Introduction 16: Reassembly ® How to reassemble the Fluorolog -3 after it has been moved. Instructions 17: Glossary A list of some useful technical terms related to fluorescence spectroscopy. 18: Bibliography Important sources of information.
Page 12: Disclaimer
HORIBA Jobin Yvon is also under constant development and subject to change without notice. Any warranties and remedies with respect to our products are limited to those provided in writing as to a particular product. In no event shall HORIBA Jobin Yvon be held...
Page 13 HORIBA Jobin Yvon is also in no event liable for damages on any theory of liability arising out of, or in connection with, the use or performance of our hardware or software, regardless of whether you have been advised of the possibility of damage.
Page 14: Safety Summary
Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design, manufacture and intended use of instrument. HORIBA Jobin Yvon assumes no liability for the customer’s failure to comply with these requirements. Certain symbols are used throughout the text for special conditions when operating the instruments: A WARNING notice denotes a hazard.
Page 15 Fluorolog-3 v. 3.2 (2 May 2008) Introduction Explosion hazard! Wear explosion-proof goggles, full-face shield, skin-protection Warning: clothing, and protective gloves. Risk of electric shock! This symbol warns the user that uninsulated voltage within the unit Warning: may have sufficient magnitude to cause electric shock.
Page 16 Fluorolog-3 v. 3.2 (2 May 2008) Introduction Wear an appropriate face-shield to protect the face. Read this manual before using or servicing the instrument. General information is given concerning Note: operation of the equipment.
Page 17: Risks Of Ultraviolet Exposure
Fluorolog-3 v. 3.2 (2 May 2008) Introduction Risks of ultraviolet exposure Caution: This instrument is used in conjunction with ultraviolet light. Exposure to these radiations, even reflected or diffused, can result in serious, and sometimes irreversible, eye and skin injuries.
Page 18 Fluorolog-3 v. 3.2 (2 May 2008) Introduction are reduced. This should not lessen the concern over lens damage however, because cataracts are the direct result of lens damage. Burns to the eyes are usually more painful and serious than a burn to the skin. Make sure your eye protection is appropriate for this work.
Page 19: Additional Risks Of Xenon Lamps
Fluorolog-3 v. 3.2 (2 May 2008) Introduction Additional risks of xenon lamps Warning: Xenon lamps are dangerous. Please read the following precautions. Among the dangers associated with xenon lamps are: • Burns caused by contact with a hot xenon lamp.
Page 20 Fluorolog-3 v. 3.2 (2 May 2008) Introduction The skin and eyes absorb infrared radiation (IR) as heat. Workers normally notice excessive exposure through heat sensation and pain. Infrared radiation in the IR-A that enters the human eye will reach (and can be focused upon) the sensitive cells of the retina.
Page 21: 1: Requirements & Installation
Fluorolog-3 v. 3.2 (2 May 2008) Requirements & Installation 1: Requirements & Installation Surface requirements • A sturdy table- or bench-top. • Table size varies according to the system configuration; an average size of 38″ × 60″ (96.5 cm × 152.4 cm) is usually sufficient.
Page 22: Environmental Requirements
Fluorolog-3 v. 3.2 (2 May 2008) Requirements & Installation Environmental requirements • Temperature 72 ± 5°F (22 ± 3°C) • Humidity level ~70% • No special ventilation. Note: ® The standard xenon lamp provided with the Fluorolog -3 is ozone-free. The lamp housing contains an electrically powered fan...
Page 23: Electrical Requirements
Fluorolog-3 v. 3.2 (2 May 2008) Requirements & Installation Electrical requirements • 115 V, 20 A or 220 V, 20 A; factory-set. • As an extra measure of caution, plug the xenon lamp into a circuit separate from Note: For the computer, HORIBA the other components.
Page 24: Installation
Subsequent assembly because of relocation either can be performed by a HORIBA Jobin Yvon Inc. engineer for a specified fee, or by the user. Re-assembly instructions and diagrams are provided in Reassembly Instructions.
Page 25: 2: System Description
Fluorolog-3 v. 3.2 (2 May 2008) System Description 2: System Description Overview General operation ® All Fluorolog -3 spectrofluorometers have common features: A source of radiation produces photons. The beam of light is filtered by an Controller excitation (SpectrAcq) monochromator that...
Page 26 Fluorolog-3 v. 3.2 (2 May 2008) System Description Basic components Monochromators ® The Fluorolog -3 comes equipped with either a single- or double-grating monochromator in the excitation and emission positions. Double-grating monochromators offer a significant increase in sensitivity, resolution and stray-light rejection.
Page 27: Configurations
Fluorolog-3 v. 3.2 (2 May 2008) System Description Configurations ® The different configurations and various accessories available for the Fluorolog -3 system allow you to customize a system specific for today’s needs, while the interchangeability of the components and the inherent design enable the system to grow and change as new applications arise.
Page 28 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-11 ® The Fluorolog -3 Model FL3-11 is an economical system designed for routine fluorescence measurements. The standard model FL3-11 comes equipped with: • 450-W light source • single-grating excitation monochromator •...
Page 29 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-12 ® The Fluorolog -3 model FL3-12 provides optimum performance for highly scattering samples such as proteins, membranes, and solid samples. ® Like the Model FL3-11, the Fluorolog -3 Model FL3-12 has a single-grating excitation monochromator;...
Page 30 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-21 ® The Fluorolog -3 model FL3-21 includes a double-grating monochromator at the excitation position. Features of the Model FL3-21 are: • 450-W light source • double-grating excitation monochromator •...
Page 31 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-22 ® Because of the double-grating excitation and emission monochromators, the Fluorolog model FL3-22 offers unsurpassed performance in resolution, sensitivity, and stray-light rejection. This system is perfect for highly scattering samples like lipids and proteins, or solids like powders, semiconductors, or phosphors.
Page 32 Fluorolog-3 v. 3.2 (2 May 2008) System Description Standard options The previously described systems represent the standard configurations. Each system, however, can be customized by selecting different components. Available options are listed below. For additional information, or for a list of the most recently developed products, ®...
Page 33 Fluorolog-3 v. 3.2 (2 May 2008) System Description Custom configurations With custom configurations, you can change the layout of the system to a T-format or add an imaging spectrograph at the emission port of the system. Using single-grating, double- grating, or the imaging spectrometer, you can create a system for almost any application.
Page 34 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-XXX Systems with the T-configuration design were developed for T-format polarization or anisotropy and dual-emission spectroscopy. Models FL3-XXX (where the Xs are the type of spectrometer positioned at the excitation, first-emission, and second-emission positions, respectively) are available in numerous configurations.
Page 35 Fluorolog-3 v. 3.2 (2 May 2008) System Description ® Fluorolog -3 Model FL3-12-320M The fully automated TRIAX 320M imaging spectrograph can be a part of a custom ® Fluorolog -3 configuration. The imaging spectrograph offers the latest advances in optical design and automation.
Page 36 Fluorolog-3 v. 3.2 (2 May 2008) System Description Custom options Each system can be customized further by selecting different options. Available options are ® listed below. For additional information, or for a list of the newest options, contact a Spex Fluorescence Product Specialist.
Page 37: 3: System Operation
Fluorolog-3 v. 3.2 (2 May 2008) System Operation 3: System Operation Turning on the system Start the lamp. ® The lamp must be turned on prior to the Fluorolog accessories, and peripheral equipment. On the back of the lamp housing, turn on the switch marked POWER.
Page 38 Fluorolog-3 v. 3.2 (2 May 2008) System Operation On the rear of the SpectrAcq, switch on the power button to start. Immediately below the mµA logo on the front of the Spec- trAcq, the LED indicator lamp should illuminate. ®...
Page 39: Checking System Performance
® the Fluorolog -3. HORIBA Jobin Yvon Inc. recommends checking the system calibration before each day of use with the system. Scans of the xenon-lamp output and the Raman- scatter band of water are sufficient to verify system calibration, repeatability, and through- put.
Page 40 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Excitation calibration check This calibration check verifies the wavelength calibration of your excitation monochroma- tor, using the reference photodiode located before the sample compartment. It is an excita- tion scan of the xenon lamp’s output, and should be the first check performed.
Page 41 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Use the default parameters or adjust them. Default monochromator parameters for the xenon-lamp scan Monochromator Initial wave- Final wave- Increment Slits (bandpass) (1200 length length grooves/mm) Excitation 200 nm 600 nm 1 nm...
Page 42 Fluorolog-3 v. 3.2 (2 May 2008) System Operation calibration structure ~ 450 nm peak at 467 broadband This is an uncalibrated ® FluoroMax lamp Note: scan. The main peak Your lamp scan may appear differ- ought to be at 467 nm,...
Page 43 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Cursor Wave- length in This example shows the peak actually at 477 nm, which is 10 nm too high. Therefore we must recalibrate the monochromator. Click the Previous Experiment button. The Experiment Setup window appears.
Page 44 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Click the RTC button on the lower right. The Real Time Control window opens. Click the Monos icon to view the monochromators’ index card, then click the excitation monochromator tab.
Page 45 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Enter the current, observed position of the peak in the Position Control (here, 477 nm). Click the Calibrate Excitation 1 button. The Calibrate window opens: In Peak Of Interest, enter the actual or expected position of the peak (it ought to be 467 nm).
Page 46 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Emission calibration check Note: The emission calibration of the instrument is directly affected by the calibration of the excitation monochromator. This calibration check verifies the wavelength calibration of the emission monochromator with the emission photomultiplier tube. It is an emission scan of the Raman-scatter band of water performed in right-angle mode.
Page 47 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Choose Emission., then click Next >> water-Raman experiment automatically loads. Use the default parameters or adjust them. Monochromator parameters for the water-Raman scan: Monochromator Initial wave- Final wave- Incre- Slits (band- (1200 grooves/mm)
Page 48 Fluorolog-3 v. 3.2 (2 May 2008) System Operation Detector parameters for the water-Raman scan: Detector (Signal) Integration time Units Signal (S1) 100 ms Reference (R1) 100 ms Click Run. The Intermediate Display opens. The water-Raman scan runs. ® A new Fluorolog -3 should display a water-Raman peak intensity of at least –1...
Page 49 (a signal) with a value for system noise (no signal) to show the overall performance of the instrument. Definitions At HORIBA Jobin Yvon, we define the S/N as the difference of peak and background sig- nal, divided by the square root of the background signal. −...
Page 50 44 6 11000 HORIBA Jobin Yvon feels that the first method is correct although it gives a lower S/N. The second method only takes into account the detector noise and the shot noise of the electron- ics. By using the background total intensity as a measure of noise, the HORIBA Jobin Yvon method is more representative of a real “live”...
Page 51 They can be run, after the system is switched on each day, to check the calibration ® and performance of the Fluorolog • HORIBA Jobin Yvon Inc. recommends that the number of hours of xenon-lamp use be recorded in a log (see sample sheet in Xenon Lamp Information & Record of Use Form).
Page 52: Useful Materials For Characterizing System And Samples
Fluorolog-3 v. 3.2 (2 May 2008) System Operation Useful materials for characterizing system and samples The following are materials that HORIBA Jobin Yvon Inc. has found useful in determining system sensitivity or as standards for lifetime measurements. Substance CAS Number...
Page 53: 4: Data Acquisition
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition 4: Data Acquisition This chapter presents an introduction to the special buttons used in FluorEssence™ to ® record and present data with the Fluorolog -3. These buttons, located in FluorEs- sence™’s main window, are:...
Page 54: Experiment Menu Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Experiment Menu button The Experiment Menu button chooses an overall type of experiment to run, such as an emission scan, a phosphorimeter scan, a synchronous scan, etc., based on the instru- ment and connected accessories, such as a temperature bath, MicroMax, etc. Only those scans that can be run using the available hardware configuration are active;...
Page 55 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Click the Experiment File field, and enter a new file name or select a previously saved file. Verify that experimental parameters are correct. Be sure to check all parameters under all icons in the left-hand column.
Page 56: Previous Experiment Setup Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Previous Experiment Setup button The Previous Experiment Setup button resets the experiment to the previous experi- ment used, with minor modifications to the hardware possible. Note: The Previous Experiment Setup button is active only after an experiment has already been loaded.
Page 57: Auto Run Previous Experiment Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Auto Run Previous Experiment button The Auto Run Previous Experiment Note: The Auto Run Previous Experiment but- button reruns the last ton is active only after an experiment has al- experiment loaded ready been loaded and run.
Page 58: Make Overlay File Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Make Overlay File button The Make Overlay File button creates an Note: The Make Overlay File button is active *.SPC file for use as only with an active graph. an overlay file.
Page 59: 3D Scan To 3D Profile Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition 3D Scan to 3D Profile button The 3D Scan to 3D Profile button extracts emission profiles from Note: The 3D Scan to 3D Profile button only an excitation-emission operates with excitation-emission matrix data.
Page 60 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Click the Arbitrary Line button to choose an arbi- trary profile. Grab an end of the profile line and move to the desired location on the matrix. The profiles are updated. To return to perpendicular profiles, click the...
Page 61: Run Jy Batch Experiments Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Run JY Batch Experiments The Run JY Batch Experiments button runs a series of automated experiments, in- cluding adjustable repeats and delays between experiments. Click the Run JY Batch Experiments button. The Setup batch experiments window appears.
Page 62 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition The file is saved in a .jyb format. Set up each experiment in the batch job. Select an experiment from the Execution List. In the Total Repeats: field, enter the number of times that experiment should be repeated.
Page 63: Real Time Control Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Real Time Control The Real Time Control button opens the Real Time Control window directly, so that the user can adjust experimental parameters in real time. In the toolbar, click the Real Time Control but- ton.
Page 64: Create/Use Calibration Curve From Cwa Data Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Create/Use Calibration Curve from CWA Data When the user is doing Single Point Note: This button only op- experiments (especially with the MicroMax or erates if CWA data exist. multiple-sample changers), the Create/Use Calibration Curve from CWA Data button creates a calibration curve for analytical measurements.
Page 65 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Set up the standards and unknown(s). If you have an automatic sample changer, you can change the samples manually with the SC Manual radio button. Otherwise, the software changes the samples automatically.
Page 66 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Set up the excitation and emission wave- length(s). Enter the appropriate excitation and emission wavelength set(s), one per row, in the Wavelength Sets area. To add a wavelength set, click the Insert row button.
Page 67 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Click the Create/Use Calibration Curve from CWA Data button in the toolbar. The Calibration Curve Parameters window appears. Choose a Linear fit, or a Polynomial fit with Order of the polynomial, and then the OK button.
Page 68: Intensity Map Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition 2D Intensity Map This button creates a two-dimensional intensity map from the active data. Note: This button only operates if data are displayed. Be sure that data are open in FluorEssence™. In the toolbar, click the 2D Intensity Map button...
Page 69: Gemini Alpha Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Gemini Alpha The Gemini Alpha button works with data taken via TCSPC with the MF system, transferring data from FluorEssence™ to Gemini Alpha. Gemini Alpha is a modeling program from TCSPC lifetime data. Modeling is the process by which the lifetime ac- quisition results are fitted, to predict the various lifetime components and fractional contribution of each component.
Page 70: Gemini Alpha Results Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Gemini Alpha Results The Gemini Alpha Results button returns fits based on TCSPC lifetime data taken with the MF system from the Gemini Alpha program to FluorEssence™. Gemini Al- pha is a modeling program from TCSPC lifetime data. Modeling is the process by which the lifetime acquisition results are fitted, to predict the various lifetime compo- nents and fractional contribution of each component.
Page 71: Launch Datastation Button
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Launch DataStation The Launch DataStation button closes the FluorEssence™ software, and starts Note: This function only operates with TCSPC accessories. DataStation software. In the toolbar, click the Launch DataStation but- ton. FluorEssence™ shuts down. DataStation starts.
Page 72: Running An Unknown Sample
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Running an unknown sample Often a researcher will scan a sample whose spectral characteristics are unknown. For optimal spectra, the optimal excitation and emission wavelengths must be found. The optimal excitation wavelength is the wavelength that creates the most intense emis- sion spectrum for a given sample.
Page 73 Set the scan parameters. Most of these parameters are a trade-off between speed and precision. Choose integration time, increments, and number of scans judiciously, to give an accu- rate result without excessive time spent. HORIBA Jobin Yvon suggests an in- 4-21...
Page 74 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition crement of 1.0 nm, an integration time of 0.1–0.5 s, and one scan. If unsure of an excitation wavelength, try 300 nm, at which many samples absorb light. Use S (signal detector) for the acquisition mode. Don’t forget a data file name.
Page 75 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Choose Spectra. The Experiment Type menu appears. Choose Excitation. Click the Next >> button. The Experiment Setup window appears: 4-23...
Page 76 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Set the scan parameters. Use the emission maximum determined above for the excitation, use 250 nm for starting, enter the emission maximum minus 15 nm for the end of the scan, and select two acquisition modes, S and S/R.
Page 77: Using Corrected Signals In Fluoressence
Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition Using corrected signals in FluorEssence™ Introduction Subtracting blanks, removing dark noise, and correcting for inhomogeneities in the in- strument or detector response give more accurate spectra. Take special precautions to incorporate these functions properly into a FluorEssence™ experiment.
Page 78 Fluorolog-3 v. 3.2 (2 May 2008) Data Acquisition 4-26...
Page 79: 5: Optimizing Data
Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data 5: Optimizing Data Spectra can be enhanced by optimization of data-acquisition. This chapter lists some meth- ods of optimizing sample preparation, spectrofluorometer setup, and data correction to get higher-quality data. Cuvette preparation...
Page 80: Sample Preparation
4-mL cuvette. If fluorescence is weak or if trace elements are to be determined, HORIBA Jobin Yvon recommends a capillary cell such as our 50-μL or 250-μL optional micro-sample capillary cells, which are specifically de- signed for a small volume.
Page 81 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Dissolved solids Solid samples, such as crystals, sometimes are Solid dissolved in a solvent and analyzed in solution. Sample Solvents, however, may contain organic impu- Holder rities that fluoresce and mask the signal of in- terest.
Page 82: Running A Scan On A Sample
Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Running a scan on a sample Precautions with the Solid Sample Holder Excitation monochromator Avoid placing the front face of the sample so that 60° the excitation beam is reflected directly into the emission monochromator.
Page 83 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Highly opaque samples Highly concentrated and opaque liquids often have problems with self-absorption or com- plete attenuation of the beam. Intensity measurements with the excitation beam at 90° to the emission beam may not be reproducible or detectable, and the excitation or emission spectra may appear distorted.
Page 84: Data Collection Techniques
Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Data collection techniques Select the collection method The two basic collection methods are right-angle and front-face. In right-angle detection, primarily for clear solutions, the fluorescence is collected at 90° to the incident exciting beam.
Page 85 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Determine the optimum integration time The length of time during which photons are counted and averaged for each data point is called the integration time. An unwanted portion of the signal comes from noise and dark counts (distortion inherent in the signal detector and its electronics when high-voltage is ap- plied).
Page 86 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Scan a sample multiple times Scanning a sample more than once and averaging the scans together enhances the S/N. In ½ general, the S/N improves by n , where n is the number of scans.
Page 87 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Use the appropriate wavelength increment The increment in a wavelength scan is the spacing, in nm, between adjacent data points. The spacing between the data points affects the resolution of the spectrum, and total time for ac- quisition.
Page 88 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Select the appropriate bandpass The bandpass (wavelength spread) affects the resolution of spectra. If the bandpass is too broad, narrow peaks separated by a small change in wavelength may be unresolved. For ex- ample, for two 2-nm peaks 5 nm apart, and a bandpass of 10 nm, one broad peak, instead of two well-defined ones, is visible.
Page 89 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Smooth the data Smoothing the data improves the appearance of the spectrum. Smoothing, as are most post- ® processing features, is handled by Origin Select Analysis from the main FluorEssence™ menu. A drop-down menu appears.
Page 90: Correcting Data
Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Correcting data Introduction Collecting accurate information about the luminescent properties of a sample depends upon several factors: o Equipment specifications, o Sample characteristics, and o Timing considerations. To ensure that the spectra collected indicate the actual properties of the sample and not ex- ternal conditions, data often must be corrected.
Page 91 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Correcting data during acquisition Data can be acquired either as raw data or as corrected data. A spectrum composed of raw data exhibits the effects of system parameters, while a corrected spectrum displays only the properties related to the sample.
Page 92 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data Choose Preferences , then the Instrument Correction Files icon. The Instrument Correction Files area should display a correction file for the Detector ( S or R ). If not, click the Insert button, and browse for the desired correction file.
Page 93 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data In the Signal Algebra area, a signal with appended “ c ” appears, denoting a corrected signal: Click the Add >> button to add the corrected signal to the Formulas table. The corrected signal appears in the Formulas table.
Page 94 Fluorolog-3 v. 3.2 (2 May 2008) Optimizing Data After acquisition To apply the correction factors after the data have been acquired, multiply the data file by the appropriate correction factor file ( mcorrect for the S detector, xcorrect for the R detector, or tcorrect for the T detector).
Page 95: 6: System Maintenance
-3 to record lamp usage. Each time the lamp is turned on, it constitutes one full hour of use; therefore, HORIBA Jobin Yvon Inc. suggests leaving the lamp on between brief periods of inactivity. Record the hours of use on the form in Chapter 12: Xenon Lamp Information and Record of Use Form.
Page 96 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance be installed in the lamp housing. Read all the packing material including instructions and precautions before attempting to insert the lamp into the lamp housing. Warning: Xenon lamps are an explosion hazard. Make sure the power is off and all AC power is disconnected from the system.
Page 97 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance To open the lamp housing: Remove the 10 Phillips-head screws on top of the lamp housing. Lift off the cover. This exposes the lamp assembly: Upper bracket Lamp Lower bracket Mirror Top view of the lamp housing with cover removed.
Page 98 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance To remove an existing lamp, Remove the 5/32″ cap screws from the upper and lower brackets, freeing the positive and negative power leads. upper bracket 7/64" cap screw 5/32" cap screws Remove the 7/64″ cap screw from the upper bracket.
Page 99 To insert a xenon lamp Be sure the cover of the lamp housing is removed. Note: HORIBA Jobin Yvon Inc. provides new bulbs with leads and connectors of the proper length and size. Other manufacturers’ lamps may require extra adjustment be- fore installation.
Page 100 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance The positive end should be pointing upward. Insert and tighten the 7/64″ cap screw on the upper bracket. Secure the upper lamp lead and upper power cable to the upper bracket, Power using a 5/32″...
Page 101 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Pull the excess power cables away from the lamp assembly beneath the baffle. Baffle Pull excess cable beneath internal housing away from lamp assembly Note: Make sure the cables do not block the exit slit of the lamp housing.
Page 102 ® maximize the sensitivity of the Fluorolog -3. To do this, HORIBA Jobin Yvon Inc. recom- mends running a water Raman scan to check wavelength accuracy, and then monitoring the peak intensity while adjusting the vertical height of the lamp: Insert a water-filled cuvette in the sample compartment.
Page 103 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Start Calibrate the emis- sion monochroma- Is the water Ra- tor. Go to System man peak at 397 ± 0.5 nm? Operation. Lamp is Continue Is the peak inten- properly with step sity as listed in the adjusted.
Page 104 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Set the excitation and emission monochromators to 350 nm and 397 nm, respectively. The Raman band’s emission appears at 397 nm when water is excited using 350 Make sure HV1 is on and set to 950 V.
Page 105 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance the changes while standing by the lamp housing. Insert a 5/64″ Allen wrench inside the middle port on the top of the lamp housing. Focus Horizontal adjustment Vertical adjustment Note: To reach the...
Page 106 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Record the date that the new lamp was installed, as well as its maximum intensity. Save the protective cover of the new bulb, for when the bulb must be replaced in the future.
Page 107 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Lamp housing Note: Clean the window in front of the lamp Dust reduces the excitation housing once a year, or more light transmission, resulting in lower frequently if needed. system sensitivity. Remove 10...
Page 108 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance If the cap screws were removed, replace them. Replace the baffle, and re-install the 2 screws holding it in place. Re-install the cover, and secure it with the 10 Phillips-head screws. 6-14...
Page 109: Installing An Optional New Photomultiplier Tube
Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Installing an optional new photomultiplier tube ® If your Fluorolog -3 has an optional R928P (usually on the T-side of the sample compart- ment), and you want to change the detector, this section is applicable. When the R928P pho- tomultiplier is replaced with a different detector, the emission correction factors must be up- dated (see Chapter 8: Producing Correction Factors).
Page 110: Reference Signal Detector
Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Reference signal detector The reference signal detector is a state-of-the art silicon diode that requires no routine main- tenance. 6-16...
Page 111: Gratings
Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Gratings The standard excitation and emission monochromator gratings are 1200 grooves/mm, and are blazed at 330 nm and 500 nm, respectively. If an application requires that the system be optimized for a particular region, the gratings can be changed by following a simple proce- dure.
Page 112 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Remove the existing grating Close the slits in the emission spectrometer. Turn off all instrument power. Remove the Phillips-head screws holding the lid on the monochromator. Lift the lid slowly until you feel resistance.
Page 113 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Release the grating from the stand. Loosen the thumbwheel screw securing the grating to the grating stand. Back view of grating assembly. Pull the grating away from the stand. 6-19...
Page 114 Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance To insert a new grating, Place the 3 mounting pins on the back of the grating into the matching mounting slots on the grating stand. Tighten the thumbscrew on the back of the grating stand.
Page 115: Mirrors
Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Mirrors Mirrors are aligned at the factory and usually do not need realignment. Only the Model 1692M Selection Mirror (an option) can be adjusted easily to optimize the fluorescence sig- nal from the sample. This mirror should be adjusted periodically if low signal is caused by poor alignment at the slit position.
Page 116: Automated 4-Position Turret
Fluorolog-3 v. 3.2 (2 May 2008) System Maintenance Automated 4-position turret If a circulating bath is used to regulate the turret’s temperature, periodically replace the flu- ids in the bath. Note: Over time, bacteria can grow in the temperature bath, or the water can become hard.
Page 117: 7: Troubleshooting
Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting 7: Troubleshooting ® The Fluorolog -3 spectrofluorometer system has been designed to operate reliably and predictably. Should a problem occur, examine the chart below, and try the steps listed on the following pages.
Page 118 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting No signal. Lamp is not on. Replace bad xenon lamp. Detectors are saturated. Reduce slit settings. High voltage is off. Turn on high voltage through the software. Erratic signal. Lamp unstable. Let lamp warm up 20 min before use.
Page 119: Using Diagnostic Spectra
The following spectrum shows the trace resulting from a lamp scan run with a known good lamp. 0.35 467 nm 0.25 0.15 0.05 Wavelength (nm) Scan of good quality 450-W xenon lamp in Fluorolog-3 with single excitation monochromator.
Page 120 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting The following lamp scan spectrum shows poor resolution in the area around the peak. 0.4473 Xenon-lamp peaks are unresolved 0.3355 0.2236 0.1118 Wavelength (nm) Lamp scan of 150-W Xe lamp. Note poor resolution in the area near the 467-nm peak.
Page 121 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Water Raman spectra Contaminated water Running a water Raman scan helps identify abnormalities as a result of accessory prob- lems or miscalibration. The following spectrum is normal: 397 nm Wavelength (nm) Clean water Raman scan.
Page 122 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting If the problem goes away, then the problem was due to the cuvette surface. Clean or use a different cuvette. Clean the cuvette. Fill with fresh, double-distilled, deionized water. If the problem goes away, then the problem was due to contaminated water.
Page 123 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Stray light In the following diagram, notice the high level of stray-light below 380 nm in the water Raman spectrum. Wavelength (nm) High stray light in a water Raman scan. To correct this problem, Inspect the cuvette surface for fingerprints and scratches.
Page 124: Further Assistance
Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Further assistance... ® Read all software and accessory manuals before contacting the Spex Fluorescence Service Department. Often the manuals show the problem’s cause and a method of so- lution. Technical support is available for both hardware and software troubleshooting.
Page 125 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Determine FluorEssence™’s version number. Choose the Help menu. Choose About FluorEssence..The About FluorEssence window opens. Near the bottom are the FluorEs- ® sence™ and Origin ver- sion numbers. Click the View System Info button.
Page 126 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Determine the SpectrAcq firmware version. Open the Experiment Setup window: Click the Detectors icon. Move the mouse over the detectors’ table in the Select area. The SpectrAcq firmware version appears in a small pop-up window:...
Page 127 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting Write down the software’s version numbers, along with the purchase dates, model numbers, system configuration, and serial numbers of the instrument and its accessories. ® Call the Spex Fluorescence Service Depart- ment at (732) 494-8660 × 160.
Page 128 Fluorolog-3 v. 3.2 (2 May 2008) Troubleshooting 7-12...
Page 129: 8: Producing Correction Factors
Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors 8: Producing Correction Factors Introduction Gratings, detectors and other monochromator components have response characteristics that are functions of wavelength. These characteristics are superimposed on spectra, and may yield a potentially misleading trace. For accurate intensity comparisons, such as those required for quantum-yield determinations, monochromator-response charac- teristics must be eliminated.
Page 130: Types Of Correction-Factor Kits
Producing Correction Factors Types of Correction-Factor Kits To generate emission correction factors, several items are needed: a standard lamp, ap- propriate holders, and a scatter assembly. HORIBA Jobin Yvon Inc. offers two meth- ods: 1908 Accessory The 1908 is a complete correction-factor kit, while the 1908MOD Scatter Assembly is provided for users who already have a cali- brated standard lamp and a constant-current source.
Page 131: Generating Emission Correction Factors Via 1908 Accessory
Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Generating emission correction factors via 1908 accessory Set up the accessory. Set up the sample compartment. Remove the sample-holder from the compartment, leaving only the 4 posts of the sample drawer.
Page 132 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Nipple points upward The negative (–) lead goes to the negative (black) side of the holder. The nipple of the lamp should point upward. Fix the lamp holder to the top of the sample compartment with double-sided tape.
Page 133 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors may take up to 2 min. For valid irradiance values, the lamp current must be maintained precisely at 6.500 A. Turn off the room lights, set the emission monochromator to 520 nm, and both emission slits to a 5-nm bandpass.
Page 134 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Determine the dark counts via 1908. Place the sample lid over the mask to block all light to the detector. Set up and run an emission scan. Open the Experiment Setup dialog box for an emission scan.
Page 135 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Choose the Detectors icon Enable the signal (S1) detector. Enter the following parameters in the Signals area: Cycles Integration Time (s) Accumulations Run the standard lamp spectrum. Name this file stdlamp.
Page 136 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Set up and run a blank scan, using the same parameters as in step 2, and name this file blank. Set the excitation and emission slits to 0. The data have very low intensity.
Page 137 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Choose Analysis from the toolbar. A drop-down menu appears. Choose Simple Math..The Simple Math Tool window appears. In the upper Data field, choose stdlamp. For the Math Function, choose subtract.
Page 138 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors stdlamp blank Screenshot of blank and lamp spectra. Your spectrum should appear similar to the one pictured above. Its actual ® appearance, however, depends on the configuration of the Fluorolog -3 system.
Page 139: Calculating Emission Correction Factors Via 1908 Accessory
Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Calculating emission correction factors via the 1908 accessory Introduction For more information about the theory and application of radiometric correction, con- sult Accuracy in Spectrophotometry and Luminescence Measurements, Mavrodineau, Schultz, and Menis, NBS Spec. Publ. 378 (1973), especially p. 137, “Absolute Spectro- fluorometry,”...
Page 140 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Using the Simple Math Tool window Note: under the Analysis menu, divide Naming the file IRR by stdlamp2, and name the mcorrect over- resulting file mcorrect. writes the mcor- rect file supplied mcorrect with the software.
Page 141: Generating Emission Correction Factors Via F-3026 Accessory
Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Generating emission correction factors via F-3026 accessory Set up the accessory. Attach the correction- factor light-source to its gap-bed with the brass thumbscrews. Use this orientation. Be sure the power cable doesn’t block optical apertures.
Page 142 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Run the scan. Start FluorEssence™ and initialize the instrument. Turn the xenon lamp OFF, and the lamp for the correction-factor kit ON. Warning: Wear appropriate eye-protection against UV, visible, and IR when the tungsten-halogen lamp is on and the sample-compartment is uncovered.
Page 143 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Analyze the data. Import the standard irradiance file for the calibrated lamp in the correction-factor kit: In the File menu, click Import, and choose SPC Data…. The Open window ap- pears.
Page 144 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors From the File menu, choose New..The New window appears. Choose Graph, then the OK button. A new plot appears on the FluorEs- sence™ main window. In the Analysis menu, choose Simple Math..
Page 145 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Choose the data corresponding to the irradiance file. Choose divide as the math function For Operand, select the data corresponding to the emission spectrum obtained using the correction-factor kit. Select the Add New radio button, and click the Apply button.
Page 146 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Export the normalized correction-factor graph. In the File menu, click Export, and select SPC Data…. Apply the correction factor into your system configuration by follow- ing the instructions in your instrument man- ual.
Page 147 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Note: We recommend keeping a log of the amount of time the source is on. Calibration is necessary after every 50 h of use. Remove the accessory’s sample-compartment gap-bed, and return the standard gap-bed to the sample compartment.
Page 148: Calculating Excitation Correction Factors
This can be accomplished by a simple excitation scan with rhodamine-B placed in the sample position. Note: To calibrate the reference detector out to 800 nm, use HORIBA Jobin Yvon’s calibrated photodiode accessory. Fill a cuvette with a solution of rhodamine-B.
Page 149 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Set the excitation and emission monochromators to 467 nm and 630 nm, respectively. The largest lamp peak occurs at 467 nm. Set the slit on the excitation monochromator to 0.5 mm.
Page 150 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Enter the scan parameters: Excitation Wavelength Start (nm) Excitation Wavelength End (nm) Inc (nm) Emission Wavelength Park Excitation Slit (nm) Emission Slit (nm) Use value determined in step 3. Click the Detectors icon...
Page 151 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Enable both S1 and R1 detectors. The codes S1 and R1 appear in the Formulas table. Remove both S1 and R1 from the Formulas table. In the Signal column, click S1. In the Operations column, click the division sign, /.
Page 152: Using Correction-Factor Files
Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors Using correction-factor files To use the newly acquired xcorrect and mcorrect files, you must tell FluorEs- sence™ use them when the Correction check box is activated in the Experiment Setup window.
Page 153 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors The Preferences area appears. Choose the Instrument Correction Files icon. The Instrument Correction Files area appears. If there are no active fields in the Instrument Correction Files area, click Insert. In sequence,...
Page 154 Fluorolog-3 v. 3.2 (2 Mar 2008) Producing Correction Factors The Grating column, the correct grating from the drop-down list, Then, in the File column, browse for the appropriate correction-factor file. When all necessary detectors have an associated correction-factor file, click OK.
Page 155: 9: Automated Polarizers
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers 9: Automated Polarizers Introduction Theory The measurement of polarized emission of fluorescence allows the observation of rota- tional motions in fluorophores during the lifetime of the excited state. Because the rota- tion of macromolecules depends on their size, shape, and local environment (i.e., sol- vent), several kinds of information may be extracted.
Page 156 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers ented polarizers (H) are said to be at 90°. Polarization and anisotropy are expressed as follows: − − In a real optical system, the G, or grating factor, must be included to correct for the wavelength response to polarization of the emission optics and detectors.
Page 157 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers − For single-photon excitation, the allowed values for the emission anisotropy are gov- erned by: α = 0 4 (cos ) − ( ) = where P x is the second Legendre polynomial, and α is the angle between the molecule’s absorption and emission dipoles.
Page 158 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Excitation Polarizer Excitation Light Excitation and Emission Polarizers rotated to give both components V and H. Emission Polarizer An L-format polarization measurement is illustrated schematically in Figure 1. The excitation polarizer is...
Page 159 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers when the rotational correlation time of a fluorophore is similar to the fluorescence life- time, the effect can be significant. To record spectra that are free of rotational artifacts, use polarized photoselection conditions that cause the anisotropy to be zero. These po- larization angles are called magic-angle conditions.
Page 160: Installation
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Installation ® Spex polarizers are made for easy installation and removal from the light path. All of the polarizers use pinned collars to hold the polarizers in their mounts and maintain calibration when the polarizers are removed.
Page 161: Alignment
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Alignment Checking polarizer alignment Polarizer alignment is verified by measuring the anisotropy of a dilute scattering solu- tion. Scattered light is highly polarized, and this allows a simple check of the crystal alignment in the instrument.
Page 162 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Apply the proper voltage on S (950 V for an R928P photomultiplier). Open the excitation shutter (if applicable). Click the Accessories icon. Set both polarizers to VV (0°). Check the signal on S. Set slits evenly for each monochromator to result in 1–1.5 million cps on...
Page 163 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Re-alignment of polarizers ® Fluorolog -3 autopolarizers may be aligned using a software routine called Polarizer Alignment in Experiment Setup . Using Polarizer Alignment This routine automatically calibrates ® autopolarizers. Use a sample of Ludox...
Page 164 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Click the Advanced... button. This opens the Polarizer Alignment dialog box: Choose various options: • Subtract Dark (recommended) • Reset to Mechanical Zero —only if the polarizers are definitely miscalibrated. This deletes the...
Page 165 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers When complete, the software routine displays the measured anisotropy for each emission channel (S or T). Approve or retry the measurement based on satisfaction with the result. To quit, hit Cancel any time during the proce- dure.
Page 166 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Set the polarizer crystals so that they protrude from the mounts far enough (~1/4" or ~6 mm) to allow rotation. Start the polarizers and accessory controller. ® Insert the Ludox or glycogen sample into the sample holder.
Page 167 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Open the excitation shutter (if applicable). Turn on high voltage and set appropriately for S channel (950 V for R928P; 1050 V for R1527). Set slits to 5-nm bandpass for all monochroma- tors.
Page 168 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Set polarizers to VV. Reset slits for 1–1.5 × 10 cps on S channel. Measure polarization ratio (Equation 9). If the polarization ratio > 100, then the alignment is acceptable. Otherwise, repeat steps 15–18.
Page 169: Using Automated Polarizers
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Using automated polarizers ® FluorEssence™ software with Spex polarizers provides many choices for polarization measurements. Depending on the accessories, the opportunity exists to remove polari- zation effects from the sample, measure the polarization characteristics, or analyze the decay of anisotropy using frequency-domain techniques.
Page 170 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Using FluorEssence™ To use the autopolarizers, load an instrument configuration with autopolarizers. Real Time Control Real Time Control manipulates the polarizers and other instrument settings, to observe and optimize the spectrofluorometer in real time. Under the Accessories icon, each polarizer may be set independently into or out of the optical path under its own index- card tab.
Page 171 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Experiment Setup Experiment Setup runs all scanning options for the autopolarizers. First choose the type of scan using polar- izers in the Fluorescence Main Ex- periment Menu: The Experiment Setup window appears.
Page 172 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Constant Wavelength Analysis To do a constant-wavelength analysis experiment, that is, to take polariza- tion acquisitions at fixed excita- tion/emission wavelength-pairs, choose Anisotropy from the Fluo- rescence Main Experiment Menu . The Experiment Type window opens.
Page 173 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Make sure to use the appropriate Signal in the Signal Algebra area. Add >> it to the Formulas table. Click the checkbox to measure G factor (s) during the scan, or specify G-factor(s) beforehand in the field.
Page 174: Measuring The G Factor
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Measuring the G factor Include the grating factor, or G factor, whenever polarization measurements are taken. The G factor corrects for variations in polarization wavelength-response for the emis- sion optics and detectors. A pre-calculated G factor may be used when all other ex- perimental parameters are constant.
Page 175 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers In the Experiment Setup window, click the Detec- tors icon. This shows the parameters related to detectors, including the G factor, in the Polari- zation area. Click the G Factor checkbox to include a G fac- tor in your measurements.
Page 176: Maintenance
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Maintenance Like all optics, polarizers should be handled with care and stored properly. With proper care, a polarizer should last for many years. Aside from installation, removal, and stor- age, there is no routine maintenance necessary for a polarizer. Polarizers should be re- moved and stored when not in use.
Page 177: Troubleshooting
Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Troubleshooting For difficulties with polarizers, consult the table below to see if your question is an- ® swered here. Otherwise, reach Spex Fluorescence Service at HORIBA Jobin Yvon by phone, fax, or e-mail. Before contacting us, please follow the instructions below: Note the problem and record any error mes- sages.
Page 178 Fluorolog-3 v. 3.2 (2 May 2008) Automated Polarizers Problem Cause Possible Remedy Improper sample concen- Adjust sample concentration. Poor polarization data tration Photomultiplier saturated; Check that sample signals are slits improperly set in linear region (< 2 × 10 on S or T, < 10 µA on R). Re- set slits.
Page 179: 10: Phosphorimeter Operation
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation 10: Phosphorimeter Operation Introduction ® ® The FL-1040 phosphorimeter, used with the Fluorolog -3 and Fluorolog -Tau-3 spec- trofluorometers, allows measurement of long-lived luminescence from samples, espe- cially that caused by phosphorescence. The phosphorimeter includes a pulsed xenon lamp, plus gating electronics to control the size and temporal displacement of the detec- tion window.
Page 180: Theory Of Operation
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Theory of operation A second source of illumination, a pulsed xenon lamp, is used for phosphorescence measurements. Samples are excited with pulsed light; the emitted phosphorescence is measured using an R928P photon-counting detector with a variable delay and open window between pulse and detection.
Page 181 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation saved to disk. The data then can be manipulated using the Analysis menu. Parameters involved in phosphorimetry Lamp Lamp pulse pulse Time per flash Initial delay Sample window t = 0 This sequence of excitation, delay, and sampling, is repeated for each lamp flash. The signal is accumulated for a predetermined number of excitation pulses, then FluorEs- sence™...
Page 182 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation the lamp. Initial Delay can be varied with time to yield a decay curve. Spec- tra can be scanned to isolate different phosphorescing components based on the lifetime of the luminescent species in the sample. To- gether, these two techniques can be used to create three- dimensional plots.
Page 183: Applications For The Phosphorimeter
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Applications for the phosphorimeter Phosphorescence decay curve With the Phos experiment type in the Fluorescence Experiment Menu, create a phospho- rescence-decay curve, as in the screenshot from FluorEssence™ shown above. This is...
Page 184 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Isolate components in a mixture based on lifetimes To the right are three scans of an aqueous mixture of terbium and europium chlo- rides that isolate different phosphorescent compo- nents based on their life- times.
Page 185 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Kinetic analysis of mixtures Often a sample containing a mixture of components can be analyzed through fitting its phosphorescence-decay curve. Here is a phosphorescence decay of an aqueous mixture of EuCl and TbCl , whose different lifetimes have been extracted by FluorEssence™’s...
Page 186: Installation
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Installation Warning: Untrained personnel should not attempt to install or calibrate this accessory. High voltage exists inside the lamp housing. ® The phosphorimeter must be installed and calibrated by a Spex Fluorescence Service Engineer.
Page 187: Operation Of The Phosphorimeter
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Operation of the phosphorimeter Start-up Load an appropriate instrument configuration that includes the phosphorimeter. FluorEssence™ features Experiment Setup window The Experiment Setup window, under the Detectors icon, includes a Phosphorimeter area, indicating that the phosphorimeter is available.
Page 188 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Excitation These are similar to the standard excitation, and emission synchronous scans. Among the changes are that the Integration Time field is removed, Emission and instead are data-entry fields for Sample window, Delay after flash, Time per flash, and Flash count.
Page 189: Processing Phosphorimeter Data
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Processing phosphorimeter data Open the graph to be processed. Click on the data points to be processed. In the toolbar, choose Analysis. A drop-down menu appears. Choose Fit. From the sub- menu, choose the type of analytical curve to use.
Page 190: Lamp Replacement
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Lamp replacement The xenon flash lamp (part # FL-1035) typically has a half-intensity life of at least 10 million flashes. If you hear an abnormal click with each flash or missed flash, this is an indication that the flash lamp is failing.
Page 191 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Hazards Xenon-arc lamps are an explosion hazard. Wear explosion-proof face-shield and protective clothing when opening the lamp housing and handling the lamp. Disconnect the lamp power supply from the AC power line (mains) while han- dling lamp leads.
Page 192 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Be sure that the SpectrAcq controller itself, and the lamp-housing’s two power switches, are all turned off. Warning: High voltage exists within the lamp housing. To avoid fatal shocks, follow these precautions: Be-...
Page 193 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Remove the flash lamp. Follow all safety precautions on the new lamp’s box. Pull the old flash lamp out with a steady motion. flip mirror Warning: Be sure not to hit the flip mirror with the flash lamp.
Page 194 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Gently rotate the lamp-housing cover with its cooling fans at- tached. Rotate the cover backwards, and set it behind the instrument so that electrical connections are not strained. Remove the flash lamp.
Page 195 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Replace the lamp-housing cover. Re-attach the 9 Phillips-head screws. Reconnect all cables. 10-17...
Page 196: Troubleshooting
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Troubleshooting Troubleshooting chart The phosphorimeter accessory has been designed to operate reliably and predictably. If there is a problem, examine the chart below. If all else fails try the steps in the Trouble- shooting chapter, in the “Further Assistance...”...
Page 197: Phosphorimeter Trigger
Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Phosphorimeter trigger Introduction There are two important pins to consider: Pin 5 is for the flash lamp, which provides a 12.8-µs pulse, active LOW. The acquisition delay starts on the HIGH→LOW transition (<...
Page 198 Fluorolog-3 v. 3.2 (2 May 2008) Phosphorimeter Operation Typical circuitry Input A typical input circuit to the user trigger might look like this: USER SPECTRACQ USER I/O CONNECTOR +5V (user supplied) Input opto power Pin 1 470 Ω 74HC or HCT logic...
Page 199: 11: Applications
11: Applications Introduction HORIBA Jobin Yvon Inc. realizes the importance of accurate and reproducible data. Therefore, all systems are calibrated and tested prior to shipment to a customer site and then again after installation. Performance spectra indicate the operational parameters ®...
Page 200 HORIBA Jobin Yvon spectrofluorometers have carved a niche in the scientific com- munity by consistently demonstrating the capability to perform extraordinary tasks. Some of the remarkable features of the system are outlined below.
Page 201: Detecting Sub-Picomolar Concentrations Of Fluorescein
Fluorolog-3 v. 3.2 (2 May 2008) Applications Detecting sub-picomolar concentrations of fluorescein Instrument sensitivity is often ex- pressed in terms of the limit of detec- tion of a standard substance. The supe- ® rior sensitivity of Fluorolog -3 systems is demonstrated by their ability to de-...
Page 202: Fluorescence Detection Of Highly Scattering Samples
Fluorolog-3 v. 3.2 (2 May 2008) Applications Fluorescence detection of highly scattering samples Qualitative and quantitative determinations normally are difficult to ascertain from highly scattering samples. Typically, fluorescence signals are dwarfed by stray or scat- ® tered light from the sample. The flexibility of the Fluorolog...
Page 203: Characterizing Complex Mixtures Via Synchronous Scanning
Fluorolog-3 v. 3.2 (2 May 2008) Applications Characterizing complex mixtures via synchronous scanning The fluorescence spectrum of a complex mixture often contains overlapping spectral features representative of the mixture and revealing no indications of the contents of the sample. A spectrum of this nature is all but useless. Synchronous scanning offers a so- lution to this problem.
Page 204: Phosphorescence For Time-Resolved Data
Fluorolog-3 v. 3.2 (2 May 2008) Applications found in conventional instruments. Because the most widely used photomultiplier de- tector is insensitive above 860 nm, an IR spectrofluorometer must be equipped with a red-sensitive photomultiplier, or a solid-state detector whose response is effective far into the IR region.
Page 205: Low-Temperature Scans
Fluorolog-3 v. 3.2 (2 May 2008) Applications Low-temperature scans One way to protect a sample from molecular collisions that can quench luminescence is by isolating the sample in a rigid matrix. Thus, cooling with liquid nitrogen enhances the phenomenon of fluorescence, even for seemingly dormant samples. In addition, the ®...
Page 206: Polarization To Detect Trace Quantities Of Biological Probes
Fluorolog-3 v. 3.2 (2 May 2008) Applications swing-away mirror is positioned to allow collection of sample luminescence at 90° to the excitation beam, or front-face at 22.5°. In front-face viewing, the fluorescence is collected from the sample’s surface. Polarization to detect trace quantities of...
Page 207: 12: Xenon Lamp Information & Record Of Use Form
Fluorolog-3 v. 3.2 (2 May 2008) Xenon Lamp Information 12: Xenon Lamp Information & Record of Use Form Xenon lamps typically are used in fluorescence instruments because they provide a ® continuous output from 240 nm to 600 nm. In the Fluorolog -3 spectrofluorometers, the standard xenon lamp is ozone-free.
Page 208 Fluorolog-3 v. 3.2 (2 May 2008) Xenon Lamp Information 12-2...
Page 209: Xenon Lamp Record Of Use
Fluorolog-3 v. 3.2 (2 May 2008) Xenon Lamp Information Xenon Lamp Record of Use Page _____ of _______ In Service Current Time Time Total Time Date Operator Date (Hours/Min.) Total Hours 12-3...
Page 210 Fluorolog-3 v. 3.2 (2 May 2008) Xenon Lamp Information 12-4...
Page 211: 13: Triax Operation With The Fluorolog ® -3
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 13: TRIAX operation with the Fluorolog ® Introduction The TRIAX series of imaging spectrometers is a standard building block in a ® Fluorolog -3 spectrofluorometer. Using a TRIAX imaging spectrometer on the emis- sion side of the sample mount offers the option of detection with a CCD, to create an image of the dispersed fluorescence for subsequent analysis.
Page 212: Hardware
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Hardware Electrical power Have a free AC (mains) outlet available for the TRIAX. A 24-V adapter and power supply plugs into the AC circuit, and connects to the back of the TRIAX. The power supply contains a voltage converter that automatically adjusts to the input AC voltage.
Page 213 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Turret installation Reverse steps Caution: Leave clear- 1 to 3. ance under the collar when replacing the top The turret is self- shim-washer. This en- indexing, so the initial position does not sures free rotation of matter.
Page 214 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Attachment to sample compartment Lateral entrance port (with ® Fluorolog -3 users typically choose the lat- baseplate) eral entrance port as the path for lumines- cence from the sample compartment into the TRIAX.
Page 215: Software
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Software Experiment Setup To run a CCD acquisition with an instrument layout that includes a TRIAX, On the main FluorEssence toolbar, select the Ex- periment Menu button: The Fluoresence Main Ex- periment Menu appears: Choose Spectra.
Page 216 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Use the default parameters or adjust them. This is an example using an emission scan-type. Under the Monos icon, func- tions specific to the TRIAX are available, such as:...
Page 217 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Monos icon The Monos icon controls many TRIAX-related parameters for various scan-types. Mirrors The Mirrors area controls the entrance and exit flip-mirrors’ positions. Choose Axial or Lateral positions for each mirror, from drop-down menus.
Page 218 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Gratings The Gratings area allows you to choose, from a Note: To choose a grating, the grat- drop-down menu, which ings must have been previously en- grating to use.
Page 219 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Multi Channel Detectors icon To adjust parameters related to the CCD detector, click on the Multi Channel Detectors icon in the Experiment Setup window. Acquisition Parameters Under Acquisition Parameters, change the Exposure Time, and view the Gain, and the precision of Analog-to-Digital Conversion (ADC).
Page 220 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 choosing which pixels are used on the CCD: graphically or tabular. To choose the active pixel areas graphically, click the Free Form Area tab, and draw with the cursor or enter the coordinates under the X and Y columns. Choose the number of active areas with the Areas field.
Page 221 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Real Time Control In Real Time Control, many TRIAX parameters can be adjusted in real time. To gain access to the TRIAX in the Real Time Control, click the Monos icon. The TRIAX, when used as an emission spectrometer in L-format, is listed under the Emission 1 tab.
Page 222 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Monitoring temperature of the detector ® Click the Symphony button to access the functions for the Symphony controller. In the Acquisition Parameters area, click the Advanced button to open the Multichannel Detector Advanced Parameters window.
Page 223: System Configuration Window
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 System Configuration window To set the cooled detector’s temperature, use the System Configuration window. In the FluorEssence toolbar, choose Collect. A drop-down menu appears. Choose Advanced Setup. A sub-menu appears.
Page 224 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Enter the desired temperature for the detector, then click OK. The Configure [Symphony] window closes. Click OK to confirm and close the System Con- figuration window. 13-14...
Page 225: Correcting Data With The Triax
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Correcting data with the TRIAX Introduction General comments about data correction are found in Chapter 5: Optimizing Data. This section deals with the procedures that are different when using the TRIAX.
Page 226 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Mcorrect.spc The default emission correction-factor file for an instru- ment, with no TRIAX, with only one emission grating and detector. MC9286x300.spc An emission correction-factor file for an R928P PMT, with a grating of 600 grooves/mm and a blaze at 300 mm.
Page 227 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Choose Advanced Setup, then System Configuration. The System Configuration window appears. Choose Preferences, then the Instrument Correction Files icon. 13-17...
Page 228 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 The Instrument Correction Files area should display a correction file for the Detector (Symphony or other special TRIAX-related controller and detector). If not, click the Insert button, and browse for the desired correction file (mc…...
Page 229 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Choose Setup. Choose the Detectors tab, S, from the Available Devices area, and confirm that the Symphony or other special detector/controller combi- nation is correct in the Available Slots area.
Page 230 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 The Configure [Emission 1] window appears. Click the AccessoryConfig button. The Monochromator Accessories Configuration window opens. (FluorEs- sence™ considers gratings within a TRIAX to be accessories.) Check the Turret checkbox to activate the grating turret in the TRIAX, then click OK.
Page 231 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Click OK to close the System Configuration window. Choose correction when setting up the experi- ment. In the Experiment Setup window, choose the Detectors icon to display the detectors’ parameters.
Page 232 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Correcting data after acquisition To apply the correction factors after the data have been acquired, multiply the data file by the appropriate correction factor file (mc… for the S detector, xc… for the R detector, or tc…...
Page 233: Triax 320 Specifications
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 TRIAX 320 Specifications Focal length 0.32 m Entrance-aperture ratio f/4.1 Grating size 68 mm × 68 mm Image magnification at exit 1.00 Scanning range 0–1500 nm (with 1200-grooves/mm grating) Multichannel coverage 79.2 nm over 30-mm array width (with 1200-...
Page 234: Troubleshooting
Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Troubleshooting Should there be a problem with the TRIAX spectrograph, check the following chart for ® possible problems. Try the remedies listed on these pages before contacting Spex Fluorescence Service. See the Troubleshooting chapter for information on how to con- ®...
Page 235 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 Signal is too weak. Try to increase signal strength at the detector. Signal is too noisy Light leak. Follow directions above for detecting light leaks. Improper grounding. Exit FluorEssence™.
Page 236 Fluorolog-3 v. 3.2 (2 May 2008) TRIAX operation with the Fluorolog-3 13-26...
Page 237: 14: Introduction To Lifetime Measurements
14: Introduction to Lifetime Measurements Introduction ® The standard Fluorolog -3 spectroscopy system is designed to perform steady-state measurements. HORIBA Jobin Yvon offers two methods of upgrading your ® Fluorolog -3 system to lifetime capability. ® 1) Fluorolog -Tau-3, which uses the frequency domain for lifetime detection;...
Page 238: Lifetime Measurements
Fluorolog-3 v. 3.2 (2 May 2008) Introduction to Lifetime Measurements ® TCSPC on Fluorolog lifetime system ® The wide range of pulsed sources with the TCSPC on Fluorolog system allow you to measure slow decays of phosphorescence to rapid picosecond fluorescence lifetimes.
Page 239 Fluorolog-3 v. 3.2 (2 May 2008) Introduction to Lifetime Measurements φ . In a fluorescence-lifetime measurement, the phase angle, , and demodulation fac- tor, m, are measured at each frequency and used to calculate the phase lifetime (τ ) and modulation lifetime (τ...
Page 240: Types Of Lifetime Scans
Fluorolog-3 v. 3.2 (2 May 2008) Introduction to Lifetime Measurements Types of lifetime scans The type of scan defines which measurement will be acquired. In lifetime operation, four scan types are available: • Lifetime • Lifetime-resolved • Dynamic depolarization or anisotropy-decay •...
Page 241: 15: Technical Specifications
Fluorolog-3 v. 3.2 (2 May 2008) Technical Specifications 15: Technical Specifications ® Each Fluorolog -3 system consists of: • An excitation source • An excitation monochromator • A sample compartment with reference detector • At least one emission monochromator • At least one emission detector.
Page 242: Spectrofluorometer System
Fluorolog-3 v. 3.2 (2 May 2008) Technical Specifications Spectrofluorometer system ® The Fluorolog -3 spectrofluorometer consists of modules and components controlled by the specialized software. Although the system can be configured in various ways for ® a variety of applications, the basic (standard) Fluorolog...
Page 243 Fluorolog-3 v. 3.2 (2 May 2008) Technical Specifications Solid-state detectors for higher wavelength emissions. CCD multichannel detector for instant emission spectra and sample spatial information. Frequency domain: Lifetime Options Lifetime range: 10 picoseconds to 10 microseconds Frequency range: 0.2 to 310 MHz.
Page 244: Minimum Computer Requirements
Fluorolog-3 v. 3.2 (2 May 2008) Technical Specifications Minimum computer requirements Pentium IV or higher recommended Microprocessor ® Windows 2000 or XP Pro Operating system and envi- ronment 1.4 MB, 3½″ floppy-disk drive Floppy drive At least 80 GB of free storage...
Page 245: 16: Components & Accessories
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 16: Components & Accessories ® The Fluorolog -3 can be configured to obtain optimum results for a variety of applica- tions. The basic system, regardless of configuration, consists of slits, detectors, a xenon lamp with power supply, and a sampling module.
Page 246: Itemized List
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Itemized List Item Model Page Assembly, Liquid Nitrogen Dewar FL-1013 16-3 Cell, HPLC Flow 1955 16-4 Cell, Micro F-3011 16-4 Cell, Micro F-3012 16-4 Cell, Quartz 1925 16-4 Cell, Sample 1920...
Page 247: Fl-1013 Liquid Nitrogen Dewar Assembly
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1013 Liquid Nitrogen Dewar Assembly Warning : Refer to your Material Safety Data Sheet (MSDS) for information on the hazards of cryogenic materials such as liquid nitrogen. For phosphorescence or delayed fluorescence...
Page 248: Sample Cells
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Sample Cells 1955 HPLC Flow Cell With a sample capacity of 20 μL, this non-fluorescing fused silica cell is ideal for on-line monitoring of fluorescent samples. The cell maintains high sensitivity because...
Page 249: F-3026 Correction Factor Kit
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-3026 Correction Factor Kit The F-3026 Correction Factor Kit is an NIST-traceable accessory for the ® ® FluoroMax and Fluorolog spec- trofluorometers for calibrating the relative spectral response of the spectrofluorometer, and includes a ®...
Page 250: Cm-Mh Monolayer Coverslip
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories CM-MH Monolayer coverslip The CM-MH system is designed to assist researchers in recording cation measurements of live cells grown on the special CM-MH monolayer coverslip, or other multiple-wavelength applications. A diagram of the CM-MH accessory is shown here. The coverslip is approximately 5 mm ×...
Page 251: F-3023 Janis Cryostat
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-3023 Janis cryostat The F-3023 Janis cryostat is a liquid-nitrogen variable- temperature cryostat with the sample located in flowing vapor. Ideal for experiments with samples that are difficult to thermally anchor e.g., liquids or powders, the F-3023 features a top-loading sample chamber for rapid sample-exchange, and four-way f = 1.0...
Page 252: 1967 Photodiode Reference Detector
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1967 Photodiode Reference Detector The Photodiode Reference Assembly monitors the xenon lamp up to 1 μm. This acces- ® sory is standard in the Fluorolog -3 package, but a photomultiplier can be substituted instead (necessary for phosphorescence measurements).
Page 253: Ccd Detectors
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories CCD Detectors For multichannel spectral acquisition, many charge- coupled devices are available to suit the researcher’s needs. Both air-cooled and liquid-nitrogen-cooled ® CCDs can be inserted into the Fluorolog -3. Available...
Page 254: 1911F Room Temperature Signal Detector
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1911F Room Temperature Signal Detector ® The Fluorolog -3 includes a room temperature R928P emission signal detector. This detector is mounted to the emission monochromator and operated in the photon- counting mode.
Page 255: 1914F Thermoelectrically Cooled Signal Detector
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1914F Thermoelectrically Cooled Signal Detector As a rule, cooling a detector improves the S/N ratio by reducing the inherent dark count or noise. For the standard signal detector, 1911F (described above), cooling reduces the dark count from 1000 cps to 20 cps;...
Page 256: Fl-1030 Thermoelectrically Cooled Near-Ir Photomultiplier Tube
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1030 Thermoelectrically Cooled Near- IR Photomultiplier Tube For spectral measurements extending into the near-infrared, the FL-1030 Thermoelec- trically Cooled PMT is perfect. Included in the FL-1030 is the thermoelectrically cooled housing. The InGaAs detector has a spectral range from 250 nm all the way to 1050 nm.
Page 257: F-3000 Fiber Optic Mount And 1950 Fiber Optic Bundles
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-3000 Fiber Optic Mount and 1950 Fiber Optic Bundles Now you can study marine envi- ronments, skin and hair, or other large samples in situ! For those us- ers who want to examine samples...
Page 258: 1938 And 1939 Cut-On Uv-Visible Filters
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1938 and 1939 Cut-On UV-Visible Filters The 1938 Cut-On Filter Set consists of 5 filters with dimensions of 1″ × 2″ (2.5 cm × 5 cm). To properly position the filter,...
Page 259: Fl-1010 Cut-On Filter Holder
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1010 Cut-On Filter Holder Cut-on filters are used to eliminate second-order effects of the gratings. The sample compartment has three slots that can hold the FL-1010 Filter Holder. Refer to 1938 or 1939...
Page 260: Fl-1001 Front-Face Viewing Option
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1001 Front-Face Viewing Option Designed to examine fluorescence from the surface of solid samples, the FL-1001 Front-Face Viewing Option includes a swing-away mirror. This allows the researcher to change from front-face and right-angle data collection instantly. In the front-face col- lection mode, the viewing angle is 22.5°.
Page 261: Gratings
• 250 nm • 330 nm • 500 nm • 750 nm • 1000 nm All gratings are classically ruled, and measure 50 mm × 50 mm. For details and model numbers, contact a HORIBA Jobin Yvon Inc. Sales Representative. Grating. 16-17...
Page 262: Fl-1011 Four-Position Thermostatted Cell Holder
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1011 Four-Position Thermostatted Cell Holder The FL-1011 Four-Position Thermostatted Cell Holder keeps a sample at a constant temperature from –20°C to +80°C. The temperature is maintained by an ethylene- glycol–water mixture pumped through from an external circulating temperature bath (not included).
Page 263 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Place the sample in a 10 mm × 10 mm cuvette and insert a magnetic stirring bar. (The stirring bar is available from Bel-Art Products, Pequannock, NJ) Place a cuvette in each holder.
Page 264: Fl-1012 Dual-Position Thermostatted Cell Holder
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1012 Dual-Position Thermostatted Cell Holder The FL-1012 Dual-Position Thermostatted Cell Holder keeps a sample at a constant temperature from –20°C to +80°C. The temperature is maintained by an ethylene- glycol–water mixture pumped through from an external circulating temperature bath (not included).
Page 265 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Place your sample in a 10 mm × 10 mm cuvette and insert a magnetic stirring bar. (The stirring bar is available from Bel-Art Products, Pequannock, NJ) Place a cuvette in each holder.
Page 266: Fl-1027 Single-Position Thermostatted Cell Holder
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1027 Single-Position Thermostatted Cell Holder The FL-1027 Single-Position Thermostatted Cell Holder keeps a sample at a constant temperature from –20°C to +80°C. The temperature is maintained by an ethylene- glycol–water mixture pumped through from an external circulating temperature bath (not included).
Page 267 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Place your sample in a 10 mm × 10 mm cuvette and insert a magnetic stirring bar. (The stirring bar is available from Bel-Art Products, Pequannock, NJ) Place the cuvette in the holder.
Page 268: 1933 Solid Sample Holder
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1933 Solid Sample Holder The 1933 Solid Sample Holder is designed for samples such as thin films, powders, pellets, microscope slides, and fibers. The holder consists of a base with graduated dial, upon which a bracket, a spring clip, and a sample block rest.
Page 269 FF (front-face). Note: Before scanning a solid sample, HORIBA Jobin Yvon Inc. rec- ommends running a water Raman scan in a cuvette with front-face detection. This ensures accurate alignment of the 1692M Selection Mirror.
Page 270: Fl-1039 Xenon Lamp Housing
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1039 Xenon Lamp Housing The FL-1039 is the standard lamp housing for the 450-W Xenon Lamp. The power supply is included internally. FL-1040 Dual Lamp Housing The FL-1040 Dual Lamp Housing contains both the standard continuous 450-W xenon lamp and a UV xenon flash tube.
Page 271: F-3005/6 Autotitration Injector
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-3005/6 Autotitration Injector For controlled, automatic injection of aliquots into the sample of your choice, the F-3005/6 Autotitration Injector is just the thing, available in both 110-V (F-3005) and 220-V (F-3006) models.
Page 272: Microscope Interface
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Microscope Interface The Microscope Interface eases the ® use of the Fluorolog -3 system for fluorescence-microscopy measurements. The accessory in- cludes fiber-optics to bring excitation light to the microscope’s stage and...
Page 273: 1907 450-W Xenon Lamp
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 1907 450-W Xenon Lamp The 1907 450-W xenon lamp delivers light from 240 nm to 850 nm for sample excita- tion. The lamp has an approximate life of 2000 hours, and is ozone-free. The lamp is designed to fit into the FL-1039 Xenon Lamp Housing and the FL-1040 Dual Lamp Housing.
Page 274: Fc-Op-Lio1 And Fc-Op-Lio2 Laser Input Optics Accessories
LIO2 enables you to perform anisotropy and polarization measurements, with the FC- OP-LIO1. It includes a Babinet-Soleil compensator, and two extra prism polarizers in mounts. Contact your HORIBA Jobin Yvon Sales Representative for details. Note that the laser itself is not included in these accessories.
Page 275: F-3004 Sample Heater/Cooler Peltier Thermocouple Drive
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-3004 Sample Heater/Cooler Peltier Thermocouple Drive For rapid control of the sam- ple’s temperature ® Fluorolog -3’s sample com- partment, choose the F-3004 Peltier Drive. Instead messy fluids, the Peltier de-...
Page 276: Phosphorimeter Accessory
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Phosphorimeter Accessory The phosphorimeter adds a programmable, pulsed excitation source and selectable sig- nal gating from the signal photomultiplier tube. This provides time-discrimination ca- pability to sort out the lifetimes of simultaneous, competing luminescence emissions.
Page 277: Micromax 384 Microwell Plate Reader
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories MicroMax 384 Microwell Plate Reader The MicroMax 384 Microwell Titer-Plate Reader allows multiple samples to be scanned in one experiment. The MicroMax 384 is controlled through the FluorEs- sence™ software via a serial port to the host computer. The titer plate moves beneath a stationary optical beam, and fluorescence measurements are collected with top-reading geometry.
Page 278: Fl-1044 L-Format Polarizer & Fl-1045 T-Format Polarizer
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1044 L-Format Polarizer & FL-1045 T- Format Polarizer For L-format spectrofluorometers, the FL-1044 dual polarizer is ideal. The kit includes two polarizers, to be placed at the entrance and the exit of the sample compartment.
Page 279: Fl-1015 Injector Port
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories FL-1015 Injector Port For the study of reaction kinetics, such as Ca measurements, the FL-1015 Injector Port is ideal. This accessory allows additions of small volumes via a syringe or pipette to the sample cell without removing the lid of the sample compartment.
Page 280: Quantum-Yield Accessory
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Quantum-Yield accessory ® The integrating sphere is used in the Fluorolog spectrofluorometer to study fluorescence from solid and liquid samples. The sphere has an inter- nal diameter of 4″ (10 cm). Of special interest is...
Page 281: Sfa-20/Spex Stopped-Flow Accessory
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories SFA-20/SPEX Stopped-flow accessory The SFA-20/SPEX series of stopped-flow rapid-kinetics accessories offers versatility for spectroscopic monitoring of fast reactions in solution. In addition to the conventional two-syringe mixing system, there is also a three-syringe...
Page 282: Tcspc Upgrades Fl-1054, Fl-1057, Fl-1065, And Fl-1066
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories TCSPC upgrades FL-1054, FL-1057, FL- 1065, and FL-1066 Now you can have The World’s Most Sensitive Spectrofluorometer you’ve ® come to expect in the Fluorolog with the bonus of pico- and nanosecond lifetime capability.
Page 283: F-1000/1 Temperature Bath
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories F-1000/1 Temperature Bath For studies of samples whose properties are temperature-dependent, use the F-1000/1 Temperature Bath. The controller circulates fluids externally, with tubes leading to the sample chamber. The temperature range is from –25°C to +150°C. Sensor and all ca- bles are included with the F-1000/1.
Page 284: Trig-15/25 External Trigger Accessory
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories TRIG-15/25 External Trigger Accessory The TRIG-15/25 accessory permits the fluorescence system to be operated with almost any external trigger stimulus. Data acquisition can be synchronized with external events, either automatically following a voltage pulse (minimum 3 V above ground), or manually by pushing a button on a trigger-release cable.
Page 285: Fl-1024 Windows On The Fluorolog
Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories ® FL-1024 Windows on the Fluorolog sample compartment The FL-1024 windows are useful when the sample compartment contains a sample chilled with liquid-nitrogen, in order to prevent condensation on the optics. With the windows installed, the sample compartment can be purged with dry nitrogen gas.
Page 286 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Replace the beam-splitter assembly. Remove old beam-splitter assembly from external wall of sample compartment. Insert two 6-32 × ⅜″ cap screws in the side holes of the new beam-splitter assembly. New beam-splitter assembly.
Page 287 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Replace mirror and baffles. Replace mirror on mount. Replace cap screw on back of mirror. Tighten set screw on top of mirror. Center the excitation beam’s image on photodiode. Loosen two cap screws.
Page 288 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Screw in manually the new metal purge port. Tighten with a 7/16″ wrench. Remove the plastic cap whenever a hose is to be attached to the new purge port. Note: The plastic cap also pre- vents light from entering the sample compartment.
Page 289: 17: Reassemby Instructions
Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions 17: Reassembly Instructions ® The Fluorolog -3 system consists of four main components: Caution: HORIBA Jobin • Personal computer Yvon Inc. does not rec- • Color monitor ommend reassembly by • Expanded keyboard the user.
Page 290: Spectrofluorometer Assembly
Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions Spectrofluorometer assembly ® Once the computer has been assembled, the external components of the Fluorolog ® spectrofluorometer must be connected. The modules of the Fluorolog -3 fit together in a seamless configuration. Each module and the lamp housing has alignment studs,...
Page 291: Cable Connections
Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions Cable connections These connectors connect spectrofluorometer components with BNC Connector SpectrAcq (SAC) and D-shell Connector the computer system. MHV Connector SHV Connector 4-Pin Circular Connector The jacks on the rear of the SpectrAcq...
Page 292 Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions The cable diagram below shows the path of each cable. During installation, refer to the schematic to ensure proper system interconnections. mains mains mains Lamp System housing controller (SAC, Mono drive SpectrAcq)
Page 293 Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions Connecting the reference detector to the SpectrAcq ® The Fluorolog -3 spectrofluorometer system comes standard with a silicon-photodiode reference detector inside the sample-compartment module. The connections to the ref- erence detector are on the outside front of the sample compartment module. The sample compartment module (reference detector) is joined with the SpectrAcq via a split cable;...
Page 294 Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions Take the end of the #33977 cable that has two connectors. Plug the end with the 4-pin circular connector into the POWER jack of the DM302, and the BNC plug into the OUT jack of the DM302.
Page 295: Connecting Power Cables
Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions Connecting power cables Several components in the system have AC power cables. These items include: • SpectrAcq • CCD-detector power supply • Host computer (and peripherals) • Xenon lamp • Accessories (such as MicroMax, temperature bath, etc.)
Page 296 Fluorolog-3 v. 3.2 (2 May 2008) Reassembly Instructions 17-8...
Page 297: 18: Glossary
Fluorolog-3 v. 3.2 (2 May 2008) Glossary 18: Glossary 3D excitation/emission This maps a specified emission-scan wavelength range using various display excitation wavelengths. 3D synchronous/offset A scan that maps a specified synchronous scan using various offset scan wavelengths between the spectrometers.
Page 298 Fluorolog-3 v. 3.2 (2 May 2008) Glossary detector signal is ratioed to the reference signal, which provides 90% of the correction. To obtain a completely correct scan, the excitation scan acquired in the manner described above is multiplied by correc- tion factors.
Page 299 Fluorolog-3 v. 3.2 (2 May 2008) Glossary Fluorescence lifetime The average length of time that a molecule remains in the excited state before returning to the ground state. Front-face detection A mode of detection in which fluorescence is collected off the front surface of the sample.
Page 300 Fluorolog-3 v. 3.2 (2 May 2008) Glossary resolved emission spectra. Raman scattering Scattering caused by vibrational and rotational transitions. Raman bands generally appear red-shifted relative to the incident electro- magnetic radiation. The primary characteristic of Raman scatter is that the difference in energy between the Raman peak and the inci- –1...
Page 301 Fluorolog-3 v. 3.2 (2 May 2008) Glossary Time-base scan Scan in which the sample signal is monitored while both the excita- tion and the emission monochromators remain at fixed wavelengths. Time-base data are used to monitor enzyme kinetics, dual wave- length measurements, and determine the reaction rate constant.
Page 302 Fluorolog-3 v. 3.2 (2 May 2008) Glossary 18-6...
Page 303 Fluorolog-3 v. 3.2 (2 May 2008) Bibliography 19: Bibliography P.M. Bayley and R.E. Dale, Spectroscopy and the Dynamics of Molecular Biological Systems, Academic Press, London, 1985. R.S. Becker, Theory and Interpretation of Fluorescence and Phosphorescence, Wiley-Interscience, New York, 1969. I.B. Berlman, Handbook of Fluorescence Spectra in Aromatic Molecules, 2 ed., Vols.
Page 304 Fluorolog-3 v. 3.2 (2 May 2008) Bibliography M.A. Konstantinova-Schlezinger, ed. Fluorometric Analysis, Davis Publishing Co., New York, 1965. Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 3 ed., Springer, New York, 2006. _______, ed., Topics in Fluorescence Spectroscopy, Vols. 1–5, Plenum Press, New York, 1991–1998.
Page 305 Fluorolog-3 v. 3.2 (2 May 2008) Bibliography T. Vo-Dinh, Room Temperature Phosphorimetry for Chemical Analysis, Wiley- Interscience, New York, 1984. I.M. Warner and L.B. McGown, ed., Advances in Multidimensional Luminescence, Vols. 1 & 2, JAI Press, Greenwich, CT, 1991–1993. E.L. Wehry, ed., Modern Fluorescence Spectroscopy, Vol. 1–4, Plenum Press, New York, 1975–1981.
Page 306 Fluorolog-3 v. 3.2 (2 May 2008) Bibliography 19-4...
Page 307 Fluorolog-3 v. 3.2 (2 May 2008) Index 20: Index Key to the entries: Times New Roman font..subject or keyword Arial font ........command, 400108 ............. 17-6 menu choice, or data-entry field Arial Condensed Bold font..dialog box 9-CA ............3-16 Courier New font....file name or extension About FluorEssence window ......
Page 308 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories TCSPC upgrade ........ 16-38 baffle....6-7, 6-13–14, 16-41, 16-43 temperature bath........ 16-39 bandpass ....5-6–7, 5-10, 8-5, 9-2, 9-13 thermoelectrically cooled near-IR signal baseplate adapter ......13-4, 13-24 detector.......... 16-12 batch job ..........4-9–10 ®...
Page 309 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories circular frequency ........14-2 Czerny-Turner ......... 15-2 cleaning............9-22 Clear all button ........4-14 click..........10-12, 10-18 CM-MH ...........16-6 damage............0-6 Collect .....5-13, 8-24, 13-13, 13-16 danger to fingers........0-7 ® colloidal silica ......See Ludox dark counts ..
Page 310 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Device drop-down menu... 13-13, 13-19 Exit ............13-11 dimensions ..........15-3 exit port............13-1 Expand button ..........3-6 disclaimer..........0-4 dispersion ..........5-10 Experiment File ........4-24 Experiment File field ......4-3, 4-21 dissolved solids ......... 5-3 divide............
Page 311 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories File Name: field........4-9 fluorophore ...... 9-1, 9-5, 9-15, 14-4 Formula box ........... 8-23 file read error ..........7-2 Formulas list .......... 4-25 Filter Holder........16-14–15 filters ........5-4, 7-2, 8-2, 16-14 Formulas table..5-15, 8-23, 9-19, 13-21 final wavelength.......
Page 312 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories host computer...0-1, 1-3, 2-1, 3-2, 9-24, 13-4, 16-33–34, 17-1–3, 17-6 Janis cryostat ...........16-7 hot equipment..........0-7 HPLC Flow Cell ........16-4 .jyb file ..........4-10 humidity level ........... 1-2 HV............ 9-8, 9-13 kinetics.............9-15...
Page 313 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories low polarization ratio .......9-24 modulation lifetime ......... 14-3 low signal...........7-1 monitor ............1-3 Monitor checkbox ......... 13-12 low-temperature scans ......11-7 ® Ludox ..... 3-16, 9-7, 9-9–10, 9-12, 9-24 Mono column .......... 8-25 mono drive connector......
Page 314 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Open button..........8-15 POLAR.LOG ..........9-9 Open window .......... 8-15 Poor polarization ........9-24 Operations ..........8-23 polarization 2-2, 2-10, 9-1–5, 9-7–8, 9-14–15, Operations tab ........5-11 9-18, 9-20, 9-23, 11-8 optical filter........7-2, 16-15 Polarization area........9-21...
Page 315 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories Run button 3-5, 3-9, 3-12, 4-3–4, 4-10, 4-14, 4-22, 4-24, 8-23, 9-19, 13-6 R .........5-12, 5-14, 9-24, 15-2 Run JY Batch Experiments button .4-1, 4-9 〈r〉 ........9-1, 9-3, 9-7–8, 9-15 R1 ...........3-5, 3-12, 8-23 R1527............9-13...
Page 316 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories ® serial numbers ......3-1, 7-11, 9-23 Spectralon ..........16-5 Service Department 1-4, 6-17, 6-21, 7-1–2, 7- standard...........4-13, 4-15 Standard..........4-13 8, 7-11, 8-1, 9-23, 10-8, 10-18, 13-24 Settings tab ..........5-11 standard lamp....8-1–3, 8-5, 8-7, 8-11 Setup ............
Page 317 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories T-Format Polarizer.........16-34 Thermoelectrically Cooled Near-IR ventilation..........1-2 Photomultiplier Tube ......16-12 Thermoelectrically Cooled Signal Detector version number .........7-9, 7-11 VH ..........9-3, 9-8, 9-13 ............16-11 View System Info button......7-9 thin film..........16-24–25 voltage converter ........
Page 318 Fluorolog-3 v. 3.2 (2 May 2008) Components & Accessories 20-12...
Page 319 3880 Park Ave., Edison, New Jersey 08820-3012, U.S.A. http://www.jobinyvon.com [Design Concept] The HORIBA Group application images are collaged in the overall design. Beginning from a nano size element, the scale of the story develops all the way to the Earth with a gentle flow of the water.

horiba Fluorolog-3 Operation Manual

1 : Requirements & Installation

2 : System Description

3 : System Operation

4 : Data Acquisition

5 : Optimizing Data

6 : System Maintenance

7 : Troubleshooting

8 : Producing Correction Factors

9 : Automated Polarizers

10 : Phosphorimeter Operation

11 : Applications

12 : Xenon Lamp Information & Record of Use Form

13 : TRIAX Operation with the Fluorolog ® -3

System Configuration Window

14 : Introduction to Lifetime Measurements

15 : Technical Specifications

16 : Components & Accessories

17 : Reassemby Instructions

18 : Glossary

Quick Links

Troubleshooting

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Summary of Contents for horiba Fluorolog-3