Horiba Scientific Deltaflex System Manual

Modular fluorescence lifetime system

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DeltaFlex

Modular Fluorescence Lifetime System

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Page 1 DeltaFlex Modular Fluorescence Lifetime System System guide...
Page 3: Table Of Contents
User guide DeltaFlex Modular Fluorescence Lifetime System Table of contents 1. Safety precautions..........................5 1.1 Safety symbols ..........................5 1.2 DeltaFlex safety notice........................6 1.3 DeltaHub safety notice........................6 1.4 NanoLED safety notice ........................7 1.5 DeltaDiode safety notice........................ 7 1.6 SpectraLED safety notice ......................
Page 4 User guide DeltaFlex Modular Fluorescence Lifetime System 4.4 Histogram streaming measurements..................36 4.4.1 Analysis for streamed histogram data..................37 4.4.2 Experimental considerations for streamed histogram measurements ........38 4.5 Photon streaming measurements ....................39 4.6 Automated scripts ........................43 4.6.1 Time-resolved anisotropy measurements.................. 43 4.6.1.1 Anisotropy data analysis ......................
Page 5: Safety Precautions
1. Safety precautions 1.1 Safety symbols Terms and symbols described in this section are used throughout the HORIBA Scientific product line. These symbols may appear in this user guide only, or they may be marked on the outside of the product or other equipment that is intended for use with the product.
Page 6: Deltaflex Safety Notice
1.2 DeltaFlex safety notice WARNING On opening the DeltaFlex sample chamber, care should be taken as the light source can be operative. Practice care when inserting objects into the chamber because of the possibility of reflected or scattered excitation light. Also see safety notices relating to light sources.
Page 7: Nanoled Safety Notice
User guide DeltaFlex Modular Fluorescence Lifetime System Confirm that the product’s input voltage, as indicated next to the IEC mains power inlet, is appropriate for your local mains voltage supply before using for the first time. Different fuse ratings are used for 110V 60Hz and 240V 50Hz. If...
Page 8: Spectraled Safety Notice
If this product is used in a manner not specified by the manufacturer then any safety protection provided by the product may be impaired. This product has no user serviceable parts. Please contact HORIBA Scientific if this product requires servicing. Do not attempt to open the casing. Opening the casing is dangerous and will invalidate your warranty.
Page 9 (e.g. direct laser illumination) at all times. Always ensure that the product is switched off while connecting or disconnecting cables attached to it. If in doubt, please contact your HORIBA Scientific representative for advice. IBH.710.UG.3481.E Page 9 of 77 © HORIBA Scientific...
Page 11: Introduction
UV to the near infrared (NIR). The modular nature of the design means that it can be easily extended and upgraded using the comprehensive range of products available from HORIBA Scientific. This makes DeltaFlex an easy to use, versatile system in the field of lifetime determination, with the high sensitivity associated with the TCSPC technique.
Page 12 User guide DeltaFlex Modular Fluorescence Lifetime System Optical DeltaPro – Platform Filter based, independent sample chamber “DeltaPro” “DeltaPro” DeltaFlex – Fully modular, baseplate mounted optics, eg “T-format” “T-format” “L-format” “L-format” FluoroHub-A+ FluoroHub-A+ DeltaHub DeltaHub Timing Electronics General purpose, high speed...
Page 13 The previous illustration showed the DeltaPro sample chamber, this is a filter based entry level system coupled with DeltaHub timing electronics. It has similar capabilities to DeltaFlex systems, except that the emission is selected using a filter rather than a monochromator. The DeltaPro sample is designed for simplicity and is not suitable for some temperature control options, such as cryostats.
Page 15: Unpacking And Installation
DeltaFlex Modular Fluorescence Lifetime System 3. Unpacking and installation The DeltaFlex fluorescence lifetime system is a sensitive instrument and should be handled with care at all times. Inspect the packaging for signs of rough handling and report any damage to HORIBA Scientific immediately.
Page 16: Mounting
Carefully unpack each component carefully removing any protective packaging material and identify it according to the list above. 3.1 Mounting To prepare the DeltaFlex fluorescence lifetime system for use, place the modules on a flat surface next to your PC. An example is illustrated below (Figure 1), including optional components (*). Avoid positioning the system in a location where, ...
Page 17: Deltadiode / Nanoled / Spectraled Pulsed Light Source
User guide DeltaFlex Modular Fluorescence Lifetime System 3.1.1 DeltaDiode / NanoLED / SpectraLED pulsed light source These sources connect directly to the excitation port and are fastened using a hex key provided. 3.1.2 PPD picosecond photon detection module First remove the protective PVC cap and attach the module by slotting it onto the emission port and then tightening the three grub screws, using the hex key provided, to hold it in position.
Page 18: Your Computer (Usb)
User guide DeltaFlex Modular Fluorescence Lifetime System 3.2.2 Your computer (USB) The Timing electronics connects to the USB port on your PC using the cable provided. Note that the Timing electronics will not function correctly if connected to a USB 1.1 port. The PC requirement is for Windows 7 (32 or 64 bit) or Windows 10, English language version.
Page 19: Simplified Schematic Connection Schemes (Viewed From The Rear)
User guide DeltaFlex Modular Fluorescence Lifetime System 3.2.6 Simplified schematic connection schemes (viewed from the rear) It should be noted, that it is possible to have both (DeltaDiode or NanoLED) and SpectraLED sources connected electrically simultaneously (although only one can be operated at a given time) and the required source then selected in EzTime.
Page 20: Software Installation (Not Necessary If A Computer Has Been Supplied)
3.3 Software installation (not necessary if a computer has been supplied) The DeltaFlex with EzTime acquisition and analysis software is designed for use with Windows 7 (32 or 64 bit) and Windows 10, operating systems (English language version only). During the installation process your equipment does not need to be connected.
Page 21: Operation
DeltaFlex Modular Fluorescence Lifetime System 4. Operation The main interface for performing measurements is the EzTime software, which controls the DeltaFlex through the DeltaHub. The DeltaHub is software driven and does not feature any front panel controls; locations of the signal connections for lifetime measurements with DeltaDiode, NanoLED and SpectraLED sources can be seen in section 3.2.6.
Page 22: General Considerations
Advanced Settings, Start Inputs tab. Note that it is possible to exchange light source heads with the DeltaFlex powered on, although it is recommended to put the key switch on the DD-C1 or NL-C2 controllers to standby first.
Page 23 User guide DeltaFlex Modular Fluorescence Lifetime System (c) The wavelength can be changed by selecting the Optics dialogue on the Instrument page, which will activate a drop down form. Wavelength can be changed either by using the arrows or by typing in the number, followed by Apply or OK.
Page 24 User guide DeltaFlex Modular Fluorescence Lifetime System (h) If the sample requires stirring make sure that an appropriate stirrer bar is present in the cuvette. The stirring speed and direction can be controlled by selecting the dialogue under Sample. Note that if the stirring bar becomes trapped, the “capture” button can be used, which momentarily stops the stirrer and then ramps up the speed to aid coupling between the “stirring”...
Page 25: Performing Time-Resolved Measurements Using Eztime
Launch the EzTime software by clicking on the icon on the desktop. This software should automatically find and initialise any accessories present. If it does not load check the connections and retry. Leave the DeltaFlex until the power indicator on the DeltaHub becomes constant before launching this application.
Page 26: Data Storage Concept
User guide DeltaFlex Modular Fluorescence Lifetime System 4.3.1 Data storage concept Data is stored in “containers” primary “LiveData” container (always positioned uppermost left) is the only container that can be used to collect new data. Any other visible containers are “locked” - no new data can be collected into them.
Page 27: Measurement Set Up - Instrument Page
User guide DeltaFlex Modular Fluorescence Lifetime System 4.3.2 Measurement set up – Instrument page The conditions, such as time range, optical parameters and count rate can be set up in the Instrument page. This is selected by clicking (tapping) on Instrument on the top on the right of the EzTime form. It is recommended that these parameters are set up and optimised prior to performing a measurement.
Page 28 User guide DeltaFlex Modular Fluorescence Lifetime System (b) Time Range control This dropdown control selects the time window to be measured. Changing the Time Range also selects the allocated excitation source (chosen in Advanced Settings) and sets the optimal operating parameters for that source to suit the newly selected Time Range e.g.
Page 29: Data Collection - Data Page
User guide DeltaFlex Modular Fluorescence Lifetime System (d) The count rate can be seen using the Ratemeter tab. The alpha indicator which relates to the stop to start ratio will change colour (red from green) if the recommended 2% ratio is exceeded.
Page 30 User guide DeltaFlex Modular Fluorescence Lifetime System (a) Configuring peak or run time presets Selecting to the right of the start/stop measurement button opens the Measurement Settings form. Here the presets for new lifetime and steady measurements may be entered.
Page 31 User guide DeltaFlex Modular Fluorescence Lifetime System (c) Saving data and making new measurements New measurement nodes can created by: (i) Adding individual nodes (IRF or decay) - right click or tap measurement to open radial menu... (ii) Adding measurement pairs i.e. a measurement node with IRF and decay sub-nodes (iii) Selecting New from File menu (left) will open a new measurement container with its own tree view.
Page 32: Data Analysis - Data Page
User guide DeltaFlex Modular Fluorescence Lifetime System 4.3.4 Data analysis – Data page (a) Using Autofit (must be selected prior to measurement) With Autofit the decay data will automatically be analysed using 1, 2 and 3 exponential models. Fits that are saved by user appear under the appropriate decay node in the Data tree.
Page 33: How Do I Know If I Have A Good Data Fit
User guide DeltaFlex Modular Fluorescence Lifetime System (c) Using selected data from the Data Tree – manual fit limits As before select the data that you wish to analyse so that it is present in the Data View. Select the configure icon to the right of the Analyse button and put in the ranges for decay and prompt (if different).
Page 34: Interpretation Of And Exporting Fitted Data
User guide DeltaFlex Modular Fluorescence Lifetime System 4.3.4.2 Interpretation of and exporting fitted data Once the data has been analysed the results can be accessed either by selecting the “view results” option just after the fit has completed or by selecting the fit node in the tree and using a right click or tap to bring up a radial menu with “view results”.
Page 35 User guide DeltaFlex Modular Fluorescence Lifetime System The resultant graphics may be copied directly by using a right click / tap on the selected chart to bring up a radial menu. It is also possible to export data in a spread sheet (work book) format. This can be done by selecting the tabulate function, either from the radial menu when accessing the fit results or when viewing the results, see below.
Page 36: Histogram Streaming Measurements
User guide DeltaFlex Modular Fluorescence Lifetime System 4.4 Histogram streaming measurements (Appropriate hardware required) This mode, selected from the Preview tab on the Instrument page, permits the acquisition and analysis of up to 26,000 decays measured sequentially, without any delay between individual decays. The time per measurement can range from 1 ms to 60 seconds and the measurement can be triggered directly using EzTime or using an external (TTL) trigger connected to the Timing electronics.
Page 37: Analysis For Streamed Histogram Data
User guide DeltaFlex Modular Fluorescence Lifetime System When the data has been collected the corresponding data file will be written and only after Done has been selected should any further measurements or opening of the file be attempted. The length of time for writing the file will depend on the number of decays it contains and when the maximum number of 26,000 is used this can take several seconds and mean that EzTime may not respond.
Page 38: Experimental Considerations For Streamed Histogram Measurements
User guide DeltaFlex Modular Fluorescence Lifetime System The node, as well as giving an indication of changes with time, allows for data manipulation of the histogram stream. Two “sliders” either side of the intensity - time plot allow truncation of the time range.
Page 39: Photon Streaming Measurements
4.5 Photon streaming measurements (Appropriate hardware required) This mode requires the appropriate hardware to be present on the DeltaFlex. If it is not, the Photon Streaming function (see below), found on the Preview tab, will advise if selected. Photon streaming, also known as “time-tagging” or “FIFO mode” records individual photon events rather than sorting them out into histograms.
Page 40 User guide DeltaFlex Modular Fluorescence Lifetime System Enter file name and presets Enter file name and presets Presets are run time and file size. Presets are run time and file size. Selecting “Run” starts the measurement The measurement will stop...
Page 41 User guide DeltaFlex Modular Fluorescence Lifetime System It is from this chart that data can be manipulated and histograms generated, that can be either analysed in a global or batch manner. The bottom intensity – time chart has “sliders” that can be used to select a certain time period, which is then displayed in the upper left panel.
Page 42 User guide DeltaFlex Modular Fluorescence Lifetime System The upper left panel showing the selected time period is scaled so that it contains a maximum of 10,000 points in the chart. This determines the display (not data) resolution and adjacent bins summed to restrict the number of points plotted.
Page 43: Automated Scripts
User guide DeltaFlex Modular Fluorescence Lifetime System 4.6 Automated scripts An easy to use script language is included with EzTime allowing measurements to be made in a predetermined manner. However for common measurements such as anisotropy and time-resolved emission spectra (TRES) there is an automated script feature that guides the user through these simple measurements automatically generating a script to control the experiment.
Page 44 User guide DeltaFlex Modular Fluorescence Lifetime System Selecting Anisotropy brings up the following dialogue, enabling the presets and optical conditions to be set up for the anisotropy measurement. Selects optical path, presets and wavelengths for the decays Conditions for instrumental...
Page 45: Anisotropy Data Analysis
User guide DeltaFlex Modular Fluorescence Lifetime System 4.6.1.1 Anisotropy data analysis It is recommended that the appropriate manual is consulted for full details of the significance of the data and its analysis as only a brief summary is given here.
Page 46: Time-Resolved Emission Spectra (Tres) Measurements
User guide DeltaFlex Modular Fluorescence Lifetime System 4.6.2 Time-resolved emission spectra (TRES) measurements The measurement can be commenced by selecting the required node in the automated script function. It is recommended to limit the measurement to 100 wavelength dependent decays, which are acquired at fixed wavelength increments.
Page 47: Tres Data Analysis
5 linked common lifetimes can be used to describe the data set. This form of analysis also allows for automatic determination of the decay associated spectra. The user is recommended to see the appropriate manual and application notes (in the Fluorescence Lifetime section of the HORIBA Scientific website).
Page 48: Turret Scan Measurements
User guide DeltaFlex Modular Fluorescence Lifetime System 4.6.3 Turret scan measurements To be accessible this automated script requires the sample turret to be present. This feature allows the programmed acquisition from (up to a maximum of) each of the 4 sample positions. By running over several cycles it is also possible to add (append) data into that already acquired for a particular sample position.
Page 49: Steady State Measurements
User guide DeltaFlex Modular Fluorescence Lifetime System 4.7 Steady state measurements Steady state measurements made using the pulsed excitation source can be simply performed by adding an additional node from the radial menu obtained in the Data Tree. This adds a steady state node to that of the IRF and decay.
Page 50: Advanced Functions / Settings
This is an important section as it shows what components are present in the DeltaFlex system and can provide useful diagnostic information. An example of this form is shown below. The population of this form will depend on the system configuration.
Page 51: Other Light Sources
4.10 Initial system check  After the instrument has been set up we recommend that the DeltaFlex is tested using a standard sample of a known lifetime on an appropriate time range and taken to 10000 counts in the peak channel. We would recommend POPOP in methanol with excitation at 375nm and emission at 425nm, which should return a single exponential lifetime of 1.32 ±...
Page 53: Maintenance And Troubleshooting
DeltaFlex Modular Fluorescence Lifetime System 5. Maintenance and troubleshooting 5.1 General The DeltaFlex component parts (Timing electronics, sources, detection module) contain no user serviceable parts. Please contact your local HORIBA Scientific representative if you require technical support. Note – the LED indicators on both the optical system (SDB) and components, principally the DeltaHub and excitation source controller are indicative of the state of the equipment and can be used to assist in trouble shooting any problem, along with the F-Link form (see section 4.7).
Page 54: If No Stop (And / Or Hit) Ratemeter Signal Seen When Expected
If only a “detector noise” signal seen – (a) check DeltaDiode / NanoLED turned on (b) check appropriate filters in sample chamber, ie not too many ND filters and correct emission wavelength. If in doubt, contact your HORIBA Scientific representative for assistance. IBH.710.UG.3481.E Page 54 of 77...
Page 55: Accessing Log Files
EzTime creates a log file that contains details about the particular session of the program. If anything goes wrong with the application then these log files can be a great help to HORIBA Scientific support staff in tracing the problem.
Page 57: Specifications
DeltaFlex Modular Fluorescence Lifetime System 6. Specifications A typical DeltaFlex fluorescence lifetime system consists of a sample chamber, DeltaHub, PPD detection module and NanoLED / DeltaDiode (and / or SpectraLED) source(s) – see component user guides for full specifications. The dimensions (footprint) and weight below are based on a DeltaFlex with DeltaDiode excitation.
Page 59: Appendix A. Luminescence And Techniques To Measure It
User guide DeltaFlex Modular Fluorescence Lifetime System Appendix A. Luminescence and techniques to measure it Background The term luminescence originates from the latin (lumen for light) and was first used as luminesenz by Wiedemann in 1888 for “all those phenomena of light which are not solely conditioned by the rise in temperature”...
Page 60 User guide DeltaFlex Modular Fluorescence Lifetime System Measurement techniques These can be roughly divided into steady state or time-resolved measurements. The former can be used to provide information concerning the wavelength, quantum yield, Stokes’ shift and molecular interactions. Time-resolved measurements also give a measure of how long a molecule spends in the excited state and have the advantage that (within certain limits) they are independent of concentration, as each molecule has its characteristic decay time, independent of how many are present.
Page 61 User guide DeltaFlex Modular Fluorescence Lifetime System fluorescence technique fluorescence technique information information molecular “freedom” or energy molecular “freedom” or energy depolarisation. r depolarisation. r = 0.4 (or -0.2 = 0.4 (or -0.2 detector detector detector detector if perpendicular dipoles), r if perpendicular dipoles), r ...
Page 62 DeltaFlex Modular Fluorescence Lifetime System Fluorescence measured using time-correlated single-photon counting This is the method used to measure fluorescence lifetimes on the DeltaFlex systems and has become the method of choice for those working with short lifetimes, weak fluorophores and high repetition rate excitation sources (>10kHz).
Page 63: Appendix B. Deltapro Sample Chamber
The DeltaPro is a filter based sample chamber with motorised focusing optics and a shutter on the excitation channel. Although it makes use of the same electronics as the DeltaFlex, because it does not comprise of a monochromator, with built in shutter, on the emission channel it is only recommended for use with PPD detectors.
Page 64 User guide DeltaFlex Modular Fluorescence Lifetime System If a PPD-650 is used as the detector, then this can be powered directly from the Aux Power socket on the Timing electronics. If a PPD-850 or PPD-900 is used then a DPS-1 power supply is required and the Interlock cable connected.
Page 65: Appendix C. Fluorohub-A+ Timing Electronics
The FluoroHub-A+ meets these requirements (time bins from 0.3ps and jitter < 10ps FWHM). It can be used in place of the DeltaHub on the DeltaFlex system. The EzTime software will function in the same manner as with the DeltaHub, although there are some different features present on both the Data and Instrument pages.
Page 66 User guide DeltaFlex Modular Fluorescence Lifetime System Instrument page On this page to accommodate the functionality of the FluoroHub-A+ the advanced settings dialogue has some additional features. Time range selection Advanced settings can be used to change sources for TCSPC and Phos plus Stop channel inputs etc.
Page 67 User guide DeltaFlex Modular Fluorescence Lifetime System Histogram size / ADC window In the “General” tab there is the ability to select the histogram size (number of time bins) and for maximum resolution 16k is recommended. In the histogram because of the nature of TAC-based electronics it is advisable to remove time bins at both the beginning and end of the histogram to obtain the best quality data.
Page 68: Appendix D. Detector Options
NIM signal can be obtained from the PHV unit. When considering the inclusion of a MCP in a DeltaFlex system it is recommended that for optimum performance it is used in conjunction with FluoroHub-A+ timing electronics. A schematic of the principal electrical connections are shown below for an example of a DeltaFlex with DeltaDiode controller and FluoroHub-A+ in reverse mode, viewed from rear.
Page 69: Appendix E. Temperature Control Options
DeltaFlex Modular Fluorescence Lifetime System Appendix E. Temperature Control Options There is a choice of temperature control options for the DeltaFlex system and the principal ones are given below. Note only the water bath is compatible with the DeltaPro sample chamber.
Page 70 User guide DeltaFlex Modular Fluorescence Lifetime System In order to set the temperature control device, go to the Instrument page and use the Sample drop down to locate the controller and temperature settings (see right). This enables the setting of the temperature in the software of the supported device.
Page 71 User guide DeltaFlex Modular Fluorescence Lifetime System When using temperature control devices, it is advised that they are turned on before starting the EzTime software and turned off after the software has been closed. If a device is not used, it can be disconnected using the hardware dialogue, so that the EzTime software does not report it as not connected.
Page 72: Appendix F. Sample Holder Options
DeltaFlex Modular Fluorescence Lifetime System Appendix F. Sample Holder Options There is a choice of sample holders available as accessories for the DeltaFlex fluorescence lifetime system, which enable a variety of different sample types to be measured There is a choice of sample holders available as accessories for the DeltaFlex fluorescence lifetime system, which enable a variety of different sample types to be measured.
Page 73: Appendix G. Accessories - Mounting Polarisers
User guide DeltaFlex Modular Fluorescence Lifetime System Appendix G. Accessories – Mounting polarisers The polarisers can either be manually controlled or in motorised mounts. They are supplied in barrel mounts of diameter 30mm and length 25mm as shown in the figure below. Note that the polarising element is located closer to one end of the mount than the other.
Page 74: Appendix H. Computer Settings (Usb / Power)
(USB) settings to avoid them “sleeping” and hence an interruption in communication between the computer and the DeltaFlex system. Also, note that, the USB from the computer should be connected to the Timing electronics, and any subsequent “daisy chained” connection made from this device.
Page 75 2. Open Device Manager and expand the section “Universal Serial Bus Controllers” 3. Select the device “HORIBA Scientific DeltaHub” 4. In the View menu select “Devices by connection”. 5. This view shows a tree with all the USB connections that the hub is attached to.
Page 77: Notes
User guide DeltaFlex Modular Fluorescence Lifetime System Notes IBH.710.UG.3481.E Page 77 of 77 © HORIBA Scientific...

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