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PicoQuant MicroTime 100 User Manual And Technical Data

Fluorescence lifetime microscope
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MicroTime 100
Fluorescence Lifetime
Microscope
User's Hardware Manual and Technical Data
Version 1.3

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Summary of Contents for PicoQuant MicroTime 100

  • Page 1 MicroTime 100 Fluorescence Lifetime Microscope User's Hardware Manual and Technical Data Version 1.3...

  • Page 2: Table Of Contents

    PicoQuant GmbH MicroTime 100 Table of Contents 1. Safety Instructions............................4 2. Introduction..............................6 2.1. Pre-Installation Requirements:......................7 3. System Layout.............................. 9 3.1. Microscope............................10 3.2. Excitation Subsystem.......................... 12 3.2.1. Diode Laser Drivers........................12 3.2.2. Laser Heads..........................13 3.2.3. Laser Combining Unit........................14 3.3.
  • Page 3 PicoQuant GmbH MicroTime 100 6.1. Hardware configuration gets lost or software needs to be installed again...........40 6.2. Instrument is loosing sensitivity......................40 6.3. No image is displayed during measurement..................41 7. Recommended literature..........................41 8. Abbreviations.............................. 42 9. Support and Warranty..........................43...

  • Page 4: Safety Instructions

    PicoQuant GmbH MicroTime 100 1. Safety Instructions LASER Warning! Lasers for two photon excitation deliver infra red emission which is in most cases not visible for the eye. These lasers emit light of laser class 4 / IV. Laser class 3B / IIIb and 4 / IV requires to wear special laser safety glasses.

  • Page 5: Safety Labels On The System

    Nevertheless, please check that the actual line voltage corresponds to the value set on these instruments! If pulsed lasers from PicoQuant are present at the system, please check that the actual line voltage corresponds to the value set on the PDL-800-D or -B laser driver! Never connect or disconnect any cable while the data acquisition and control electronics are ON.

  • Page 6: Introduction

    Laser Combining Unit - Manual (LCU - Manual): This manual should be consulted for information • about the laser combining unit, which contains the lasers of your MicroTime 100, if the system is equipped with more than 1 laser. It explains setting the correct intensities and also contains a detailed description for realignment.

  • Page 7: Pre-Installation Requirements

    PicoQuant GmbH MicroTime 100 Light Sources: In the PicoQuant folder you'll also find detailed information about the properties of your • pulsed diodes lasers. Router (PHR800): The router is needed in systems equipped with a PicoHarp 300 and more than one •...
  • Page 8 70 x 25 x 55 The delivered instruments are preset by PicoQuant to be operated with either 115V or 230 V AC. This has to be specified by the customer before shipping of the instrument. The system is shipped together with all necessary power distribution blocks and has therefore a single IEC 320-C14 type male power inlet.

  • Page 9: System Layout

    Hand-held laser power meter LASER Warning! All lasers offered by PicoQuant are at least class 3B lasers and therefore require the installation room of the LSM Upgrade kit to be labeled as laser area. For class 3B lasers, a laser safety officer has to be announced to meet the laser safety regulations.

  • Page 10: Microscope

    PicoQuant GmbH MicroTime 100 Fig. 3.1: Scheme of the MicroTime 100 3.1. Microscope The Olympus BX-series are versatile, research grade upright microscopes. A detailed description of its components as well as instructions how to use their controls can be found in the dedicated microscope manual.
  • Page 11 PicoQuant GmbH MicroTime 100 Fig. 3.2: Switches and elements of the Olympus BX microscope: 1) Focussing knob to adjust the sample height. 2) Transmission illumination power switch. 3) Intensity illumination control. 4) Switch for changing between ocular and confocal optics 5) Objective The microscope is equipped with a transmission illumination unit by default.

  • Page 12: Excitation Subsystem

    PicoQuant GmbH MicroTime 100 Fig. 3.4: Settings for the slider inside the UDP-unit. The UDP-unit allows to insert a mirror into the beam path and direct the light to different outputs. When the slider is in (left), a mirror is inserted into the beam path and the laser light is directed onto the sample.

  • Page 13: Laser Heads

    PDL 800-D or the PDL 828 "Sepia II" driver. The family of PicoQuant diode lasers is continuously growing. The up-to-date list of laser heads is available on the PicoQuant website www.picoquant.com. The current spectral coverage is shown in Fig. 3.7.

  • Page 14: Laser Combining Unit

    PicoQuant GmbH MicroTime 100 Fig. 3.7: PicoQuant pulsed diode lasers. The complete list of available laser diiode heads, which also span beyond 700nm is available on the PicoQuant webside. Please inquire for special lines in the infrared region. The exact specification of your diode laser heads can be found in the Laser delivery report.

  • Page 15: Main Optical Unit (Mou)

    Gaussian beam profile at the fiber output is preserved. The optical fiber has FC/APC connectors at both ends. It is suitable for any wavelength between 375 nm and 650 nm. Of course, other wavelengths beyond 650nm can also be fiber coupled; contact PicoQuant to discuss your specific needs.
  • Page 16 (FC/APC standard). The detection fiber has usually a diameter of 50 µm. The tube lens' z-position is pre-set by PicoQuant and should not be changed, because of the distance from the fiber, which serves as a pinhole remains constant. Furthermore, a filter can be inserted before the tube lens, in case a self built detection unit is used that does not contain its own filter inset as PicoQuant's detection units.

  • Page 17: Excitation Segment

    The beam is then focused by an achromatic tube lens to the multi mode fiber (diameter 50µm), which functions as a pinhole. The tube lens' Z-position is pre-set by PicoQuant, but it remains adjustable. When the objective is not apochromatic corrected, the lens position should be optimized for the spectral range of detected emission.

  • Page 18: Detection Unit

    The standard operation in scanning and FCS applications of the MicroTime 100 is based on a special data acquisition mode called Time-Tagged-Time-Resolved (TTTR) mode. This is an advanced and very versatile version of the well known Time-Correlated Single Photon Counting (TCSPC).

  • Page 19: Router And External Synchronization Unit (Optional)

    MicroTime hardware, data acquisition, as well as to provide various data analysis tools. In this sense, it is an integral component of the MicroTime 100 system. The so-called Analysis mode operation is particularly useful for off-line data analysis, since the sample’s lifetime is usually limited and the large amount of information-rich TTTR data can be analyzed conveniently later.

  • Page 20: High Precision Piezo Scanner (Optional)

    Alternate illumination schemes like mercury illumination for brightfield fluorescence may be restricted due to the size of the MicroTime 100. Before undertaking substantial changes in the setup, please contact PicoQuant. Don't apply any changes that might tamper with the laser safety of the system 3.7.1.

  • Page 21: Widerange Scanner (Optional)

    PicoQuant GmbH MicroTime 100 capacitive position sensor, thus the system always knows the actual XYZ focus position. The piezo-controller is based on a 32-bit digital signal processor and communicates with the SymPhoTime 64 software through an RS-232 interface (COM serial port of the PC). Detailed information can be found in the separate user manual of the piezo-controller.

  • Page 22: Operation

    4.1. Safety Instructions Laser Warning The MicroTime 100 is fitted with one or more pulsed diode lasers. To avoid hazardous radiation exposure you should carefully obey the safety instructions that are provided with your PDL diode laser operation manual. If your instrument uses another excitation system, follow the safety instructions of the relevant manual.
  • Page 23 PicoQuant GmbH MicroTime 100 3. Place the sample on the MT100 and focus using wide field illumination. When the sample is placed in focus, switch off the widefield illumination first and set the U-DP to reflect the laser light onto the sample (mirror 1 is slided in).
  • Page 24 PicoQuant GmbH MicroTime 100 6. Alternatively, the focus position can also be determined at a single point. Therefore, switch to “Point/Time Trace, click on “select point” and mark a point in the image. Then start the point measurement. Switch the display to the “Time Trace”-tab and mointor the intensity over time. You can now focus manually, or –...
  • Page 25 PicoQuant GmbH MicroTime 100 in most cases, avoid these problems. Avoiding them in bidirectional scanning mode may involve thorough refining the scan speed values. (b) Widerange scanner Please ensure the stage can move freely during initialization and over the complete selected scanning range, and make sure that sample or objective cannot be damaged during this procedure.

  • Page 26: Optimise The Detected Photon Count Rate

    PicoQuant GmbH MicroTime 100 4.3.2. Optimise the detected photon count rate Adjust the photon count rate by changing the intensity of the pulsed laser at the fiber combining • unit LCU or – if you don't have a LCU, but a directly fiber coupled laser – in an analogous way at the lasers scaffold with the intensity regulation knob.

  • Page 27: Choose The Correct Laser Repetition Rate

    4.3.3. Choose the correct laser repetition rate For PicoQuant's pulsed diode lasers the laser repetition rate can be set at the PDL laser driver. In general the repetition rate should be as high as possible to achieve the highest possible photon count rate. However, the time window after a laser pulse should be still large enough to allow the population of excited dye molecules to deplete completely before the next laser pulse.

  • Page 28: Measure An Instrument Response Function (Irf)

    PicoQuant GmbH MicroTime 100 Every raw date file is stored together with the actual settings. It contains all actual measurement • parameters known by the SymPhoTime software. To access, double click on the raw (“.ptu”)-file. data file contains actual measurement...

  • Page 29: Point Measurements E.g. As Used In Fluorescence Correlation Spectroscopy

    Also, sensitive detectors (SPAD detectors or Hybrid PMTs) are necessary If FCS is performed on an upright system like this MicroTime 100, the sample solution must be placed into a closed chamber with the cover slide on top. The chamber must be free from air bubbles, as the working distance of these objectives is limited.
  • Page 30 PicoQuant GmbH MicroTime 100 Your sample is already placed onto the microscope and brought into focus. Also it is assumed that • the correct filter is placed in front of the detection unit and laser power and repetion rate are set correctly.

  • Page 31: Performing A Point Measurement

    PicoQuant GmbH MicroTime 100 Fig. 4.11: FCS preview of the test mode. Here only a single autocorrelation curve is shown. Optimize the detected photon count rate: • Similar guidelines as described for FLIM-measurements apply. Additionally for certain analysis • modes some special requirements are of interest: Too high photon count rates can lead to artefacts and falsify the fluorescence lifetime analysis.
  • Page 32 PicoQuant GmbH MicroTime 100 Fig. 4.12: Online display for a point measurement, here a FCS measurement. 3 online analysis are simultaneously shown: a FCS calculation (top), the count rate display (lower left) and the TCSPC histogram. The online preview can be configured by clocking at the icons on the lower left.

  • Page 33: Point Measurements Without Scanner Present

    PicoQuant GmbH MicroTime 100 Fig. 4.13: Setting multiploint measurements in the SymPhoTime 64. 4.4.3. Point measurements without scanner present If no z-scanner is attached to the system, focussing and selection of the measurement point is completely manual. Still, the necessary steps are very similar as for a normal FCS measurement.

  • Page 34: Measurements With Stacked Emission Filters

    Do not detach the fiber and at the MOU unless absolutely necessary, because it may affect the beam alignment inside the MOU! PicoQuant guarantees a minimum coupling efficiency of at least 25 % into the optical fiber when using the delivered fiber coupling instrumentation. To measure this light intensity, PicoQuant recommends purchasing a suited power meter.

  • Page 35: Standard Maintenance - Main Optical Unit (Mou)

    It may take several iterations of the above steps to achieve the maximum. If the coupling efficiency is still too low, most probably the Z-focusing and/or the polarization axis should be aligned too. This goes, however, far beyond the standard maintenance procedure. Contact PicoQuant for further support in this case.
  • Page 36 PicoQuant GmbH MicroTime 100 This procedure is a quick check of the optical hardware and evaluates the overall system performance. It takes typically less than 30 minutes and allows to easily maintain the overall alignment. Due to the stability and rigidity of the system, it is not necessary to carry out a MOU maintenance every day. We recommend to do it when a drop in system performance is observed, or after changing any substantial part of the optical layout, like the major dichroic (e.g.
  • Page 37 PicoQuant GmbH MicroTime 100 Fig. 5.3: Centering the beam on the optical axis: attach the alignment screws (upper left) to the objective turret. Switch on the laser. If the laser is of axis, move the collimator by slightly untighening the screws indicated in red.
  • Page 38 Toggle between these two tasks until the count rate can no more be increased. If your MicroTime 100 system utilizes a PMA detector, then no further adjustment is needed. Switch to TCSPC histogramming mode (by selecting the TCSPC Histogram tab) to check the width and the time- position of the IRF.

  • Page 39: Exchange Of The Major Dichroic

    PicoQuant GmbH MicroTime 100 In case of multiple detection channels, the time trace in the PicoHarp (or TimeHarp) window may show the summed count rate of all open detector channels. Similarly, the on-line TCSPC histogram may be built from the summed photon events. Selective monitoring of a detection channel of interest is possible by configuring the online preview window.

  • Page 40: Troubleshooting

    In case the hardware equipment is changed, the *.pfs file needs to be changed in order to adapt the software to the respective hardware changes. Please contact PicoQuant in this case. As the system is delivered already configured, it is not recommended to change settings without PicoQuant's supervision.

  • Page 41: No Image Is Displayed During Measurement

    Select to show the markers which should then appear in the time trace window. If no marker signals are present, check cable connection. If no marker signals are displayed in spite of a present connection, contact PicoQuant. 7. Recommended literature...

  • Page 42: Abbreviations

    Olympus FV 300 and FV 1000 laser scanning microscopes Wahl M.: Time-correlated single photon counting in fluorescence lifetime analysis Wahl M.: Time tagged time resolved fluorescence data collection Numerous measurement examples are published on the PicoQuant website. Please visit the MicroTime 100 section of http://www.picoquant.com/products/category/fluorescence-microscopes. 8. Abbreviations...

  • Page 43: Support And Warranty

    In any case, we would like to offer you our complete support. Please do not hesitate to contact PicoQuant if you would like assistance with your system. General questions about hard- and software can also be put to the user community forum ( http://forum.picoquant.com/...
  • Page 44 PicoQuant GmbH MicroTime 100 PicoQuant GmbH Unternehmen für optoelektronische Forschung und Entwicklung Rudower Chaussee 29 (IGZ), 12489 Berlin, Germany Telephone: +49 / (0)30 / 6392 6560 Fax: +49 / (0)30 / 6392 6561 e-mail: info@picoquant.com WWW: http://www.picoquant.com Page 44...

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