Fluorescence Lifetime Imaging and Fluorescence Correlation for Laser Scanning Microscopes

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1 Fluorescence Lifetime Imaging and Fluorescence Correlation for Laser Scanning Microscopes Direct approach to fluorescence resonance energy transfer (FRET) Probing cell parameters by environment-dependent lifetime Co-localisation experiments by combined FRET and fluorescence correlation spectroscopy (FCS) Separation of fluorophores by lifetime Four-dimensional TCSPC technique Superior time resolution Multi-wavelength capability Simultaneous FCS / FCCS and lifetime recording Ultra-high sensitivity Near-ideal counting efficiency Works at any scan rate of the microscope Lifetime images up to 1024 x 1024 pixels Steady state images up to 4096 x 4096 pixels 03 Lifetime, ns 12 CFP/YFP FRET 05 FRET Intensity 20 Becker & Hickl GmbH Nahmitzer Damm Berlin, Germany Tel +49 / 30 / Fax +49 / 30 / info@becker-hicklcom wwwbecker-hicklcom US Representative: Boston Electronics Corp tcspc@boseleccom wwwboseleccom UK Representative: Photonic Solutions PLC sales@psplccom wwwpsplccom SPC-830 TCSPC FLIM Module Covered by patents DE and DE

2 Introducing a New Dimension into Laser Scanning Microscopy Fluorescence Lifetime When a dye molecule absorbs a photon it goes into an excited state from which it can return by the emission of a fluorescence photon, by converting the absorbed energy internally, or by transferring the energy to the environment, see figure below Laser Pulse Excited State Population e -t/ Emission + Internal Conversion e -t/ 0 Energy transferred to Environment Return of molecules from the excited state The probability that one of these effects occurs is independent of the time after the excitation If a large number of similar molecules with similar local environment is excited by a short laser pulse the fluorescence decay function is therefore single exponential As long as no energy is transferred to the environment the lifetime is the natural fluorescence lifetime, τ 0 which is a constant for a given molecule and refraction index of the solvent The fluorescence lifetime of the fluorophores commonly used in microscopy are of the order of a few ns Fluorescence Quenching If energy is transferred to the environment the actual fluorescence lifetime, τ, is less than the natural lifetime, τ 0 Therefore the lifetime of markers can be used as an additional probe technique in cells A wide variety of chromophores including CFP, GFP and YFP clearly show variations in their fluorescence lifetimes Although most of these effects are not investigated in detail yet there is probably a large potential for intelligent markers based on lifetime changes Resonance Energy Transfer A particularly efficient energy transfer process is fluorescence resonance energy transfer, or FRET FRET occurs if the molecules two different dyes are closely coupled and the emission band of one dye overlaps the absorption band of the other In this case the energy from the first dye, the donor, goes immediately into the second one, the acceptor Donor Laser Donor Acceptor Acceptor Absorption Emission Absorption Emission D D A A Wavelength Fluorescence Resonance Energy Transfer (FRET) The strong energy transfer results in an extremely efficient quenching of the donor fluorescence and, consequently, decrease of the donor lifetime Intensity Laser pulse Intensity Laser pulse -t/ e 0 -t/ e 0 unquenched lifetime e -t/ quenched donor FRET unquenched donor e -t/ quenching Fluorescence quenching For almost all dyes the energy transfer rate depends more or less on the concentration of ions, on the oxygen concentration, on the ph value or on the binding to proteins in a cell Therefore, specifically designed dyes can be used to probe the concentration of biological relevant ions such as Na +, Mg ++, or Ca ++, the oxygen concentration or the ph value inside a cell There is a direct relation between the lifetime and the quencher concentration Fluorescence markers used to reveal particular protein structures in cells often bind to a variety of slightly different targets Although this often does not cause significant changes in their spectral behaviour the lifetime can be clearly different due to different quenching efficiency Decrease of donor lifetime in case of FRET The most intriguing feature of FRET is that it works only over a distance shorter than a few nm Therefore, it can be used to probe the distance between different sub-units in the cell It is difficult to obtain quantitative FRET results from steady-state images The fluorescence intensity does not only depend on the FRET efficiency but also on the unknown concentration of the dyes Moreover, some of the acceptor molecules are excited directly, and the donor emission band extends into the acceptor emission Up to eight measurements at different excitation wavelength and in different emission wavelength bands are required to obtain calibrated FRET results from steady state data FRET results can also be obtained by measuring the donor fluorescence, then photobleaching the acceptor, and measuring the donor once more The FRET efficiency is then given by the ratio of the two donor images Although this procedure looks

3 reliable at first glance photobleaching products may induce damage in the cell, and in a living cell diffusion may replace a fraction of the photobleached acceptor molecules In lifetime data, however, FRET shows up as a dramatic decrease of the donor lifetime In practice the fluorescence decay function contains the fluorescence of quenched and of unquenched donor molecules and is therefore doubleexponential Intensity b quenched b e -t / FRET a unquenched e -t / 0 a Fluorescence decay components in FRET systems time Therefore, quantitative measurements require double exponential decay analysis that delivers the lifetimes, τ 0 and τ FRET, and the intensity factors, a and b, of the two decay components The relative numbers of quenched and unquenched molecules is given by the ratio of the two intensity components, b/a, while the average coupling efficiency of the FRET pairs is given by τ 0 / τ FRET In principle, both the relative numbers of quenched and unquenched molecules and the coupling efficiency can be derived from a single donor lifetime measurement The image below shows the FRET efficiency in an HEK cell containing CFP and YFP in different sub-units of the cell The ratio a/b is represented by the colour Separation of DNA stained with Hoechst and tubulin stained with Alexa 488 by the lifetimes of the two fluorophores Especially autofluorescence images of cells and tissue show a wide variety of fluorescence components with poorly defined, variable, and often unknown spectra When spectral unmixing fails the components can usually be distinguished by their different lifetimes The relative concentration of two components can be determined by double-exponential decay analysis Even if only a single exponential approximation, ie an average lifetime is used, the contrast in the fluorescence images can be considerably improved Moreover, changes in the relative concentration and the lifetime of autofluorescence components can possibly be used as diagnostic tools The figure below shows an autofluorescence lifetime image of human skin with lifetimes varying from 05 ns to 25 ns FRET in HEK cell containing CFP and YFP in different sub-units Colour represents FRET intensity b/a, blue to red corresponds to 05 to 20 Red indicates strong FRET Separation of Different Fluorophores The fluorescence lifetime can be used as an additional parameter to separate the fluorescence signals of fluorophores with similar or overlapping spectra The figure below shows a cell stained with Hoechst and Alexa 488 linking to the DNA and the tubulin, respectively The two fluorophores are clearly separated only by their different lifetimes Autofluorescence lifetime image of human skin, colour represents lifetime, Colour scale: blue to red = 05 ns to 25 ns Size about 60 um x 60 um Multi-Wavelength TCSPC FLIM The Becker & Hickl fluorescence lifetime imaging (FLIM) modules employ a novel multi-dimensional time-correlated single photon counting (TCSPC) technique in conjunction with a new analog-to-digital conversion principle The new technique records a full fluorescence decay curve for each pixel of the image, allows a time resolution down to a few 10 ps, has a near-ideal counting efficiency, works with the full scan rate of the microscope, and is able to record in several wavelength intervals simultaneously The TCSPC FLIM technique requires a laser scanning microscope with pulsed laser excitation and is typically used in multi-photon microscopes The principle of the technique is shown below At the input of the system are several photomultipliers detecting in different wavelength intervals At the typical count rate of a two-photon microscope it is very unlikely that several detectors detect a photon within the same laser pulse

4 period Therefore, the detector electronics combines the single photon pulses of all detectors into a common timing pulse line Simultaneously, the detector electronics delivers a digital signal that indicates in which of the detectors a particular photon was detected Electronics s from Laser from Microscope Router Channel Timing Start Stop Frame Sync Line Sync Pixel Clock Channel register CFD CFD Measurement TAC Counter Y Scanning Interface Counter X TCSPC Module ADC y x n t Channel / Wavelength channel 1 within decay curve channel 2 Location within scanning area channel 3 Multi-wavelength TCSPC lifetime imaging channel 4 The recording electronics consists of a time measurement channel, a scanning interface, a detector channel register, and a large histogram memory The time measurement channel contains the usual TCSPC building blocks (CFDs, TAC, ADC) in the reversed start-stop configuration For each photon, it determines the detection time (t) with respect to the next laser pulse The scanning interface is a system of counters which receive the scan control signals (frame sync, line sync and pixel clock) from the microscope It determines the current location (x and y) of the laser spot in the scanning area Synchronously with the detection of a photon, the detector channel number (n) for the current photon is read into the detector channel register If the light is split into different wavelength intervals in front of the detectors n represents the wavelength of the detected photon The obtained values for t, x, y and n are used to address the histogram memory in which the distribution of the photons over time, wavelength, and the image coordinates builds up The result can be interpreted as separate data blocks for the different wavelength intervals Each block can be interpreted as an image containing a full fluorescence decay curve in each pixel The data acquisition runs at any scanning speed of the microscope As many frames can be accumulated as necessary to collect enough photons Due to the synchronisation via the scan clock pulses, the regular zoom and image rotation functions of the microscope act automatically on the TCSPC recording and can be applied in the usual way It should be pointed out that the histogramming process does not use any time gating, detector multiplexing or wavelength scanning Therefore, the method yields a near perfect counting efficiency and a maximum signal to noise ratio for a given fluorescence intensity and acquisition time Due to the short dead time of the TCSPC imaging electronics (125 ns) there is virtually no loss of photons for count rates up to about 10 6 /s For comparison, living cells under twophoton excitation typically deliver count rates of some 10 4 /s, cells stained with very stable high efficiency fluorophores of a few 10 5 /s Combined Lifetime and FCS Recording Fluorescence Correlation Spectroscopy (FCS) exploits intensity fluctuations in the emission of a small number of excited molecules in a femtoliter sample volume The fluctuations are on a time scale from some 100 ns to several ms To record intensity changes such fast, the SPC-830 TCSPC FLIM modules has an FCS mode that records the information about each individual photon The entry for each photon contains the time of the photon within the fluorescence decay curve, the time from the start of the experiment, and the number of the detector that detected the photon The data structure is shown below ps time from TAC / ADC Laser FIFO Buffer Photon Laser resolution 25 ps of picoseconds Fluorescence decay curves Channel Det No Det No Det No Det No Readout Hard disk time from start of experiment Start of experiment Photons resolution 50 ns Autocorrelation of ns to seconds Fluorescence correlation spectra Simultaneous FCS / lifetime data acquisition For each detector an individual correlation spectrum and an individual fluorescence decay curve can be calculated Or, if the detectors are recording in different wavelength intervals, the signals from several chromophores can be crosscorrelated The advantage of the TCSPC FCS technique is that FCS, FCCS, and lifetime data are obtained from the same cell, from the same spot inside the cell, and at the same time Therefore lifetime and correlation data can directly be compared Options In general, the SPC-830 FLIM module works with a wide variety of microscopes FLIM detectors and cable sets are available for the Zeiss LSM 510 NLO, the Leica SP 2, the Biorad 1024 and Radiance 2100, the Olympus FV 300, and the Nikon PCM 2000 PMH-100 detector Cables to detector Scan Control Cable SPC-730 TCSPC Module Cable to laser FLIM upgrade kit with PMH-100 detector and interface cables

5 FLIM detectors for different budget, time resolution and detection efficiency are shown below The PMH-100 is a general purpose detector module with internal power supply and preamplifier The width of the instrument response function (IRF) is 150 ps The Hamamatsu H is a detector with ultra-high sensitivity and an IRF width of 250 ps It is exceptionally suitable for FCS measurements The ultimate in time resolution is the Hamamatsu R3809U with 30 ps IRF width Overview of Features Superior Resolution The width of instrument response function of the fastest available detectors is about 30 ps The time channels of the TSCPC recording can be selected down to less than 1 ps This allows the application of standard fit and deconvolution techniques for lifetime analysis Single and double exponential decay functions can be resolved - with decay components down to less than 10 ps Multi-Wavelength Capability Up to four FLIM detection channels can be operated simultaneously in different wavelength intervals Compared to consecutive acquisition runs for different wavelength the overall radiation dose at the sample is substantially reduced Multi-wavelength operation does not only reduce photobleaching in general, it also reduces errors due accumulated photobleaching in consecutive acquisition runs FLIM detectors: Left to right, PMH-100, IFR width 150 ps, H7422, IRF width 250 ps, ultra-high sensitivity, R3809U, IRF width 30 ps, with preamplifiers For the LSM 510, the SP 2 and the Radiance 2100 FLIM detector modules with ready-to use optical adapters are available An R3809U and a PMH-100 detector module for the non-descanned output of the Zeiss LSM 510 is shown below Both modules contain a shutter for detector overload protection FLIM detectors for LSM 510 NDD output A shutter is used for overload protection For other microscopes detector assemblies with standard C- mount adapters are available Two dual-wavelength detection modules with shutters for overload protection are shown below General purpose dual wavelength detector assemblies with shutter and dichroic beamsplitter Left: R3809U, IRF width 30 ps Right: PMH-100, IRF width 150 ps Near Ideal Recording Efficiency - Every Photon Counts There is no time-gating, wavelength scanning or detector multiplexing Every photon counts - regardless in which of the PMTs it was detected This high recording efficiency is maintained even for short lifetimes - down of the order of the instrument response width And it does not substantially drop up to count rates of 10 6 photon per second The high efficiency allows to reduce the excitation power and, in turn, reduce photobleaching effects Crisp Images from Deep Tissue Layers Multi-photon imaging was originally developed because it allows to obtain images from deep tissue layers Because there is no substantial excitation outside the focus the multiphoton microscope delivers sharp images with depth resolution However, the localised excitation is only one half of the story Before and image is built up, the fluorescence light must get out of the sample - and on the way out it is heavily scattered At the surface it emerges from a fuzzy spot The reason that a multi-photon microscope delivers crisp images from deep tissue layers is that it collects the photons from a large sample area and then assigns them to the correct image coordinates Obviously, this works only for single channel detectors - but not with a camera acquiring an image of the sample surface Consequently, in scanning microscopes TCSPC lifetime imaging is superior to gated or modulated image intensifiers in terms of image quality High Pixel Resolution The SPC-830 TCSPC imaging module is able to acquire a single image as large as 1024x1024 pixels with 16 time channels per pixel Four images with 512x512 pixels and 16 time channels can be recorded simultaneously Four images with 256x256 pixels are recorded with 64 time channels

6 Steady State Photon Counting The SPC-830 module is flexible enough to be operated with only one time channel This turns the TCSPC microscope into an ultra-high sensitivity high resolution multiwavelength photon counting imager A single image up to 4096 x 4096 pixels can be recorded Four simultaneously acquired images can be as large as 2048 x 2048 pixels each Single Pixel Analysis Fluorescence decay data of a single pixel can be acquired if the scanning of the laser beam is stopped in a defined location Sequences of decay curves can be recorded with acquisition times of less than 1 ms per curve Combined lifetime and FCS / FCCS Capability An individual correlation spectrum and an individual fluorescence decay curve is calculated If the detectors are operated in different wavelength intervals, the signals from several chromophores can be cross-correlated The advantage of the TCSPC FCS technique is that FCS, FCCS, and lifetime data are obtained from the same cell, from the same spot inside the cell, and at the same time Sample-Limited Acquisition Due to its near-ideal counting efficiency TCSPC imaging gives the shortest possible acquisition time for a given count rate obtained from the sample and for a given lifetime accuracy Unfortunately that does not actually mean that the acquisition times are short For single exponential lifetime analysis a few 100 photons per pixel are sufficient For samples stained with highly stable and highly efficient fluorophores a single exponential lifetime image can be recorded within less than 10 s However, double or even triple exponential decay analysis requires much more photons Moreover, count rates as low as a few 10 4 photons per second are not unusual for samples of poor photostability To obtain high accuracy data from such samples acquisition times of the order of 10 to 20 minutes can be required It should be pointed out that the reason for the long acquisition time is not the TCSPC technique - the sample itself is the bottleneck It is an unique feature of the TCSPC imaging technique that it delivers accurate decay data under such odd conditions Wolfgang Becker, Axel Bergmann, Christoph Biskup, Thomas Zimmer, Nikolaj Klöcker, Klaus Benndorf, Multi-wavelength TCSPC lifetime imaging Proc SPIE 4620 (2002) König, K, Femtosecond Laser Microscopy in Biomedicine Laser Opto 32 (2/2000) Wolfgang Becker, Klaus Benndorf, Axel Bergmann, Christoph Biskup, Karsten König, Uday Tirplapur, Thomas Zimmer, FRET Measurements by TCSPC Laser Scanning Microscopy, Proc SPIE 4431 (2001) W Becker, A Bergmann, K Koenig, U Tirlapur, Picosecond fluorescence lifetime microscopy by TCSPC imaging Proc SPIE 4262 (2001) K Koenig, C Peuckert, I Riemann, U Wollina, Optical tomography of human skin with picosecond time resolution using intense near infrared femtosecond laser pulses, Proc SPIE Tirlapur, UK, König, K (2002) Targeted transfection by femtosecond laser Nature 418, S Ameer-Beg, PR Barber, R Locke, RJ Hodgkiss, B Vojnovic, GM Tozer, J Wilson, Application of multiphoton steady state and lifetime imaging to mapping of tumor vascular architecture in vivo Proc SPIE 4620 (2002) A Schönle, M Glatz, S W Hell, Four-dimensional multiphoton microscopy with time-correlated single photon counting Appl Optics 39 (2000) A Rück, F Dolp, C Happ, R Steiner, M Beil, Fluorescence lifetime imaging (FLIM) using ps pulsed diode lasers in laser scanning microscopy Proc SPIE (2003) L Kelbauskas, W Dietel, Internalization of aggregated photosensitizers by tumor cells: Subcellular time-resolved fluorescence spectroscopy on derivates of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitation Photochem Photobiol 76 (2002) Wolfgang Becker, Axel Bergmann, Christoph Biskup, Laimonas Kelbauskas, Thomas Zimmer, Nikolaj Klöcker, Klaus Benndorf, High resolution TCSPC lifetime imaging Proc SPIE (2003) Contact Becker & Hickl GmbH Nahmitzer Damm 30, Berlin, Germany wwwbecker-hicklcom Tel +49 / 30 / Fax +49 / 30 / Dr Wolfgang Becker Dr Axel Bergmann becker@becker-hicklcom bergmann@becker-hicklcom Literature SPC-134 through SPC-730 TCSPC Modules, Operating Manual and TCSPC Compendium Becker & Hickl GmbH, wwwbecker-hicklcom W Becker, A Bergmann, Lifetime Imaging Techniques for Optical Microscopy wwwbecker-hicklcom TCSPC Laser Scanning Microscopy - Upgrading laser scanning microscopes with the SPC-730 TCSPC lifetime imaging module, Becker & Hickl GmbH, wwwbecker-hicklcom Wolfgang Becker, Axel Bergmann, Assemblies for Picosecond Microscopy wwwbecker-hicklcom Wolfgang Becker, Axel Bergmann, Georg Weiss, Lifetime Imaging with the Zeiss LSM-510 Proc SPIE 4620 (2002) 30-35