FLIM on a wide field fluorescence microscope

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1 FLIM on a wide field fluorescence microscope L.K. van Geest, K.W.J. Stoop Lambert Instruments, Turfweg 4, 933TH Leutingewolde, The Netherlands. Phone: , Fax: , lkvgeest@lambert-instruments.com Presented at : Fifth Australian Peptide Conference, Daydream Island, October ABSTRACT FLIM is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modulair structure. Lifetime images showing FRET in MCF7 cells with ErbB-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg will be shown. Keywords: FLIM, fluorescence, lifetime, FRET, microscopy, LED. INTRODUCTION Quantitative fluorescence microscopy uses the intensity of the fluorescence to extract information about the local concentrations of molecules that are labeled with fluorescent probes. This technique suffers from the fact that the fluorescence of the probe is permanently destructed by light-induced conversion of the probe material to a non-fluorescent compound. This photochemical process is called "bleaching" and makes it necessary to regulate the excitation dose in an economical way. Another physical property of fluorescent molecules is the fluorescence lifetime. The fluorescence lifetime is the decay time of the emission after the excitation has been stopped. The fluorescence lifetime depends on the local concentrations of certain molecules or ions. Changes in fluorescence efficiency as caused by bleaching are not accompanied by changes in fluorescence lifetime. Intensity variations caused by ununiform illumination do not effect the lifetime either. Fluorescence lifetime imaging microscopy (FLIM) merges the information of the spatial distribution of the probe with the lifetime to enhance the reliability of the concentration measurements. Additionally, FLIM enables the discrimination of fluorescence coming from different dyes, including autofluorescent materials, that exhibit similar absorption and emission properties but showing a difference in fluorescence lifetime. More recently FLIM is used in combination with FRET (Fluorescence Resonance Energy Transfer) using GFP, CFP, YFP and related fluorescent proteins fused in constructs to study macro molecular interactions. In this article the time domain and the frequency domain method for lifetime imaging are explained; for a more detailed background about FLIM methods, experiments and results is referred to the literature []. 2. FLIM Upon excitation fluorescent molecules exhibit delayed emission. The integrated emission of a large number of molecules shows an exponential time-coarse (see Figure ). In FLIM, the time constants in each pixel of the image are determined from the decay of the emission. Two approaches have been described to implement FLIM: the pulse method and the frequency method [2,3].

2 Fluorescence decay after pulsed excitation excitation fluorescence with a lifetime of 0.2 ns fluorescence with a lifetime of.0 ns 0.6 intensity (au) time (ns) Figure : Upon excitation with a short light pulse (black line) of fluorescence molecules the time-coarse of the emission intensity shows an exponential decay. The blue and red lines describe the simulated mono-exponential emission of dyes with a decay constant (lifetime) of respectively 0.2 and.0 ns. With the pulse method (see Figure 2) the specimen is excited with a short pulse and the emitted fluorescence is integrated in two or more time-windows. The relative intensity capture in the time-windows is used to calculate the decay characteristics. The determination of prompt fluorescence with lifetime in the range of 0. to 00 ns requires elaborate fast excitation pulses and fast gated detection circuits. Currently, rather expensive pulsed laser systems and scanning imaging systems are used to meet these requirements [2,8,9,0]. intensity (au) Fluorescence lifetime measured in the time domain average lifetime is function of ratio of integrated intensities Intensity in time-window Intensity in time-window 2 fluorescence with a lifetime of.0 ns time (ns) Figure 2: With the pulse method the sample is excited with a short pulse and the emitted fluorescence is integrated in two or more timewindows (hatched areas). The decay characteristics can be calculated from the integrated signals captured in each time-window. As an alternative to the time domain method, the LIFA fluorescence lifetime imaging system as described here, uses the frequency domain method to measure the lifetime. This method uses a homodyne detection scheme and requires a modulated light source and a modulated detector. The excitation light is modulated in a sinusoidal fashion. The fluorescence intensity shows a delay or phase-shift with respect to the excitation and a smaller modulation-depth (see Figure 3). The phase-shift and modulation-depth depend on the decay constants of the fluorescent material and the modulation frequency. It can be shown that the lifetime of a fluorescent material can be determined from the phase-shift by the following relation: τ ϑ = tan ω where ω is the angular frequency of the modulation and ϑ is the phase-shift. Equally the lifetime can be determined from the modulation depth m which is the relative modulation depth of the emission signal as compared to the excitation, by: ( ϑ) τ m = ω 2 m In case of a mono-exponential material, the lifetime derived from the phase should be equal to the lifetime derived from the modulation depth. 2

3 sine excitated fluorescence intensity (au) time (ns) excitation Fluorescence shows delayed phase and less modulation fluorescence with lifetime of 0.5 ns Lifetime is a function of phase delay and relative modulation. fluoresence with lifetime of 2 ns Figure 3: With the frequency method the excitation intensity is modulated. The fluorescence intensity shows a modulation with the same frequency but a shifted phase and a smaller relative modulation. The red and blue lines describe the simulated emission of dyes with a mono-exponential decay constant (lifetime) of respectively 0.5 and 2.0 ns. The decay characteristics can be calculated from the phase shift and relative modulation. To extract the phase-shift and the modulation-depth of the fluorescence light an image intensifier is used as a detector. The sensitivity of the intensifier is modulated with the same frequency as the excitation modulation (see Figure 4), but with an adjustable phase-shift. The detector signal level depends on the phase-shift of the fluorescence signal relative to the modulated sensitivity of the detector. A series of measurements is made at a number of phase-shift values. The detector signal height is plotted as a function of the measure phase (ϕ) and a sine is fitted through the series of data-points. The modulation-depth (m) and phase-shift (ϑ) of the sine as shown in Figure 4E, are used to extract the decay constants that are present in the fluorescence emission []. In the case of a mono-exponential material, the lifetime is extracted according to the formulas for τ ϑ or τ m as given above. To create the complete fluorescence lifetime image, a series of images is captured each taken at a different intensifier phase-shift. For each pixel a sine is fitted through the data-points and from the modulation-depth and phase-shift the lifetime is calculated. fluorescence signal detector sensitivity resulting signal A: 0 degrees time (ns) pixel intensity detected B: 60 degrees C: 20 degrees relative intensity ϑ a*m a D: 80 degrees ϕ measure phase (degrees) E: pixel intensity detected Figure 4: (A): The modulated fluorescence signal (red) is detected by the intensifier with modulated sensitivity (blue). The resulting detector signal (green) is demodulated and averaged in time by the intensifier to obtain the pixel intensity (single headed arrow). (B,C,D): The modulated detector sensitivity is phase-shifted relative to the phase of the excitation modulation to obtain a series of measurements of the pixel intensity. (E): The series of measurements is plotted as a function of the measure phase (ϕ) and a sine is fitted through the data points. The fluorescence decay constants can be calculated from the phase-shift (ϑ) and the modulation depth (m) of this sine. To measure fluorescence decay times in the range of 20 ns down to 0.5 ns, modulation frequencies of 5 to 00 MHz respectively will be used. It can be derived from the formulas for τ ϑ and τ m as given above, that the theoretical optimal 3

4 frequency for measuring τ ϑ and τ m at the best time resolution is: f = 2 πτ, respectively 2 f = 2πτ. However due to ϑ m the frequency dependent characteristics of the LED and the image intensifier, it turns out that in practice the frequency for best results is lower than the theoretical values especially for the shorter lifetimes. 3. SYSTEM COMPONENTS The LIFA system uses a standard fluorescence microscope. In order to make the microscope suitable for FLIM, a special light source and camera have to be installed. The complete setup of the Lambert Instruments LIFA system and microscope is shown in Figure 5. Figure 5: FLIM system setup: Wide field fluorescence microscope and LIFA attachment for FLIM. A lamp housing with an LED light source is attached to the excitation port of the microscope. An image intensifier in combination with a digital CCD camera is attached to the camera port. A signal generator delivers modulation signals to the LED and to the image intensifier. Intensifier, camera, LED and signal generator are computer controlled. The images from the camera are transferred via an IEEE-394 ( FireWire ) interface. Remote operation enables the computer to set all important parameters. By using dedicated EZ-FLIM software a sequence of images is acquired at a selected set of phase differences between both modulation signals. With this sequence made of the sample and a similar sequence made of a reference object the software calculates a lifetime image. The lifetime image is then available for further processing or storage on the computer. The raw set of phase images are stored as well and can be used for further analysis or for processing at different parameter values (threshold, filtering etc.). 3.. Light source In many experimental FLIM systems described in the literature a laser in combination with an external modulator is used to excite the fluorescent molecules in a modulated fashion. For example an Ar/Kr gas laser with acousto-optic modulator (AOM) is used by Gadella []. The use of the laser and AOM combination is rather critical and requires special attention to obtain the required stability. In order to develop an easy-to-use FLIM system we have looked at light sources that can be directly RF modulated. As an alternative to the laser, ultra bright LEDs with different wavelengths are now available and have been applied successfully in the LIFA system. It has been reported that LEDs are also used for cuvette based lifetime measurements [4]. LEDs are easy to modulate and thanks to their non-coherency do not cause unwanted interference patterns. Additional advantages are long life and low cost and ongoing development of new types featuring higher brightness and more different wavelengths. The LED is mounted on an adapter that fits in the standard arc-lamp housing connected to the excitation light port of the microscope. Changing to a different wavelength LED is just as easy as replacing an arc-lamp. Suitable LED wavelengths available now are: 440nm (CFP), 470nm (GFP) and 50nm (YFP). Modulated solid state laserdiodes can be used alternatively as described by Boddeke [5]. Also it is possible to use a gas laser as a light source that is modulated with an AOM. The signal generator provides the modulation signal to drive the AOM. 4

5 3.2. Camera/Detector The modulated fluorescence coming from the sample and containing the lifetime information of each pixel has to be detected in order to retrieve the spatial and lifetime information. This is achieved by imaging the sample with the microscope on the input of an image intensifier that demodulates and intensifies the image. The demodulated image then is projected on the CCD image sensor, part of a complete CCD camera. This is done by either using relay lenses or via a tapered fiber-optical image guide, matching the image size to the format of the CCD. The Lambert Instruments LIFA system uses the best available modulated 8 mm Gen II proximity focused image intensifier that is optically coupled (Figure 6) to the CCD camera. The intensifier is mounted with the relay lens in a separate housing that can be connected via C-mount to the camera port of the microscope. The output of this unit is connected via C-mount to the CCD camera. The camera uses a progressively scanned 2/3 inch interline CCD having.3 million pixels. The camera offers 2x2 binning and a programmable window size. The 2 bit image data are transferred to the computer via an IEEE- 394 interface. photocathode MCP Anodescreen fiber optic output Relaylens Input window CCD modulated input 0 to -200V 0V 800 V variable 6 kv Figure 6: Proximity focused Gen II image intensifier lens coupled to CCD. The Gen II proximity focused intensifier is selected for the FLIM application for the following reasons: - the gain of the intensifier can be modulated relatively easy by either cathode or MCP voltage modulation. - photocathodes can be selected with optimised spectral response to the fluorescence to be detected. The image intensifier gain is modulated at the same frequency (-00 MHz) as the excitation of the object is modulated with. The image is demodulated by the relatively long decay time (~ms) of the phosphor anode screen of the intensifier. Additional time integration of the signal is done on the CCD. The integration time of the camera can be varied over a wide range and can be selected for optimal S/N or speed (trade-off) Signal generator The signal generator produces two sine waves at frequencies from to 00 MHz with increments of 0.00 MHz. The maximum frequency error is 00 ppm. The phase difference is selectable from 0 to 359 with increments of. The phase error between the two signals is negligible. Both output signals are amplified and superimposed on the variable DC bias values for LED and image intensifier. The signal generator is remote controlled via a serial line protocol (RS-232). The control software is integrated in the FLIM software. 4. CALIBRATION In the lifetime image acquisition procedure, a number of images are acquired at different measure-phase settings. From these images, the phase and modulation depth images are estimated. To relate this relative phase image and absolute modulation depth image to an actual lifetime image a reference is necessary. The reference for the phase compensates for path length differences, frequency dependent phase-shifts in the electronics (amplifier, intensifier driver, LED driver) and a possible phase lag towards the center of the photocathode. The reference for the modulation compensates for the modulation depth of the LED and the image sensor and for the decreasing modulation depth towards the center of the photocathode. The references are frequency dependent and vary over the image (spatial low-pass filtering of the photocathode). The references for the phase and modulation depth are acquired with the normal lifetime image acquisition procedure while the microscope is focused on a reference object with a fixed known lifetime (measured by TCSPC, time correlated single photon counting). As a reference object a piece of fluorescent plastic can be used. It is hard to find plastics with a single exponential decay however. An alternative way of calibration as described by Hanley et al.[6] is using a set of Rhodamine 6G solutions quenched with varying concentrations of Iodide, leading to lifetimes of ns, as a Multi-point calibration method. Figure 7 shows the reference object (fluorescent plastic) of which 2 images are recorded at 2 different phases at a modulation frequency of 35 MHz. The images are presented in an image stack and as tiles. 5

6 Figure 7: Image stack and tiled presentation of 2 phases of the reference image 5. SAMPLE IMAGES After the reference image has been recorded, the microscope is focused onto the sample and a fluorescence image of the sample is recorded. Figure 8 shows the fluorescence intensity image of the sample. Figure 8: Intensity image of fluorescent sample The sample shows convalaria (image taken with the Lambert Instruments system at the laboratory of Dr. T. Jovin, Max Planck Institute in Göttingen, Germany) with different lifetimes in the center, middle and outer range. In the same way as for the reference image a series of 2 images is recorded at 2 different phases at the same modulation frequency. These images are shown in the image stack and tiled presentation in Figure 9. Figure 9: 2 phases of sample in image and tiled presentation Figure 0 shows the value of a single pixel plotted as a function of the phase and the sine that has been fitted through the data points. This is done for the same pixel in sample and in reference. It can be discerned that the sample plot is shifted approximately phase-step equal to 30º as compared to the reference. 6

7 0,9 0,9 signal 0,8 0,7 Sample measured fitted signal 0,8 0,7 Reference measured fitted 0,6 0,6 0, phase 0, phase Figure 0: Sine fit in one pixel; Sample and Reference 6. LIFETIME MEASUREMENTS From the reference and sample data as described in the previous paragraphs, the lifetime image is automatically calculated and presented in a way as shown in Figure. By using the colored presentation, lifetimes are easily read from the color scale. Other examples of biological samples that have been imaged with the LIFA FLIM system are Hela cells with GFP and human bone cells with the protein SP00. The lifetime image of those objects showed a time resolution between 0, and 0,2 ns measured by the standard deviation of the lifetime over an area of 024 pixels. Figure : Lifetime image Intensity Lifetime Lifetime + Intensity Figure 2: MCF7 cells with ErbB.-GFP as donor and Py72/Cy3 as acceptor (EMBL Heidelberg) In Figure 2 the MGF7 cells are showing a decrease in lifetime at the membrame. This indicates the occurrence of FRET (fluorescence resonance energy transfer) between the GFP donor molecule and the Cy3 acceptor. In Figure 3 a uniform lifetime is measured in the tubulin alexia with golgi with Cy3 as probe. In the intensity image many details can be resolved that are not visible in the lifetime image because the lifetime is independent of the intensity. Therefore the combination of lifetime (color) and intensity in one image can be useful. This combination image is presented as well in Figure 2 and Figure 3. 7

8 Intensity Lifetime Lifetime + Intensity Figure 3: tubulin alexia + golgi Cy3 (EMBL Heidelberg) 7. SOFTWARE Methods and algorithms that are used to retrieve lifetime information from the acquired images following the frequency domain method are described in Boddeke s thesis [5], being the basis of the system described in this paper. Further reference is given to the publications of Gadella et al. [7] treating the subject extensively. The system is easy to use and contains enough intelligence to acquire automatically a series of sample images and reference images at different phase shifts between excitation modulation and detector modulation at the optimal frequency to compute and present the lifetime images. Camera control and control of the signal generator are integrated in the EZ-FLIM software to fully automate the measurement and calculation sequence, making the system very user friendly. The program offers various tools for analyzing the images, intermediate results and lifetime data. Digital gain control of the intensifier and a protection circuit are realized in a separate module. The lifetime software has been tested by using a simulation program []. The software has been designed in such a way that operation of the whole system is not complicated and easy for the user. Within one window the settings can be made and image acquisition can be started after selection of either Reference or Sample. When Sample is selected, images of the sample are collected and calculation of the lifetime image is automatically done by using sample and reference data. The intensifier gain, cathode bias voltage and LED bias current are controlled in a separate window. The intensifier protection circuit is monitoring the output current as a reference for the light level. When a certain threshold value is passed, the intensifier automatically is switched in its safe gate closed status. This status can be used also manually for leaving the instrument on stand-by. When starting the software the camera is initialized and full resolution or 2x2 binning, 8 or 6 bit mode and frame rate can be selected. During operation, settings like, integration time, acquisition area, and electronic gain are chosen in the Acquire settings window of EZ-FLIM. REFERENCES. T.W.J. Gadella, Fluorescence Lifetime Imaging Microscopy (FLIM), Microscopy and analysis, pp. 3-5, May E.P. Buurman, R. Sanders, A. Draaijer, H.C. Gerritsen, J.J.F. van Veen, P.M. Houpt and Y.K. Levine, Fluorescence lifetime imaging using a confocal laser scanning microscope, Scanning 4, pp , D.W. Piston et al., Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser scanning microscopy, Proc. SPIE-Int. Soc. Opt. Eng., 640, pp , T. Araki, and H. Mishawa, Light emitting diode-based nanosecond ultraviolet light source for fluorescence lifetime measurements, Review of Scientific Instrumentation, 66(2) pp , F.R. Boddeke, PhD-thesis Quantitative Fluorescence Microscopy, Delft University Press, Delft, The Netherlands, Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin and T. M. Jovin, Fluorescence Lifetime Imaging: Multi-point Calibration, Minimum Resolvable Differences and Artifact Suppression, Cytometry 43, pp , T.W.J. Gadella, Jr, R.M. Clegg, and T.M. Jovin, Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data, Bioimaging, 2, pp , Ni. T. and Melton, L.A. (99) Fluorescence Lifetime Imaging: An approach for Fuel Equivalence Ratio Imaging. Applied Spectroscopy 45: , Wang, X.F., Uchida, T., Coleman, D. and Minami, S. (99) A two-dimensional fluorescence lifetime imaging system using a gated image intensifier. Applied Spectroscopy 45(3): , F.R Scully, A.D., Ostler, R.B., Phillips, D., O Neill, P.O., Townsend, K.M.S., Parker, A.W. and MacRobert, A..J. (99 7) Application of fluorescence lifetime imaging microscopy to the investigation of intracellular PDT mechanisms. Bioimaging 5(): 9-8, L.K. van Geest, F.R. Boddeke, P.W. van Dijk, A.F. Kamp, C.J.R. van der Oord and K.W.J. Stoop, System for Fluorescence Lifetime Imaging Microscopy, Proc. SPIE-Int. Soc. Opt. Eng. 3605, pp ,