High frame rate fluorescence lifetime imaging

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Jul 1, 2003 - in ion concentration and the polarization/depolarization of ... emission spectra upon changes in the biological conditions, the ratio .... In deriving equation (1) it is assumed that the ... exponential fluorescence decay equation (1) yields only an .... cathode and requires an expensive, non-standard high power.
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High frame rate fluorescence lifetime imaging

This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2003 J. Phys. D: Appl. Phys. 36 1655 (http://iopscience.iop.org/0022-3727/36/14/301) View the table of contents for this issue, or go to the journal homepage for more

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INSTITUTE OF PHYSICS PUBLISHING

JOURNAL OF PHYSICS D: APPLIED PHYSICS

J. Phys. D: Appl. Phys. 36 (2003) 1655–1662

PII: S0022-3727(03)55709-6

High frame rate fluorescence lifetime imaging A V Agronskaia1 , L Tertoolen2 and H C Gerritsen1,3 1

Department of Molecular Biophysics, Debye Institute, Utrecht University, PO Box 80000, Princetonplein 1, 3508 TA Utrecht, The Netherlands 2 KNAW Hubrecht Laboratory, Netherlands Institute for Developmental Biology (NIOB), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands E-mail: [email protected]

Received 6 November 2002 Published 1 July 2003 Online at stacks.iop.org/JPhysD/36/1655 Abstract A fast time-domain based fluorescence lifetime imaging (FLIM) microscope is presented that can operate at frame rates of hundreds of frames per second. A beam splitter in the detection path of a wide-field fluorescence microscope divides the fluorescence in two parts. One part is optically delayed with respect to the other. Both parts are viewed with a single time-gated intensified CCD camera with a gate width of 5 ns. The fluorescence lifetime image is obtained from the ratio of these two images. The fluorescence lifetime resolution of the FLIM microscope is verified both with dye solutions and fluorescent latex beads. The fluorescence lifetimes obtained from the reference specimens are in good agreement with values obtained from time correlated single photon counting measurements on the same specimens. The acquisition speed of the FLIM system is evaluated with a measurement of the calcium fluxes in neonatal rat myocytes stained with the calcium probe Oregon Green 488-Bapta. Fluorescence lifetime images of the calcium fluxes related to the beating of the myocytes are acquired with frame rates of up to 100 Hz.

1. Introduction A variety of fast biological processes take place at a subsecond timescale. Examples of such processes are changes in ion concentration and the polarization/depolarization of the cell membrane. Fluorescence microscopy can be used to image such processes at the microscopic level. In recent years, the frame rate of special high-speed cameras employed in fluorescence microscopy reached the millisecond range [1, 2]. This frame rate is sufficient to monitor fast biological processes. However, it is not straightforward to obtain quantitative information from fluorescence intensity measurements. The interpretation and quantification of the measurements is hampered by, for instance, non-homogeneous staining of the cells, variations in the dye concentration over the cells, and photo bleaching of the dye. Due to the great importance of intracellular ion concentrations a lot of effort has been put into the development 3

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© 2003 IOP Publishing Ltd

of fluorescent dyes and methods to obtain quantitative data from fluorescence images. Ratiometric imaging [3–5] is one of the most commonly used quantitative imaging methods. Here, probes are employed that exhibit distinct shifts in their spectral properties upon binding to, e.g. ions. Two fluorescence images are acquired, either at two different excitation wavelengths using the same detection wavelength band or at one excitation wavelength using two different detection wavelength bands. When probes exhibit characteristic shifts in the excitation or emission spectra upon changes in the biological conditions, the ratio of the two fluorescence images can be used to reconstruct the intracellular conditions. Ratiometric probes for imaging intracellular concentrations of ions such as Ca2+ [3], K+ [6], Na+ [7], Mg2+ [5], and H+ [5, 8] are available. The calibration of the response of ratiometric probes is not straightforward and often needs to be carried out on the specimen under investigation. Calibration curves obtained from buffer solutions may differ significantly from calibration curves made in cells. Moreover, many of the ratiometric

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A V Agronskaia et al

probes, calcium probes in particular, require UV excitation. UV excitation is potentially harmful for cells and in addition may cause high levels of autofluorescence. Visible light excited probes usually do not show changes of their excitation or emission spectra. In general, only changes of their quantum yield and therefore of their fluorescence intensity take place. A few methods have been developed to extract quantitative data from measurements with these dyes [9]. The disadvantage of all these methods is the requirement of a calibration procedure for every measured sample. The fluorescence lifetime of a fluorescent dye can also be employed for imaging. The fluorescence lifetime of many fluorescent dyes is affected by their microenvironment. It is in general independent of factors that influence the fluorescence intensity such as the dye concentration and fading due to photo bleaching. Fluorescence lifetime imaging (FLIM) can be employed for the quantitative imaging of ion concentrations when ion sensitive dyes are employed. Here, the (average) lifetime is a direct measure of the ion concentration. For some of these dyes it was demonstrated [10, 11] that fluorescence lifetime based ion sensing does not require a calibration in cells, i.e. a calibration carried out on a series of buffers suffices. So far a drawback of FLIM for biological studies is the comparatively slow detection rate. Typical times required for the acquisition of fluorescence lifetime images are on the order of seconds. This is often caused by the detection schemes that are employed. There are two dominant methods in use to acquire fluorescence lifetime images: frequency domain, and timedomain based methods. Both approaches are suitable for imaging and are used in wide-field fluorescence microscopes [12–19] and in scanning microscopes [20–27]. The frequency domain based methods require either a sinusoidal modulated excitation or a pulsed excitation source and employ phase sensitive detection. The (average) fluorescence lifetime is calculated from a series of at least three images obtained at different detector phases. This series of images is recorded sequentially and usually a number of series is averaged to cancel out the effect of fading due to photo bleaching on the fluorescence lifetime. By carrying out a number of such measurements at different modulation frequencies multiple lifetime components can be resolved. The time-domain based method requires pulsed excitation in combination with time resolved detection such as time gating. The average fluorescence lifetime can be calculated from a minimum of two time-gated images obtained at different delays with respect to the excitation pulse. Multiple lifetime components can be resolved by extending the number of gates. In wide-field time gating based lifetime microscopy the gated images are recorded sequentially and again averaging is employed to avoid photo-bleaching effects on the fluorescence lifetime. We previously implemented time gating based FLIM in a confocal [25] and a multi-photon excitation [27] microscope. Despite the fact that these are scanning microscopes, they have rather short frame acquisition times: lifetime images can be recorded in as little as one second [28]. This comparatively high acquisition speed can be achieved because there is no need of averaging and the high efficiency of the method. All the gates (2–8) are enabled sequentially after each and 1656

every excitation pulse. This makes the microscopes insensitive to intensity variations introduced by instabilities of the light source, fading due to photo bleaching and movements of the specimen [29]. In wide-field microscopy all the pixels are acquired in parallel and although the pixel dwell time in scanning microscopes can be very short, wide-field microscopes are much faster than scanning microscopy. However, all widefield based lifetime imaging methods presented so far require the recording of a series of images at different gate settings (time-domain) or detector phase angles (frequency domain). This slows down the acquisition rate and makes the methods prone to errors. In this paper, a fast wide-field FLIM microscope is presented that employs two time gates. Both time gates are detected simultaneously after every excitation pulse by the same detector. To this end, the fluorescence image is divided into two parts of which one is delayed optically. This fast FLIM microscope was tested by imaging calcium fluxes in beating neonatal rat myocytes at frame rates of up to 100 Hz.

2. Experimental details 2.1. FLIM set-up The fluorescence decay time of a molecule can be employed for fluorescence imaging. In general fluorescence lifetimes are on the order of a few nanoseconds. Therefore, fast detection schemes are required for FLIM. In time-gated FLIM pulsed excitation is employed after which the fluorescence emission is detected in two (or more) time-gates each delayed by a different time relative to the excitation pulse (see figure 1). In the case of a two-gate detection scheme, the ratio of the signal acquired in the two gates is a measure of the fluorescence lifetime. For a mono-exponential fluorescence intensity decay the fluorescence lifetime is given by τfl. =

t , ln(IB /IA )

(1)

where t is the time-offset between the two windows and IA and IB are the corresponding integrated fluorescence intensities. In deriving equation (1) it is assumed that the two gates are of equal width (T ). In the case of a multiexponential fluorescence decay equation (1) yields only an effective fluorescence lifetime. The number of time gates

Figure 1. The principle of time-gated detection using two time gates. t is the time-offset between the two windows that are of equal width T . IA and IB are the corresponding integrated fluorescence intensities.

High frame rate FLIM

Figure 2. The scheme of the apparatus: AOM—acousto-optical modulator, DM—dichroic mirror; BS—beam splitter; lenses: L1 (f = 100 mm), L2 = L5 (f = 200 mm), L3 = L4 (f = 400 mm); T.g.II—time-gated image intensifier, PMT—photomultiplier tube, CCD—charged coupled device camera.

can be easily extended to enable the recording of multiexponential decays [26]. The multi-exponential analysis, however, requires a comparatively slow fitting procedure and is at present not suitable for visualizing lifetime information during image acquisition. In contrast, the ratio IA /IB , which is a measure of the lifetime, can be displayed in real time even at high frame rates. In the fast FLIM set-up presented here, IA and IB are detected simultaneously using a single-gated intensified CCD camera. To this end, the fluorescence image is optically divided into two parts by a beam splitter (BS). One is optically delayed with respect to the other and both parts are imaged onto two different halves of a gated image intensifier. The width of the gate pulse, T , and the optical delay between the two images, t, are chosen in such a way that both the early part of the fluorescence decay of the delayed image (IA ) is detected and the late part of the non-delayed image (IB ). The delayed image contains the integrated fluorescence emission intensity in the time interval from 0 to T (IA ) and the non-delayed image contains the fluorescence integrated in the time interval from t to (t + T ) (IB ). A schematic diagram of the fast FLIM system is presented in figure 2. A commercial Ti : sapphire laser (Spectra-Physics, Tsunami) generates picosecond pulses (pulse width