Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

Abstract

We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1-43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4-64, an efficient energy acceptor for FM1-43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high-resolution two-dimensional images of lifetime distributions in living neurones.

Fig. 1

A schematic illustration of the multi-dimensional TCSPC microscope. The recording electronics consists of a time measurement channel, a scanning interface and a large histogram memory. The time measurement channel contains the usual TCSPC building blocks. Two constant fraction discriminators, CFD, receive the single photon pulses from the detector and the reference pulses from the laser. The time-to-amplitude converter, TAC, measures the time from the detection of a photon to the next laser pulse. The analog-to-digital converter, ADC, converts the TAC output voltage into an address for the memory. The scanning interface is a system of counters. It receives the scan control pulses from the microscope and determines the current position of the laser beam in the scanning area. When a photon is detected the device determines the time, t, within the fluorescence decay curve and the location of the laser spot within the scanning area, x,y. These values are used to address the histogram memory. Consequently, in the memory the photon distribution vs. t, x and y builds up. The result can be interpreted as a stack of images for different times after the excitation pulse or as an array of pixels containing a complete fluorescence decay function each.

Fig. 2

A plot showing the acquisition time as a function of the number of pixels in the image. (a) The dependencies of scan time vs. pixel number for a count rate of 10⁶ s^-1. Count rates of this order require highly fluorescent samples of good photostability. (b) For a count rate of 10⁴ s^-1; such count rates are typical for cellular autofluorescence or for samples of poor photostability.

Fig. 3

TCSPC imaging of a PC12 cell expressing ECFP. Data were acquired as described, using a 10 s recording time, a long pass 470 nm emission filter and a Zeiss C-Apochromat 1.2 NA 63× water-corrected immersion objective lens. (a) The non-descanned TPE intensity image, acquired using the TCSPC card, showed a fluorescence distribution as seen in HEK293 cells, in Fig. 2. (b) The photon count over 256 time bins plotted against the x-y coordinates. (c) The fluorescence decay data from three-binned pixels from a 128 × 128 pixel image (of a 512 × 512 pixel scan: original pixel dimensions 146 nm × 146 nm) were fit to a bi-exponential curve as described in Materials and methods. The fit residuals for a mono-exponential and a bi-exponential fit are shown below the decay curve. A frequency distribution plot revealed two lifetime populations in the image; a fast component, τ₁ (open circles), and a slow component, τ₂ (filled circles); see text, (c) The pixels containing these lifetimes were reconstructed into 2-D FLIM maps, showing the short (τ₁) lifetime and the long (τ₂) lifetimes to be distributed throughout the cells with no specific accumulations, (d,e) In these FLIM images, colour corresponds to the fluorescence lifetime indicated by the false colour scale, and brightness indicates photon count.

Fig. 4

Multi-dimensional TCSPC analysis of intramolecular FRET between tandem ECFP and EYFP chromophores in HEK293 cells (CY24; the two fluorescent proteins are separated by 24 amino acids). (a,b) The non-descanned TPE intensity images for CY24-expressing HEK293 cells, in the 435-458 nm and the LP530 nm channels, respectively. (c) A FLIM map for the donor, 435-458 nm channel. (d) The normalized fluorescence decay data for ECFP alone (black filled triangles) and CY24 (open circles). ECFP data were fit to a bi-exponential curve as described (black line; see text and Table 1); FRET data were also fit to a bi-exponential curve (red line). These data were plotted from three-binned TCSPC pixels. (e) The fluorescence lifetime vs. pixel frequency distribution for HEK 293 cells expressing CY24, showing a major peak centred around the $\bar{\tau }$ lifetime mean of ∼1300 ps, with a minor peak at ∼1400-1500 ps (see text). (f-j) The respective intensity images, $\bar{\tau }$ FLIM map and frequency distribution for the same cell with a region of interest photo-bleached in the acceptor, EYFP (514 nm laser line excitation) channel. Two FLIM maps are shown (h,i), each indicating that the donor lifetime has increased in the region where the acceptor was photo-bleached. This was emphasized particularly by applying discrete colours to the FLIM map (i). In these FLIM images, colour indicates the τ lifetime, and brightness indicates photon count. Images were made using a Zeiss Plan NeoFLUAR 1.4 NA 63× oil-immersion objective lens.

Fig. 5

Living cerebellar granule cells (CGCs) were stained with FM1-43 and imaged using 800 nm TPE. The dye partitioned into membrane structures as expected (a). (b) The fluorescence spectra for membrane-intercalated FM1-43 and FM4-64, predicting that FM4-64 would act as an acceptor for FM1-43 energy; FM4-64 normalized absorbance, green filled circles, FM1-43 normalized emission, red filled circles, FM4-64 normalized emission, open circles. The line plot is the integrand, namely the product of the acceptor absorption and the donor emission, multiplied by the donor wavelength, λ⁴. (c) Decay curves from a 128 × 128 pixel FLIM image, using a 435-485 nm BP filter to dissect FM1-43 emission (filled circles, two-binned pixels underneath the cross in a), fit to a bi-exponential curve (black line). The same pixel was sampled after the same cells were counter-stained with FM4-64 (open filled circles) and the normalized data fit to a bi-exponential decay (red line, six-binned pixels). (d) The resulting donor mean fluorescence lifetime, $\bar{\tau }$, frequency distributions before (black filled circles and line) and after (red filled circles and line) FM4-64 counter-staining. (e,f) The FLIM maps generated from the distribution data for non-FRET and FRET images, respectively. In these FLIM maps, the image data brightness values have been equalized to reveal a donor-specific decrease in $\bar{\tau }$ without a loss of image clarity due to intensity quenching. 800 nm TPE was used as before, with a 500-550 BP emission filter to resolve FM1-43 (i.e. donor) fluorescence. Images were made using a Zeiss Plan NeoFLUAR 1.3 NA 40× oil-immersion objective lens.