Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy

Abstract

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Fig. 1

(a) Principle of wide-field TCSPC data acquisition with an MCP intensifier and a CMOS camera. After each excitation pulse, a sequence of frames is acquired. Software searches the frames for position of the photons and their arrival time (i.e. their frame number). These are used to build a histogram of arrival times for each pixel. A lifetime image is obtained by fitting an appropriate exponential decay function to the arrival time histogram in each pixel, and encoding the lifetime in a pseudocolour scale , . (b–d) Wide-field TCSPC decays of ruthenium compound Ru(dpp) in water and glycerol solutions, obtained using the principle in (a). (b) Single exponential fits to the data sets with all pixels binned yield lifetimes of 1.35 and $505\mathrm{\mu }\mathrm{s}$ for the water and the glycerol sample, respectively. (c) Lifetime maps of Ru(dpp) in water (top) and glycerol (bottom). (d) Histograms of individual pixel lifetimes in (c). The acquisition time was less than 1 s. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Exploiting the phosphor decay for photon arrival timing within the frame exposure time. (a) Enlarged areas of single photons events arriving at the beginning (top row), in the middle (middle row) and towards the end (bottom row) of the frame exposure time of the 3${}^{rd}$ frame, and (b) a schematic representation of the scenarios in (a). (c) Experimental calibration plot in linear (top) and semi-logarithmic (bottom) scale. (d–g) Lifetime measurement of ruthenium-based luminescent probe Ru(dpp) using the method shown in (a–c): wide-field TCSPC intensity (d) and lifetime (e) images of four Ru(dpp) ＋ water/glycerol solutions, labelled with glycerol % in (d). (f) Decays, fits (black lines) and residuals for the four different areas in (e). (g) Histograms of the individual pixel lifetimes of the different solutions in (e). The dashed line shows histogram of the whole image. The data set colours in (f) correspond to colours in (g).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

(a) Lifetime images of beads with iridium complexes Ir(ppy)₃, Ir(BMes) and Ir(fppy)₃ (from left to right, respectively), imaged with direct TCSPC measurement with 675,000 Hz camera frame rate. (b) Histograms of the individual pixel lifetimes in (a). (c) Decays of images in (a) with all pixels binned, mono-exponential fits to the data (lines) and residuals. (d) Lifetime images of beads with Ir(ppy) (top left), Ir(BMes) (top right), Ir(fppy) (bottom left) complexes and BPEA (bottom right), imaged with phosphor decay method and 54 kHz camera frame rate. (e) Histograms of the individual pixel lifetimes in (c). (f) Decays of images in (c) with all pixels binned (data points), mono-exponential fits to the data (lines) and residuals.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

(a) In a delay line anode, the position of the photon event is obtained from the signal propagation time difference to the ends of the delay line. Multiple photon events can be discarded by a run-time check, or timed with a third delay line (hexanode). The timing can be performed with three standard TCSPC boards, one for x, one for y and the third for time, t. (b–e) TCSPC images of fixed HeLa cells, stained with membrane dye laurdan, acquired with a delay line anode detector. The measured intensity shows the cell membrane only under TIRF illumination (b), and the fluorescence lifetime of laurdan is shorter (d), while the whole cell is visible under wide-field illumination (c) and the laurdan lifetime is longer (e). (f) Histograms of the individual pixel lifetimes in (c, d). (g) Fluorescence decays in the area indicated by a white rectangle are shown in (c, d). The instrument response function, measured with reflection, has a FWHM of 344 ps . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)