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Fluorescence Lifetime Imaging with a Time-Domain FLIM System on a Widefield Microscope

Fluorescence lifetime can be recorded for every pixel in the image simultaneously with a time-domain FLIM camera. This method requires an intensified camera, a pulsed laser and a widefield fluorescence microscope. This is typically more cost-effective than alternative methods that need a confocal set-up.

One of the most popular methods for fluorescence lifetime imaging microscopy (FLIM) is time-correlated single photon counting (TCSPC). This method requires a confocal microscope with a pulsed laser and a photomultiplier tube (PMT). The sample is briefly illuminated by a laser pulse after which the PMT counts the number of emitted fluorescence photons. The intensity I of the fluorescence emission decays exponentially after the laser pulse has excited
the sample:

The fluorescence lifetime τ𝜏 quantifies the rate of decay of the fluorescence light. By scanning the sample with a focused laser beam, TCSPC systems can construct a fluorescence lifetime image of the sample one pixel at a time.

As an alternative to TCSPC on a confocal microscope, Lambert Instruments has developed a new system that brings time-domain FLIM to widefield microscopes. By carefully timing the exposure of the camera in the subnanosecond range, a light pulse profile of the fluorescence light can be captured. This method requires a pulsed laser and an intensified camera to record the raw data. Custom Lambert Instruments software then processes this data to automatically calculate the fluorescence lifetime.


Images were recorded with the LIFA-TD, which has a CCD camera with a fiber-optically coupled image intensifier. The image intensifier boosts the incoming light levels and it can achieve gate widths of less than 3 ns. A 485 nm pulsed laser (Picoquant LDH-D-C-485 laser head with a PDL 800-B laser driver) with a fiber-optical output was coupled into a widefield fluorescence microscope (Nikon Eclipse Ti) to provide 85 ps excitation pulses.


The set-up was calibrated by recording the light pulse profile of the laser by placing a highly reflective material in the sample holder of the microscope. Next, the fluorescence decay profile of a convallaria (lily of the valley) sample was recorded. The fluorescence lifetime is determined by correlating the fluorescence emission to the light pulse profile.


Figure 1 shows the fluorescence lifetime of a convallaria sample overlayed on the original image. The LIFA-TD is able to detect the small variations in fluorescence lifetime between different parts of the sample, stained with different dyes.

Figure 1: Fluorescence intensity (left) recording of convallaria sample and corresponding fluorescence lifetimes (right) overlayed on the original image.


Time-domain fluorescence lifetime imaging microscopy can be done on a widefield fluorescence microscope by using an intensified camera and a pulsed laser. The LIFA-TD is an entry-level FLIM system that offers an integrated solution.

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