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.
SET-UP
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.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
