Abstract
We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the "start" input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the "stop" input. The time between start and stop is directly converted into position, with a resolution of 200-250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.
Highlights
Imaging techniques based on fluorescence detection have found broad application in life sciences because they are extremely sensitive and are able to deliver information about biochemical interactions on the molecular scale
We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM)
Different principles differ in their photon efficiency, i.e., in the number of photons required for a given lifetime accuracy,22–27 the acquisition time required to record these photons, the photon flux they can be used at, their time resolution, their ability to resolve the parameters of multi-exponential decay functions, multi-wavelength capability, optical sectioning capability, suppression of light scattered inside the sample, and compatibility with different imaging and microscopy techniques
Summary
Imaging techniques based on fluorescence detection have found broad application in life sciences because they are extremely sensitive and are able to deliver information about biochemical interactions on the molecular scale. Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, photon counting and analog techniques, and point-scanning and widefield imaging techniques.. Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, photon counting and analog techniques, and point-scanning and widefield imaging techniques.6,12,15,17 It matters whether a technique acquires the signal waveform in a few time gates or in a large number of time channels and whether this happens simultaneously or sequentially.. Different principles differ in their photon efficiency, i.e., in the number of photons required for a given lifetime accuracy, the acquisition time required to record these photons, the photon flux they can be used at, their time resolution, their ability to resolve the parameters of multi-exponential decay functions, multi-wavelength capability, optical sectioning capability, suppression of light scattered inside the sample, and compatibility with different imaging and microscopy techniques. This leads to a wide variety of instrumental principles. Different principles differ in their photon efficiency, i.e., in the number of photons required for a given lifetime accuracy, the acquisition time required to record these photons, the photon flux they can be used at, their time resolution, their ability to resolve the parameters of multi-exponential decay functions, multi-wavelength capability, optical sectioning capability, suppression of light scattered inside the sample, and compatibility with different imaging and microscopy techniques.
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