Abstract
We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.
Highlights
The observation of cells and organelles in their native 3D environment is becoming increasingly important in biological research.[1]
For the measurement of the lightsheet thickness and the instrument response function (IRF), a mirror was placed in the sample plane at at an angle of 45◦, and the emission filter replaced with a strong neutral density filter
Lightsheet microscopy enables optical sectioning of thick biological samples embedded in 3D environment while reducing photobleaching of the sample by illuminating only the imaged plane, whereas fluorescence lifetime imaging microscopy (FLIM) provides contrast based on the microenvironment of the probe or proximity of other fluorophores (i.e. FRET)
Summary
The observation of cells and organelles in their native 3D environment is becoming increasingly important in biological research.[1]. No other method can study molecules in living cells with anything remotely approaching its combination of spatial resolution, selectivity, sensitivity and dynamics. In a conventional fluorescence microscope, the sample is typically placed on a glass coverslip, and imaging is restricted to a region relatively close to the coverslip, as resolution and contrast decrease rapidly with imaging depth, which can be problematic for larger samples. Multiphoton excitation microscopy helps to address these issues, and can image relatively large samples when using a mesolens.[2,3] This is a scanning approach where the image is acquired pixel by pixel. To acquire the whole field of view in a single exposure using a camera while still addressing the photobleaching and depth issue, lightsheet illumination can be used
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