Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.
We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) timestamps from
32×32 CMOS SPAD array. Custom firmware was written to select 64 pixels out of 1024 available for fast transfer of
TCSPC timestamps. Our 64 channel TCSPC is capable of acquiring up to 10 million TCSPC timestamps per second over
a USB2 link. We describe the TCSPC camera (Megaframe), camera interface to the PC and the microscope setup. We
characterize the Megaframe camera for fluorescence lifetime imaging (FLIM) including instrument response function,
time resolution and variability of both across the array. We show a fluorescence lifetime image of a plant specimen
(Convallaria majalis) from a custom-built multifocal multiphoton microscope. The image was acquired in 20 seconds
(with average timestamp acquisition rate of 4.7 million counts per second).
Forster/Fluorescence resonant energy transfer (FRET) has become an extremely important technique to explore
biological interactions in cells and tissues. As the non-radiative transfer of energy from the donor to acceptor occurs
typically only within 1-10nm, FRET measurement allows the user to detect localisation events between protein-conjugated
fluorophores. Compared to other techniques, the use of time correlated single photon counting (TCSPC) to
measure fluorescence lifetime (FLIM) has become the gold standard for measuring FRET interactions in cells. The
technique is fundamentally superior to all existing techniques due to its near ideal counting efficiency, inherent low
excitation light flux (reduced photobleaching and toxicity) and time resolution. Unfortunately due to its slow acquisition
time when compared with other techniques, such as Frequency-domain lifetime determination or anisotropy, this makes
it impractical for measuring dynamic protein interactions in cells. The relatively slow acquisition time of TCSPC FLIM-FRET
is simply due to the system usually employing a single-beam scanning approach where each lifetime (and thus
FRET interaction) is determined individually on a voxel by voxel basis. In this paper we will discuss the development a
microscope system which will parallelize TCSPC for FLIM-FRET in a multi-beam multi-detector format. This will
greatly improve the speed at which the system can operate, whilst maintaining both the high temporal resolution and the
high signal-to-noise for which typical TCPSC systems are known for. We demonstrate this idea using spatial light
modulator (SLM) generated beamlets and single photon avalanche detector (SPAD) array. The performance is evaluated
on a plant specimen.
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling
events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging
depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared
excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining
functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically
requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled
MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator
(SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is
employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction.
Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.