Abstract
Confocal laser scanning microscopy (CLSM) is commonly used to observe molecules of biological relevance in their native environment, the live cell, and study their spatial distribution and interactions nondestructively. CLSM can be easily extended to measure the lifetime of the excited state, the concentration and the diffusion properties of fluorescently labeled molecules, using fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), respectively, in order to provide information about the local environment and the kinetics of molecular interaction in live cells. However, these parameters cannot be measured simultaneously using conventional CLSM due to damaging effects that are associated with strong illumination, including phototoxicity, photobleaching, and saturation of the fluorescence signal. To overcome these limitations, we have developed a new camera consisting of 1024 single-photon avalanche diodes that is optimized for multifocal microscopy, FLIM and FCS. We show proof-of-principle measurements of fluorescence intensity distribution and lifetime of the enhanced green fluorescent protein expressed in live cells and measurement of quantum dot diffusion in solution by FCS using the same detector.
Highlights
C ONFOCAL laser scanning microscopy (CLSM) is universally used in biomedical research to investigate molecular mechanisms underlying vital biological functions
The camera design optimized for multifocal microscopy was based on an array of 1024 independent single photon avalanche photodiode (SPAD) produced in standard CMOS technology [7], [19], [20]
The results are divided in three sections concerning the microscopy setup, fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS)
Summary
C ONFOCAL laser scanning microscopy (CLSM) is universally used in biomedical research to investigate molecular mechanisms underlying vital biological functions. We present a new 32 × 32 SPAD camera which fulfills all the requirements indicated above, which makes it suitable for a multifocal FCS experimental setup This device implements a fast time-gating control, which enables the pixel electronics for short time periods down to 1.5 ns and at delay steps below 100 ps. This camera is capable of measuring the fluctuations of the fluorescence intensity at very high frame-rates (50 to 100 kHz) and the decay kinetics of fluorophores after illumination by a pulsed laser. We validated the proposed camera using several model experiments such as the measurement of the diffusion time of single quantum dots (QD) in solution and the fluorescence decay kinetics of genetically encoded enhanced Green Fluorescent Protein (eGFP) expressed in live cells
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