Abstract
Confocal microscopy has several advantages over wide-field microscopy, such as out-of-focus light suppression, 3D sectioning, and compatibility with specialized detectors. While wide-field microscopy is a faster approach, multiplexed confocal schemes can be used to make confocal microscopy more suitable for high-throughput applications, such as high-content screening (HCS) commonly used in drug discovery. An increasingly powerful modality in HCS is fluorescence lifetime imaging microscopy (FLIM), which can be used to measure protein-protein interactions through Förster resonant energy transfer (FRET). FLIM-FRET for HCS combines the requirements of high throughput, high resolution and specialized time-resolving detectors, making it difficult to implement using wide-field and spinning disk confocal approaches. We developed a novel foci array scan method that can achieve uniform multiplex confocal acquisition using stationary lenslet arrays for high resolution and high throughput FLIM. Unlike traditional mirror galvanometers, which work in Fourier space between scan lenses, this scan method uses optical flats to steer a 2-dimension foci array through refraction. After integrating this scanning scheme in a multiplexing confocal FLIM system, we demonstrate it offers clear benefits over traditional mirror galvanometer scanners in scan linearity, uniformity, cost and complexity.
Highlights
In confocal microscopy, an excitation focal point is raster scanned across the field of view to generate a 2- or 3-dimensional (2D or 3D) image [1]
We recently demonstrated a square multiple foci array scheme to achieve fluorescence lifetime imaging microscopy (FLIM) using a streak camera as a detector [28]
If the galvanometers are fitted with fused silica (n = 1.47) windows of thickness 5 mm, this lateral deflection can be accomplished with a tilt angle of ± 8.9°
Summary
An excitation focal point is raster scanned across the field of view to generate a 2- or 3-dimensional (2D or 3D) image [1]. The high speed gated cameras required to record time domain information using a spinning disk system suffer from poor spatial resolution that many subcellular details are lost This typically limits such systems to collecting intensity images. To take full advantage of a multiplexed confocal approach, the detection may be made by discrete single-point detectors, which offer better signal-to-noise performance and temporal resolution than a CCD array [24] For these methods, a foci array is generated by a lenslet array or spatial light modulator, and raster scanning is performed by the entire array [25, 26]. It should be noted that the applications of such array scanning techniques are not limited to FLIM, but are useful to other confocal applications where stationary foci array readout is desired
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