RESUMEN
The streak camera is a picosecond resolution photodetector with parallel input capability; however, the degree of multiplexing is limited by crosstalk and temporal uncertainty in the sweeping field. We introduced a fixed time delay between adjacent fibers to reduce crosstalk in the synchroscan mode. The fixed delay and a tunable electronic delay between the input pulse and the synchroscan unit allows robust separation modes between the streaks, while spatial and temporal nonlinearities can be calibrated in. The efficacy of the design is demonstrated through a 100-fold multiplexed confocal fluorescence lifetime imaging microscope in live cells.
RESUMEN
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.
RESUMEN
The streak camera is one of the fastest photodetection systems, while its capability of multiplexing is particularly attractive to many applications requiring parallel data acquisition. The degree of multiplexing in a streak camera is limited by the crosstalk between input channels. We developed a technique that introducing a fixed time delay between adjacent fiber channels in a customized two-dimensional to one-dimensional fiber array to significantly reduce crosstalk both at the sample plane and at the input of a streak camera. A prototype system has been developed that supports 100 input channels, and its performance in fluorescence microscopy is demonstrated.