Techniques for single-shot volumetric fluorescence imaging
Tsang Min Ching, Jean-Marc
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Rapid development of genetically encoded fluorescent indicators has provided a diverse chemical toolkit to probe complex biological systems, leading to the expansion of fluorescence microscopy for biological research and applications. However, the inherent constraints on resolution, speed and field of view have hindered the development of high speed, three dimensional fluorescence imaging over large spatial scales for biological microscopy. This thesis describes two strategies based on confocal microscopy to provide single-shot volumetric fluorescence imaging over large scales. In the first part, we describe a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique, called line-scan multi-z confocal microscopy, enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples. In the second part, we describe a fiber bundle-based endomicroscopy technique, which provides pseudo-volumetric imaging over large field of views, without the need for axial scanning. Our technique uses a gradient refractive index lens to achieve an axially extended illumination and a series of reflecting pinholes of different diameters to simultaneously probe different number of fiber cores. The fluorophores are localized from the series of acquired images using a convolution neural network. We validate our system by localizing fluorescent beads distributed in a volume sample.
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