Axial localization with modulated-illumination extended-depth-of-field microscopy

Abstract

High-speed volumetric imaging represents a challenge in microscopy applications. We demonstrate a technique for acquiring volumetric images based on the extended depth of field microscopy with a fast focal scan and modulated illumination. By combining two frames with different illumination ramps, we can perform local depth ranging of the sample at speeds of up to half the camera frame rate. Our technique is light efficient, provides diffraction-limited resolution, enables axial localization that is largely independent of sample size, and can be operated with any standard widefield microscope based on fluorescence or darkfield contrast as a simple add-on. We demonstrate the accuracy of axial localization and applications of the technique to various dynamic extended samples, including in-vivo mouse brain.

Automatic Cell Segmentation by Adaptive Thresholding (ACSAT) for Large-Scale Calcium Imaging Datasets

“…Spatial overlap is profound for wide-field imaging, but not for two-photon imaging or in vitro cell culture imaging with single cell layer. With increasing improvement wide-field imaging, such as volumetric imaging (Shain et al, 2018; Xiao et al, 2018), such significant overlap may be better eliminated during data acquisition step.…”

Video-rate large-scale imaging with Multi-Z confocal microscopy

“…Random access techniques [17,18] reduce the amounts of data production, but require scan pre-calibration and are sensitive to motion artifacts. Alternatively, data production can be reduced by purposefully limiting the spatial resolution, either axially [19,20] (though see [21][22][23], where axial resolution can be recovered in sparse samples by post-processing) or near-isotropically [24]. In our case, since we are primarily interested in the segmentation of individual cells in 3D, nearisotropic resolution is more appropriate.…”