High-contrast multifocus microscopy with a single camera and z-splitter prism.

Sheng Xiao,Howard Gritton,Xue Han,Xue Han,Xue Han,Jerome Mertz,Jerome Mertz,Jerome Mertz,Hua-An Tseng,Dana Zemel

doi:10.1364/optica.404678

Abstract

Optical microscopy has been an indispensable tool for studying complex biological systems, but is often hampered by problems of speed and complexity when performing 3D volumetric imaging. Here, we present a multifocus imaging strategy based on the use of a simple z-splitter prism that can be assembled from off-the-shelf components. Our technique enables a widefield image stack to be distributed onto a single camera and recorded simultaneously. We exploit the volumetric nature of our image acquisition by further introducing a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi-optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging. Because of its simplicity, versatility, and performance, we believe our system will be a useful tool for general biological or biomedical imaging applications.

Highlights

Optical microscopy has been an indispensable tool for studying 3D complex biological systems[1]
With our multifocus imaging strategy combined with extended-volume 3D (EV-3D) deconvolution, we can record multiple focal planes across extended volumes with a single camera at high speeds, enabling high contrast, large scale functional imaging
We demonstrate this by performing in vivo calcium imaging in the mouse brain over volumes encompassing hundreds of neurons

Summary

Introduction

Optical microscopy has been an indispensable tool for studying 3D complex biological systems[1]. Standard camera-based optical microscopes provide sharp, in-focus imaging only of a single 2D plane, with out-of-focus sample structure becoming increasingly blurred with in-focus sharpness because of the inherent trade-off between spatial resolution and depth-of-field[2]. For thick samples, such out-of-focus blurring produces background that undermines the contrast and signal-to-noise ratio (SNR) of the in-focus structure. Image stacks can be obtained in light-sheet geometries[6,7,8] with the benefit of reduced background generation These strategies involve multiple sequential image acquisitions, and their volumetric imaging rate is inherently reduced compared to the native frame rate of the camera

Methods

Results

Conclusion