Dual-LED-based multichannel microscopy for whole-slide multiplane, multispectral, and phase imaging

TL;DR

This paper introduces a multichannel microscopy platform capable of simultaneous multiplane, multispectral, and phase imaging for whole-slide samples, enabling rapid, multiplexed, and multimodal biological imaging with high data throughput.

Contribution

The development of a multichannel microscopy system with 6 independent channels for simultaneous multiplane, multispectral, and phase imaging, including an open-source design to expand channels.

Findings

Achieves ~1 gigapixel per second data throughput.

Simultaneously acquires 6 focal planes, 6 spectral bands, or 3 phase images.

Enables fast 3D dynamic tracking of biological samples.

Abstract

We report the development of a multichannel microscopy for whole-slide multiplane, multispectral, and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of ~1 gigapixel per second. To perform single-frame rapid autofocusing, we place two near-infrared LEDs at the back focal plane of the condenser lens to illuminate the sample from two different incident angles. A hot mirror is used to direct the near-infrared light to an autofocusing camera. For multiplane whole slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole-slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport-of-intensity equation to recover the phase information. We also provide an open-source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z-scanning may also enable fast 3D dynamic tracking of various biological samples.