Three-dimensional fluorescence imaging flow cytometry at up to million frames per second

Flow cytometry is the tool of choice for high-speed analysis of large cell populations, with the tradeoff of lacking
intracellular spatial information. Imaging flow cytometry (IFC) has emerged as a new tool that combines
advantages of microscopy with the high speed of flow cytometry. However, they can only provide 2D images to
determine three-dimensional (3D) distribution of cellular features, have a limited field of view (FOV), and require
precise control of the fluidic system to minimize image blurring due to uncontrolled cell rotation or translation
across the FOV. The absence of 3D imaging results in ambiguity of object locations and blurring by focal depth
due to the projection of a 3D cell into a 2D image. Although in the last decades flow cytometry systems that can
actually acquire three-dimensional (3D) spatial information were developed, constraints related to resolution and
samples size remained as their biggest limitation. Therefore, the goal of this proposal is to develop the next
generation 3D imaging flow cytometers with high-throughput and high-content capabilities for 3D imaging of
hundreds to thousands of cells and spheroids per second with high resolution, for the first time. We propose to
develop such a cytometry method, using a novel microscopy method, Line Excitation Array Detection microscopy
(LEAD), that can image objects in large field of views at the rate of current 1D cytometers, but with high 3D
resolution and high signal-to-noise ratios (SNR). Our proposed LEAD cytometer is a fast-scanned light-sheet
microscope capable of MHz frame rates. We will develop the fastest MHz line-scanning method using a
longitudinal acousto-optic deflector driven by a chirped frequency signal. We will image the scanned light sheet
using a linear silicon photomultiplier array, which will provide the sensitivity required when scanning so quickly,
and the parallel readout required for such high frame rates. First, we will develop linear LEAD 3D imaging flow
cytometry at sub-micron scale resolution and small FOVs. Although our preliminary data indicates we will be
able to image at 100 kHz – MHz frame rates at such high resolution with high SNR, we will perform experiments
measuring the SNR to determine the operating range of LEAD cytometry. In the second aim, we will increase
the FOV by developing two-photon LEAD imaging flow cytometry with Bessel beams. To support the larger FOV,
we will develop a 128-channel data acquisition system using eight 16-channel data acquisition cards. In the third
aim, we will develop a state-of-the-art computational infrastructure that allows for file transfers up to 25 GB/s,
storage (>100 TB), and analysis that only takes 3x the imaging time. We will use 2 deep learning models for
analysis. If successful, this high-risk/high-reward proposal would alter the imaging flow cytometry landscape.
The proposed 3D imaging flow cytometer can offer improved cell and spheroid analysis in diverse biomedical
fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. Improved sensitivity
will help users to improve research outcomes or diagnose patients with higher statistical power.

Grant Number: 5R01GM148906-04
NIH Institute/Center: NIH
Principal Investigator: ADELA BEN-YAKAR