Three‐dimensional image cytometer based on widefield structured light microscopy and high‐speed remote depth scanning

Abstract

A high throughput 3D image cytometer have been developed that improves imaging speed by an order of magnitude over current technologies. This imaging speed improvement was realized by combining several key components. First, a depth-resolved image can be rapidly generated using a structured light reconstruction algorithm that requires only two wide field images, one with uniform illumination and the other with structured illumination. Second, depth scanning is implemented using the high speed remote depth scanning. Finally, the large field of view, high NA objective lens and the high pixelation, high frame rate sCMOS camera enable high resolution, high sensitivity imaging of a large cell population. This system can image at 800 cell/sec in 3D at submicron resolution corresponding to imaging 1 million cells in 20 min. The statistical accuracy of this instrument is verified by quantitatively measuring rare cell populations with ratio ranging from 1:1 to 1:105.

Snapshot Holographic Optical Tomography

“…Despite the development of those superresolution microscopes and other recent techniques, achieving high spatial and temporal resolution at the same time is still challenging. Cutting-the-edge imaging flow cytometers can record the 3-D image of a stationary/fixed specimen at the imaging throughput of about 50 cells/sec and at the diffraction-limited spatial resolution [23,24]. Importantly, the existing 3-D methods relying on a scanning mechanism cannot be applied to the specimens that are continuously moving/crawling on a surface or continuously flowing in a channel.…”

Advances in Chromatin and Chromosome Research: Perspectives from Multiple Fields

“…More recently, light-sheet microscopy (LSM) (Ahrens et al, 2013) and structured illumination microscopy (SIM) (Schermelleh et al, 2008) have gained popularity for 3D imaging. While LSM suffers from insufficient depth resolution, SIM could be a competitor to SDM for DNA damage imaging (Choi et al, 2015;Wadduwage et al, 2015). Interestingly, certain variations of SIM satisfy the requirements for compressive sensing (Duarte et al, 2008;Wadduwage et al, 2019), which could, in theory, reduce the measurement time by almost an order of magnitude.…”

Large population cell characterization using quantitative phase cytometer

“…As a cell analysis technique, the most significant advantage of flow cytometry is its unprecedented throughput with a typical processing speed of 10,000 cells/s; an extreme sample rate up to ∼1 million cells/s has also been demonstrated . While flow cytometry is good for extracting biochemical information from cells via fluorometric assays, it mostly cannot quantify the morphological characteristics and molecular localizations due to its low spatial resolution . We also note that flow cytometry inherently requires cells to be suspended and dissociated for flow purposes; this requires the adherent cells to be trypsinized before each measurement .…”