Three‐dimensional imaging flow cytometry through light‐sheet fluorescence microscopy

Abstract

Flow cytometry is the tool of choice for high-speed acquisition and analysis of large cell populations, with the tradeoff of lacking intracellular spatial information. Although in the last decades flow cytometry systems that can actually acquire two-dimensional spatial information were developed, some of the limitations remained though, namely constrains related to sample size and lack of depth or dynamic information. The combination of fluidics and light-sheet illumination has the potential to address these limitations. By having cells travelling with the flowing sheath one can, in a controlled fashion, force them at constant speed through the light-sheet enabling the synchronized acquisition of several optical sections, that is, three-dimensional imaging. This approach has already been used for imaging cellular spheroids, plankton, and zebra-fish embryos. In this review, we discuss the known solutions and standing challenges of performing three-dimensional high-throughput imaging of multicellular biological models using fluidics, while retaining cell and organelle-level resolution. © 2017 International Society for Advancement of Cytometry.

High-throughput multicolor 3D localization in live cells by depth-encoding imaging flow cytometry

“…Recently-developed instruments have demonstrated multiple spectral channels George et al, 2004) and other microscope modalities, e.g. phase imaging (Gorthi and Schonbrun, 2012) and optical sectioning (Gualda et al, 2017;Regmi et al, 2014). In general, because the basic operating principles of IFC and widefield microscopy are the same, tools developed for one can be applied to the other.…”

Förster Resonance Energy Transfer Pioneers Biomechanics: Molecular‐Scale Force Meters for Visualizing Intracellular Stress Fields

“…As a relatively new branch of science, biomechanics, is dealing with measurements of forces at different levels of organization of the living state and studying transmission of force through the cell membrane toward the cell interior (mechano-transduction), as well as the conversion of mechanical signals to electric and chemical ones, that is, signal transductions evoked by mechanical forces (1)(2)(3). (4)(5)(6). In order to monitor these types of signaling, an important step forward is the development of biosensors capable for mechanical force measurement at the molecular level, even inside the living cell.…”

“…Recent applications of FRET for mapping interleukin-2-interleukin-9 receptor (IL-2R-IL-9R) complexes, for revealing spatial gradients of cyclic adenosine monophosphate (cAMP) second messenger complexes, and for imaging flow cytometry by light-sheet microscopy are exemplified in Refs. (4)(5)(6). A possible application of FRET as a photoswitching agent of genetically engineered fluorescent proteins (VFPs) is described in Ref.…”

Simultaneous Analysis of Phenotype and Cytogenetics Using Imaging Flow Cytometry: Time to Teach Old Dogs New Tricks

“…The aforementioned problem of distinguishing superimposed but spatially distinct hybridization spots from truly spatially co-localized ones is a formidable problem that may only be solved through 3D imaging approaches such through the application of light-sheet fluorescence imaging (12) or the design of different hybridization probes such as the use of dual color break apart probes. The aforementioned problem of distinguishing superimposed but spatially distinct hybridization spots from truly spatially co-localized ones is a formidable problem that may only be solved through 3D imaging approaches such through the application of light-sheet fluorescence imaging (12) or the design of different hybridization probes such as the use of dual color break apart probes.…”

“…The next challenge in this field will be the application to FISH approaches that rely on the exact co-localization of hybridization sites, for example, for the detection of translocations or inversions. The aforementioned problem of distinguishing superimposed but spatially distinct hybridization spots from truly spatially co-localized ones is a formidable problem that may only be solved through 3D imaging approaches such through the application of light-sheet fluorescence imaging (12) or the design of different hybridization probes such as the use of dual color break apart probes. Depending on the specific application, the latter will have to be critically validated as was recently demonstrated for the EML4-ALK fusion (13).…”