Integrative quantitative-phase and airy light-sheet imaging

Abstract

Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free recognition of cells and organelles, and quantifies their number-density that, thermodynamically, is more representative of metabolism than size. Here, we report the fusion of these two imaging modalities onto a standard inverted microscope that retains compatibility with microfluidics and open-source software for image acquisition and processing. An accelerating Airy-beam light-sheet critically enabled imaging areas that were greater by more than one order of magnitude than a Gaussian beam illumination and matched exactly those of quantitative-phase imaging. Using this integrative imaging system, we performed a demonstrative multivariate investigation of live-cells in microfluidics that unmasked that cellular noise can affect the compartmental localization of metabolic reactions. We detail the design, assembly, and performance of the integrative imaging system, and discuss potential applications in biotechnology and evolutionary biology.

Photon-Sparse, Poisson Light-Sheet Microscopy

“…To compare Poisson with modern LSI, we imaged identical specimen locations first with a sCMOS (82% quantum efficiency, ORCA-Flash 4.0 v2, Hamamatsu) and then with the ICMOS at the same average irradiance density. We also applied the sCMOS in brightfield imaging (Figure c).…”

“…We adopted a LSI setup integrated with a standard inverted microscope, as we recently demonstrated and further detail in the Supporting Information (Figure b). The microscope was controlled with Micro-Manager with the illumination focused through a 20×/0.42 objective, yielding a ∼2 μm diameter Gaussian beam . Images were formed by laterally scanning ( y -axis, Figure b) the beam using a MEMS mirror, generating up to 200 μm × 10 μm field-of-view images.…”

“…Operation at this, semiclassical, limit relies on detectors of practically zero dark and read noise that can detect single photons (Figure b), despite the Poisson stochasticity in their arrival times (Figure a). Using a standard light-sheet microscope (Figure b), we demonstrate that operation at the Poisson limit enables brightness quantification (a proxy of protein or transcript expression) with 200-fold fewer photons than modern LSI. Further, wavelet-based image reconstruction enables object recognition at subphoton per pixel fluxes, corresponding to more than 20-fold fewer photons than conventional LSI.…”

A Review of Optical Imaging Technologies for Microfluidics

“…There have been many successful examples of microfluidic systems coupled with light-sheet fluorescence microscopy [95][96][97][98][99][100][101][102][103]. This article mainly introduces the research of Memeo et al [104] and Jiang et al [105].…”

Scattered‐light‐sheet microscopy with sub‐cellular resolving power

“…Overall, this geometrical reconfiguration has been found to reduce the total sample irradiance levels by more than one order of magnitude with respect to confocal microscopy [5,11]. Further, LSI and SPIM have been also integrated with pseudo-diffraction-free illumination sheets, such as those enabled by Bessel and Airy beams, and optical lattices, as well as photon-sparse detection to accelerate imaging rates and further decrease the levels of irradiance [12][13][14][15].…”