Functional imaging with dynamic quantitative oblique back-illumination microscopy

Abstract

Significance: Quantitative oblique back-illumination microscopy (qOBM) is a recently developed label-free imaging technique that enables 3D quantitative phase imaging of thick scattering samples with epi-illumination. Here, we propose dynamic qOBM to achieve functional imaging based on subcellular dynamics, potentially indicative of metabolic activity. We show the potential utility of this novel technique by imaging adherent mesenchymal stromal cells (MSCs) grown in bioreactors, which can help address important unmet needs in cell manufacturing for therapeutics.Aim: We aim to develop dynamic qOBM and demonstrate its potential for functional imaging based on cellular and subcellular dynamics.Approach: To obtain functional images with dynamic qOBM, a sample is imaged over a period of time and its temporal signals are analyzed. The dynamic signals display an exponential frequency response that can be analyzed with phasor analysis. Functional images of the dynamic signatures are obtained by mapping the frequency dynamic response to phasor space and colorcoding clustered signals.Results: Functional imaging with dynamic qOBM provides unique information related to subcellular activity. The functional qOBM images of MSCs not only improve conspicuity of cells in complex environments (e.g., porous micro-carriers) but also reveal two distinct cell populations with different dynamic behavior. Conclusions:In this work we present a label-free, fast, and scalable functional imaging approach to study and intuitively display cellular and subcellular dynamics. We further show the potential utility of this novel technique to help monitor adherent MSCs grown in bioreactors, which can help achieve quality-by-design of cell products, a significant unmet need in the field of cell therapeutics. This approach also has great potential for dynamic studies of other thick samples, such as organoids.

Analysis of structural effects of sickle cell disease on brain vasculature of mice using three-dimensional quantitative phase imaging

“…To recover quantitative phase information with qOBM, a pair of orthogonal DPC images are acquired and then deconvolved with the system’s optical transfer function via a Tikhonov regularized deconvolution, as previously described by Refs. 14, 15, and 18. After the deconvolution, qOBM yields clear cellular and sub-cellular contrast with quantitative image values reflective of the RI properties of the sample, denoted with the variable n.…”

Tracking cellular and subcellular dynamics with quantitative oblique back-illumination microscopy

“…2C], which signifies that indeed the frequency response of the cellular dynamics follows an exponential behavior inside 21,22 and multi-exponential behavior outside the universal semi-circle. 23 Fig. 2C also shows the average responses from two distinct regions in phasor space (indicated by the blue and green regions of interest (ROIs) in Fig.…”

Quantitative phase imaging of sickle cell disease effects on mouse brain vasculature using quantitative oblique back-illumination microscopy

“…17 To recover quantitative phase information with qOBM, a pair of orthogonal DPC images are acquired and then deconvolved with the system's optical transfer function via a Tikhonov regularized deconvolution, as previously described by Ref. 13,14,18 After the deconvolution of the four brightfield images, qOBM yields clear sub-cellular contrast with quantitative image values reflective of the refractive index properties of the sample. In this work, we use a 40X, 0.6 NA objective (Nikon S Plan Fluor LWD) and a CMOS camera (pco.edge 4.2 LT).…”