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 color-coding 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.

Keywords: dynamic; functional imaging; label-free; microscopy; quantitative phase imaging; stem cells.

Fig. 1

(a) qOBM system schematic. (b) Illustration of porous microcarrier ($\sim 150$ to $300\mu \mathrm{m}$ in diameter). (c) Timelapse qOBM stack of a microcarrier with adherent MSCs inside of a bioreactor. (d) Green: temporal phase value fluctuations from a single pixel corresponding to a cell region. Orange: log–log representation of the Fourier transform of the temporal phase value (green line).

Fig. 2

qOBM and DqOBM images of microcarriers surrounded by MSCs. (a) and (b) qOBM images of two microcarriers at 4 and 6 days of culturing, respectively. (c)–(f) Close-ups of pink and green regions in panels (a) and (b). (g)–(l) DqOBM functional images [(k) and (l)] and close-ups [(g)–(j)] of timelapses taken at [(g), (h), and (k)] 1 Hz over 8 min and [(i), (j), and (l)] 8 Hz over 1 min.

Fig. 3

Phasor analysis. (a) qOBM image of microcarrier with adherent MSCs. ROIs in green correspond to background, yellow regions correspond to the static microcarrier, and red and blue regions correspond to live cells. (b) Average phase frequency response form selected ROIs in (a). Data acquired at 1 Hz over 8 min. (c) Cumulative phasor plot of $\sim 60$ MSCs captured at 1 Hz over 8 min, mapped with $\tau =4\mathrm{s}$ (d) Log of the average signal responses of regions marked in (c).

Fig. 4

DqOBM captured at different imaging rates. (a) qOBM image of microcarrier with MSCs attached (b) Top: phasor plots of the same microcarrier imaged at 1 and 8 Hz, mapped with $\tau =4\mathrm{s}$ and $\tau =0.5\mathrm{s}$, respectively. Bottom: corresponding DqOBM functional images. Videos 1 and 2 show each respective functional image overlayed over the qOBM timelapses. Color schemes for DqOBM are shown in the phasor plots (top) (Video 1, mov, 19.9 MB [URL: https://doi.org/10.1117/1.JBO.27.6.066502.1] and Video 2, mov, 25.9 MB [URL: https://doi.org/10.1117/1.JBO.27.6.066502.2]).