Seedorf, G. J. | Search bioRxiv

bioRxiv2020

Multicolor structured illumination microscopy and quantitative control of coherent light with a digital mirror device

Structured illumination microscopy (SIM) is a broadly applicable super-resolution microscopy technique which does not impose photophysics requirements on fluorescent samples. Multicolor SIM implementations typically rely on liquid crystal on silicon (LCoS) spatial light modulators (SLMs) for precise patterning of the excitation light, but digital micromirror devices (DMDs) are a promising alternative, owing to their lower cost, increased imaging rate, and simplified experimental timings. Given these advantages, why do existing DMD SIM implementations either rely on incoherent projection, resulting in an order of magnitude lower signal-to-noise, or utilize coherent light at only a single wavelength? The primary obstacle to realizing a multicolor coherent DMD SIM microscope is the lack of an efficient approach for dealing with the blazed grating effect. To address this challenge, we developed quantitative tools applicable to a single DMD acting as a polychromatic diffractive optic. These include a closed form solution of the blaze and diffraction conditions, a forward model of DMD diffraction, and a forward model of coherent pattern projection. We applied these to identify experimentally feasible configurations using a single DMD as a polychromatic diffractive optic for combinations of three and four common fluorophore wavelengths. Based on these advances, we constructed a DMD SIM microscope for coherent light which we used to validate these models, develop a high-resolution optical transfer function measurement technique, and demonstrate SIM resolution enhancement for calibration samples, fixed cells, and live cells. This low-cost setup opens the door to applying DMDs in polychromatic applications which were previously restricted to LCoS SLMs.

biophysics↗

bioRxiv2019

High throughput imaging identifies a spatially localized response of primary fetal pulmonary artery endothelial cells to insulin-like growth factor 1 treatment

A common strategy to measure the efficacy of drug treatment is the in vitro comparison of ensemble readouts with and without treatment, such as proliferation and cell death. A fundamental assumption underlying this approach is there is minimal cell to cell variability in the response to drug. Here, we demonstrate that ensemble and non-spatial single cell readouts applied to primary cells lead to incomplete conclusions due to cell to cell variability. We exposed primary fetal pulmonary artery endothelial cells (PAEC) isolated from healthy newborn healthy and persistent pulmonary hypertension of the newborn (PPHN) sheep to the growth hormone insulin-like growth factor 1 (IGF-1). We found that IGF-1 increased proliferation and branch points in tube formation assays but not angiogenic signaling proteins at the population level for both cell types. We hypothesized that this molecular ambiguity was due to the presence of cellular subpopulations with variable responses to IGF-1. Using high throughput single cell imaging, we discovered a spatially localized response to IGF-1. This suggests localized signaling or heritable cell response to external stimuli may ultimately be responsible for our observations. Discovering and further exploring these rare cells is critical to finding new molecular targets to restore cellular function.

biophysics↗