Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

Kalita, R.; Flanagan, W.; Lightley, J.; Kumar, S.; Alexandrov, Y.; Garcia, E.; Hintze, M.; Barkoulas, M.; Dunsby, C.; French, P. M. W.

doi:10.1101/2021.04.14.437846

bioRxiv · 10.1101/2021.04.14.437846

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

Kalita, R.·Flanagan, W.·Lightley, J.·Kumar, S.·Alexandrov, Y.·Garcia, E.·Hintze, M.·Barkoulas, M.·Dunsby, C.·French, P. M. W.

View original source ↗Export metadata ↓

Abstract

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be interleaved with single-shot imaging polarimetry.

Source connections

Published article ↗

Explore related subjects

biophysics Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Kalita, R., Flanagan, W., Lightley, J., Kumar, S., Alexandrov, Y., Garcia, E., Hintze, M., Barkoulas, M., Dunsby, C., French, P. M. W.. 2021-04-14. Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast. https://doi.org/10.1101/2021.04.14.437846

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A Deformulating Multi-Attribute SEC Method for Simultaneous Quantification of Total RNA, Conjugated Antibody, and Free Antibody in Targeted Lipid Nanoparticles

Targeted lipid nanoparticles (tLNPs) hold promise for improving cell- and tissue-selective delivery of therapeutic payloads but introduce analytical challenges associated with characterizing both encapsulated cargo and surface-associated targeting ligands within the same formulation. Conventional nucleic acid and protein assays can be affected by nanoparticle scattering, matrix interference, or a limited ability to distinguish conjugated antibody from free antibody. Here, we report an online deformulating size-exclusion high-performance liquid chromatography (SE-HPLC) method that enables simultaneous quantification of total RNA, conjugated antibody, and free antibody from 5 L of tLNP sample. Following injection, exposure to a mobile phase containing sodium dodecyl sulfate and isopropanol disrupts the nanoparticles, enabling chromatographic separation of RNA, conjugated antibody, and free (unconjugated) antibody while minimizing sample handling prior to analysis. RNA was quantified by UV260 detection, whereas antibody was quantified by UV280 or intrinsic fluorescence detection to maximize sensitivity. The method demonstrated linear response over the evaluated concentration ranges, spike recoveries of 88-122%, and precision below 5% relative standard deviation. Application to purified tLNP formulations demonstrated simultaneous assessment of total RNA and conjugated antibody content, while confirming the absence of detectable free antibody following purification. Together, these results establish online deformulating SE-HPLC as a practical multi-attribute analytical method for characterization of tLNPs, enabling concurrent assessment of total RNA concentration, conjugated antibody concentration, and free antibody concentration using standard HPLC instrumentation.

biophysics↗

bioRxiv2026

Nanomechanical phenotyping uncovers a non-canonical smooth muscle-like hypercontractility program in lung squamous cell carcinoma myofibroblasts

Myofibroblast-like cancer-associated fibroblasts (myCAFs) drive the stiff, tumor-promoting microenvironment, yet their mechanical phenotype in non-small cell lung cancer (NSCLC) remains undefined. myCAFs from major NSCLC subtypes (lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC)) were examined combining atomic force microscopy and traction force microscopy with molecular and signaling analyses. LUAD-myCAFs displayed canonical myofibroblast features, including elevated stiffness, traction forces, ROCK-MYPT1 signaling, and -SMA-rich stress fibers. Unexpectedly, LUSC-myCAFs amplified these nanomechanical features despite reduced ROCK-MYPT1 signaling and comparable -SMA levels. Instead, they exhibited a smooth muscle cell (SMC)-like contractile program characterized by enhanced SM-MHC(MYH11) expression colocalized with -SMA, ERK1/2-RLC hyperactivation, and DNA methylation reprogramming including SMC-contractility regulators. LUSC-myCAF hypercontractility was linked with reduced proliferative capacity and smaller intratumoral vessel lumens, consistent with the low CAF density and enhanced hypoxia patterns reported in LUSC. SMC markers were enriched and associated with adverse outcomes selectively in LUSC, highlighting the relevance of SMC-like myCAFs in LUSC progression. These findings unveil subtype-specific myCAF programs in NSCLC, and identify -SMA/SM-MHC(MYH11) myCAFs as key determinants of tumor mechanics, providing a rationale for the survival benefits of antihypertensive drugs targeting SMC hypercontractility. Moreover, they demonstrate that nanomechanical phenotyping can uncover translationally relevant myCAF phenotypes invisible to molecular profiling.

biophysics↗

bioRxiv2026

Engineering Nanobody Conformational Switches

Conformational signaling is widely used in biology, but remains difficult to engineer into proteins. Here, we explore ligand-induced folding of nanobodies as a general strategy to generate conformational receptors for a broad range of target ligands. More specifically, we have generated partially unfolded nanobody variants that fold exclusively upon binding to their target ligand. To do so, we have introduced destabilizing mutations in the framework region, carefully tuning their stability while preserving molecular recognition. Given the high conservation of the framework across different nanobodies, this approach is broadly applicable to many other target ligands, which has enabled us to engineer conformational switches for three different important biomarkers and drugs: the pregnancy hormone chorionic gonadotropin, the cytokine tumor necrosis factor alpha, and the chemotherapy and immunosuppressant drug methotrexate. Another useful property of conformational signaling is that it can directly gate signal transduction, which we have leveraged to build an optical sensor for the quantitative detection of the pregnancy hormone in urine. Finally, conformational signaling introduces new mechanisms for the allosteric modulation of binding, which we have explored to control the affinity and dynamic range of the sensor. Our work thus establishes a rational strategy for engineering conformational signaling receptors with customizable properties and generalizable for many different target ligands.

biophysics↗