bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/718981

Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications

Mancebo, A.·DeMars, L.·Ertsgaard, C. T.·Puchner, E. M.
View original source ↗Export metadata ↓

Abstract

Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cells morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.\n\nStatement of significanceActively patterned illumination in fluorescence microscopy can reduce bleaching and phototoxicity as well as actively manipulate proteins in optogenetic applications. Matching the coordinate system of the camera and the light patterning device such as digital mirror arrays (DMA) remains a challenge. We developed a fast and easy calibration procedure that determines and corrects for the transformation between the camera and DMA coordinate system with ~50 nm precision. Using this approach, we develop spatial feedback photoactivation for Single Molecule Localization Microscopy (SMLM) to photoswitch only intracellular regions containing photoswitchable fluorophores. Our results show a 44% improvement in the possible data acquisition time before phototoxic effects become detectable and a 28% increase in detected localizations. Spatial feedback photoactivation thus significantly improves SMLM experiments.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Mancebo, A., DeMars, L., Ertsgaard, C. T., Puchner, E. M.. 2019-07-30. Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications. https://doi.org/10.1101/718981

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

KEEP EXPLORING

Related preprints

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

Frequency-Dependent Structural Responses of Amyloid-β Fibrils to Terahertz Pulses: A Nonequilibrium Molecular Dynamics Study

The abnormal aggregation of amyloid proteins into fibrils is closely associated with several neurodegenerative diseases, highlighting the importance of developing effective strategies to modulate fibril structural stability. Here, we employed nonequilibrium molecular dynamics(NEMD) simulations to examine the effects of intense terahertz-to-mid-infrared pulses on three amyloid fibrils (2LMP, 2MXU, and 5OQV), focusing on the combined effects of pulse frequency and electric-field strength. Frequency scans from 0.5 to 60 THz in 0.5 THz steps revealed frequency-dependent losses of {beta}-sheet content, with several common response patterns in these fibrils. Comparisons at 2, 23, 33, and 50 THz showed that pulse-induced changes in secondary structure differed from those in high-temperature controls. Hydrogen-bond analysis showed that total hydrogen-bond counts could partially recover even as interchain hydrogen bonds continued to decrease, with distinct responses among representative chain pairs. We therefore propose a two-threshold electric-field framework for fibril disruption: Eth1 describes the onset of structural perturbation, whereas Eth2 indicates persistent structural loss after the pulse. This framework offers a testable approach for characterizing amyloid structural responses to external fields, although the underlying energy-coupling mechanisms, quantitative thresholds, and long-term recovery require further validation. These findings may provide a theoretical basis for exploring external-field-based strategies to modulate protein aggregation.

biophysics↗
bioRxiv2026

Organised streaming of cerebrospinal fluid in the human third ventricle

Quasi-periodic pressure pulses associated with cardiac and respiratory activity generate reciprocating cerebrospinal fluid (CSF) motion in the human ventricles. Regular waves dominated by the cardiac cycle are overlaid with cycles of low-frequency respiratory waves, the latter to become stronger in forced breathing or even dominated by large successive flushes in NREM sleep. The present study investigates how such waves contribute to residual streaming within the human ventricles. Transient CFD simulations were performed in the third ventricle at varying parameters of frequency, stroke volume and wave-form asymmetry. Increasing the aqueduct peak Reynolds-number, either by small stroke volume and high frequency or large stroke volume at low frequency, transfers the flow from a reversible flow pattern to the formation of clear preferential pathways, alternating between the in- and outflow cycle. The resulting permanent streaming flux across the aqueduct relative to the mean oscillatory flow-rate, i.e. the dimensionless streaming ratio SR follows a rational saturation relation, SR [~] ReAq,peak/(C + ReAq,peak), demonstrating that increasing the peak aqueduct Reynolds number yields diminishing returns and bounds the relative contribution of residual inter-ventricular streaming. These findings demonstrate differences in hydrodynamic organisation in the third ventricle and highlight the importance of this organisation for intraventricular fluid exchange.

biophysics↗