Biology subjects

Puchner, E. M.

Publications and source records attributed to Puchner, E. M..

6 recordsLinked to original sources

bioRxiv2026

Quantitative Motion-Corrected PALM Links Endosome Structure and Dynamics in Live Cells

Quantitative structural analysis by Photoactivated Localization Microscopy (PALM) on the nanoscale is often restricted to fixed cells because motion during prolonged data acquisition distorts image reconstruction. Here, we develop motion-corrected PALM (mcPALM), a live-cell super-resolution approach combining a conventional fluorescence channel with PALM to correct motion-induced spreading of localizations. We further introduce a photoactivation-based correction to estimate molecule numbers from incomplete trajectories. Using PI3P-marked endosomes in yeast as a dynamic model system, we show that mcPALM recovers a live-cell maturation trajectory linking motion-corrected endosome size and calibrated PI3P content, consistent with fixed-cell benchmarks. Unlike fixed-cell PALM, mcPALM preserves endosome dynamics, revealing stage-dependent directed transport and maturation-associated motility shift. Thus, mcPALM extends PALM from static structural measurements in fixed samples to integrated quantification of nanoscale structure, molecular composition and dynamics in living cells. This framework is broadly applicable to other mobile organelles and biomolecular assemblies, enabling live-cell studies on how molecular organization and dynamics are coupled to biological function.

biophysics↗

bioRxiv2024

Red Light Mediated Photo-Conversion of Silicon Rhodamines to Oxygen Rhodamines for Single-Molecule Microscopy

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of microscopy experiments. Recently, far-red emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red fluorophores for two-color imaging. While this has expanded multi-color SMLM imaging capabilities, we demonstrate that silicon rhodamines can create previously unreported photoproducts with significantly blueshifted emissions, which appear as bright single-molecule crosstalk in the red emission channel. We show that this fluorescence is caused by the replacement of the central silicon group with oxygen after 640 nm illumination, turning far-red silicon rhodamines (JFX650, JF669, etc.) into their red oxygen rhodamine counterparts (JFX554, JF571, etc.). While this blueshifted population can cause artifacts in two-color SMLM data, we demonstrate up to 16-fold reduction in crosstalk using oxygen-scavenging systems. We also leverage this far-red photoconversion to demonstrate UV-free photoactivated localization microscopy (PALM) without the need for additives, and with 5-fold higher efficiency than the Cy5 to Cy3 conversion. Finally, we demonstrate multiplexed pseudo two-color PALM in a single emission channel by separating localizations by their photo-activation wavelengths instead of their emission wavelengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/595223v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@141fe66org.highwire.dtl.DTLVardef@1a4f9f5org.highwire.dtl.DTLVardef@16605dorg.highwire.dtl.DTLVardef@1109255_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

biophysics↗

bioRxiv2020

Quantitative super-resolution microscopy reveals distinct ULK1 oligomeric states and nanoscopic morphologies during autophagy initiation

Autophagy induction involves extensive molecular and membrane reorganization. Despite significant progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative Photoactivated Localization Microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1 and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

biophysics↗

bioRxiv2020

Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

Lipid droplets (LDs) are dynamic lipid storage organelles needed for lipid homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs, as well as enzymes required for LD growth and turnover. Due to LD size below the optical diffraction limit, bulk fluorescence microscopy cannot observe the density and dynamics of specific LD enzymes. Here, we employ quantitative photo-activated localization microscopy (PALM) to study the density of the fatty acid activating protein Faa4 on LDs during log, stationary and lag phases in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where the highest Faa4 densities are measured. During transition to the stationary phase LDs translocate to the vacuolar surface and lumen with a ~2-fold increased surface area and a ~2.5-fold increase in Faa4 density, suggesting its role in LD expansion. The increased Faa4 density on LDs is caused by its ~5-fold increased expression level. When lipolysis is induced in stationary-phase cells by diluting them for 2 hrs in fresh medium, Faa4 shuttles to the vacuole through the two observed routes of ER- and lipophagy. The observed vacuolar localization of Faa4 may help activating fatty acids for membrane expansion and reduces Faa4 expression to levels found in the log phase.

biophysics↗

bioRxiv2019

Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications

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.

biophysics↗