Visualizing proteins by expansion microscopy

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Aug. 5, 2022

Abstract Fluorescence imaging is one of the most versatile and widely-used tools in biology 1 . Although techniques to overcome diffraction barrier were introduced more than two decades ago, nominal attainable resolution kept improving 2, 3 , fluorescence microscopy still fails image morphology single proteins or small molecular complexes, either purified a cellular context 4, 5 Here we report solution this problem, form o ne-step n anoscale e xpansion (ONE) microscopy. We combined 10-fold axial expansion specimen (1000-fold by volume) with fluctuation analysis 6, 7 enable description cultured cells, tissues, viral particles, complexes proteins. At level, using immunostaining, our technology revealed detailed nanoscale arrangements synaptic proteins, including quasi-regular organisation PSD95 clusters. molecule upon main chain fluorescent labelling, could visualise shape individual membrane soluble Moreover, conformational changes undergone ∼17 kDa protein calmodulin Ca 2+ binding readily observable. also imaged classified aggregates cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents promising new development towards improved PD diagnosis. ONE compatible conventional microscopes can be performed software provide here as free, open-source package. This bridges gap between high-resolution structural light microscopy, provides avenue for discoveries medicine.

Language: Английский

---

Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology

The Journal of Cell Biology, Journal Year: 2023, Volume and Issue: 222(8)

Published: June 6, 2023

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal have limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions 34 20 nm, respectively, Cy3, optimal identified. Using theoretical frameworks analysis trajectories plasma membrane (PM), this successfully detected fast hop diffusion molecules PM, previously detectable only apical PM using less preferable 40-nm gold probes, thus helping elucidate principles governing organization molecular dynamics. Furthermore, as described companion paper, allows simultaneous data acquisitions PALM/dSTORM at 1 kHz, 29/19 nm 640 × pixel view-field.

Language: Английский

---

Transcription Factor Dynamics: One Molecule at a Time

Annual Review of Cell and Developmental Biology, Journal Year: 2023, Volume and Issue: 39(1), P. 277 - 305

Published: Aug. 4, 2023

Cells must tightly regulate their gene expression programs and yet rapidly respond to acute biochemical biophysical cues within environment. This information is transmitted the nucleus through various signaling cascades, culminating in activation or repression of target genes. Transcription factors (TFs) are key mediators these signals, binding specific regulatory elements chromatin. While live-cell imaging has conclusively proven that TF-chromatin interactions highly dynamic, how such transient can have long-term impacts on developmental trajectories disease progression still largely unclear. In this review, we summarize our current understanding dynamic nature TF functions, starting with a historical overview early experiments. We highlight govern dynamics dynamics, turn, affect downstream transcriptional bursting. Finally, conclude open challenges emerging technologies will further regulation.

Language: Английский

---

Accelerated MINFLUX Nanoscopy, through Spontaneously Fast‐Blinking Fluorophores

Small, Journal Year: 2023, Volume and Issue: 19(12)

Published: Jan. 15, 2023

The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little millisecond. However, current applications have so far focused on increasing this by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized a high yield and long on-times (tens milliseconds). Here, accelerated up 30-fold gain speed is presented. improvement attained designing spontaneously blinking markers remarkably fast on-times, down 1-3 ms, matching iterative process microscope. This design utilizes silicon rhodamine amide core, shifting spirocyclization equilibrium toward an uncharged closed form at physiological conditions imparting intact live cell permeability, modified fused (benzo)thiophene spirolactam fragment. best candidate microscopy (also suitable STORM imaging) selected through detailed characterization behavior fluorophores, bound different protein tags. Finally, optimization routines, customized times, renders significant commercial

Language: Английский

---

Direct optical measurement of intramolecular distances with angstrom precision

Science, Journal Year: 2024, Volume and Issue: 386(6718), P. 180 - 187

Published: Oct. 10, 2024

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular down to 1 nanometer—and in planar projections angstrom—directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- 10-nanometer polypeptides proteins. Moreover, visualized orientations immunoglobulin applied human cells, revealed specific configurations a histidine kinase PAS domain dimer. results open door examining proximities interactions direct position measurements at intramacromolecular scale.

Language: Английский

---