Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast

Abstract

Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting.

Time Resolved Cryo‐Correlative Light and Electron Microscopy

“…There are three broad categories of preparing biological and soft materials for the electron microscope: native state (no fixation), chemical fixation, and numerous variants of cryo-fixation. Taking a brief look at these is essential to understanding the possibilities [30] Native state techniques (with yellow cell) Super-resolution light microscopy -entire image, not single molecule: STED (35 ms, 62 nm), [21,72] PALM (3 s, ≈10 nm), [31,73] STORM (3 s, 60 nm), [23] SIM (≈1 ms, 60 nm), [19] (Soft)-X-ray (≈min, ≈100 nm), [32,75] Liquid cell electron microscopy (LC-EM) (3 s, 35 nm), [26,76] Atomic force microscopy (AFM) (≈80 ms, ≈2 nm), [33,74] MINFLUX (0.1 ms (for single fluorophore tracking) -40 min (for image), 1-3 nm); [34] Dynamic cryo-EM techniques: a) conventional cryo-CLEM (>10s, ≈nm), [35] b), rapid sample transfer from light microscope to cryo-arrest device (≈4s, ≈nm), [36] c) triggered stimulation and rapid cryo-arrest (10 ms, ≈nm), [37] d) fixation of sample while observation, later cryo-substitution and cryo-EM (700 ms, ≈nm), [6,77] e) cooling of sample from stable warm equilibrium to unstable cold equilibrium (1 ms, 80 nm), [38] f) cooling of sample from unstable warm equilibrium to stable cold equilibrium (5 ms, ≈nm) [39] Details in table in appendix. B) Number of publications on correlative cryo-electron microscopy based on full text search [40] C) Schematic principle of methods for dynamic process observation for cryo-CLEM, current best experimental dynamic temporal resolutions achieved a) tracing back dynamics by correlating (cryo)-LM with (cryo)-EM: timescales of minutes.…”

Multiscale X-Ray Analysis of Biological Cells and Tissues by Scanning Diffraction and Coherent Imaging

“…In this respect, three basic concepts are generally followed: (i) static chambers that typically consist of two windows glued together and containing a small, microlitersized amount of liquid (see e.g. [164]), (ii) microfluidic chambers [165,166], and (iii) jet or droplet injection systems [70].…”

X-ray phase-contrast tomography of cells manipulated with an optical stretcher

“…By contrast, hard X-ray imaging methods for suspension cells are rare. Notable examples are the study of frozen-hydrated Chlamydomonas cells by ptychographic tomography (Diaz et al, 2015) and live-cell ptychography on fission yeast Schizosaccharomyces pombe (Strelnikova et al, 2017). These examples show that the imaging of individual suspension cells in aqueous environment and in real space without ensemble averaging remains challenging.…”