Project Details
Projekt
Analysis of highly dynamic subcellular mechanisms by Lattice Light Sheet Microscopy
Subject Area
Structural Biology
Term
from 2019 to 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 413831413
Most light microscope techniques currently employed in biomedical research are based on widefield or confocal epifluorescence illumination. In these modalities, the excitation and detection axis overlap, leading to unnecessary exposure of cellular regions that are not in focus. This becomes a problem when whole, living cells are imaged at high frame rates or for long time. Pronounced phototoxicity and bleaching are the consequence. Hence, imaging of individual cells for extended time periods to monitor stochastic events at sub-cellular resolution presents a major challenge. Such capabilities, however, are critically essential to understand the kinetics of cellular and pathogenic processes at the level of molecular mechanisms. Separation of the illumination and detection axis as done in light sheet microscopy offers a solution to photo-toxicity and bleaching problems. For this, two objectives are arranged perpendicular to each other, and the illumination objective projects a thin sheet of light into the sample only illuminating the region that is actually imaged. The sheet thickness is ideally matched to the detection objective’s z-resolution, to minimize photo-damage, thus increasing the spatio-temporal window for following biological processes. Several light sheet designs with cellular resolution are currently commercially available, typically suited to analyse e.g. whole drosophila embryos or plant root development, but also to visualize cells moving in three dimensions (Fritz-Laylin et al., 2017). Recently, first light sheet systems that are capable of studying sub-cellular events emerged (Aguet et al., 2016; Cai et al., 2017). Especially the lab of Nobel laureate Erik Betzig has developed a respective method using nondiffracting Bessel beams (Gao, Shao, Chen, & Betzig, 2014; T.L. Liu et al., 2018) to generate sheets that are thin and at the same time provide a larger field of view. The latest implementation of this technique is called lattice light sheet (LLS) microscopy since it uses dithered Bessel beams (Chen et al., 2014) for sheet homogeneity. It allows the generation of sheets (0.4-0.8 μm and thinner) that are ideally sized for sub-cellular resolution while keeping a large field of view to image whole, adherent cells. The lattice sheet is thinner over a longer distance. Since the sheet thickness is matched to the detection objectives focal plane, only cellular structures in focus are illuminated. This way, cells are irradiated much less and, zresolution is improved beyond capabilities of confocal microscopes (Aguet et al., 2016; Chen et al., 2014; Legant et al., 2016; Z. Liu et al., 2014).
DFG Programme
Major Instrumentation Initiatives
Major Instrumentation
Lattice light sheet Microscope
Instrumentation Group
5040 Spezielle Mikroskope (außer 500-503)
Applicant Institution
Universität Hamburg
Leader
Professor Dr. Kay Grünewald
