Aberration-corrected cryoimmersion light microscopy.

Abstract:

:Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at [Formula: see text]C.

authors

Faoro R,Bassu M,Mejia YX,Stephan T,Dudani N,Boeker C,Jakobs S,Burg TP

doi

10.1073/pnas.1717282115

subject

Has Abstract

pub_date

2018-02-06 00:00:00

pages

1204-1209

issue

6

eissn

0027-8424

issn

1091-6490

pii

1717282115

journal_volume

115

pub_type

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