Correlative light and electron microscopy (CLEM) benefits greatly from the development of super-resolution fluorescence microscopy. With a resolution down to the 10 nm range it enables to bridge the large resolution gap between the two different microscopy techniques. However, equally important as the achievable resolution of the imaging system is the preservation of the biological structures during the sample preparation process. Imaging undisturbed structures in living cells remains very challenging for super-resolution fluorescence microscopy with its relatively long acquisition times. Typically, chemical fixation is used to immobilize the sample for achieving best technical results, but unfortunately this is associated with structural changes in the sample, especially at a level below the diffraction limit of light [1,2].
Cryo-immobilization offers a preferable alternative. Here, fast freezing techniques enable vitrification of the sample and preserve the structures in a near-native state. Although cryo-immobilization has been established as a routine technique in the fields of electron and X-ray microscopy, it was long unclear whether super-resolution fluorescence microscopy could be performed under cryo-conditions to image vitrified samples .
Recently, we demonstrated on the single molecule level that photo-switching of fluorescent proteins is possible under cryo-conditions and suitable for the super-resolution method of single molecule localization microscopy (SMLM) . Chang et al. and Liu et al. have shown the correlation of cryo-SMLM with electron cryo-microscopy [5,6]. Here, we present cryo-SMLM of vitrified biological samples and its prospects for cryo-CLEM. We demonstrate that a resolution improvement of up to 5x compared to conventional fluorescence cryo-microscopy is possible.
Super-resolution fluorescence cryo-microscopy offers the possibility to image fluorescently labelled biological samples with diffraction-unlimited resolution, immobilized, but with structural preservation in a near-native state. This is not only helping to bridge the large resolution gap in cryo-CLEM, but might also offer an alternative to the dilemma in conventional super-resolution imaging, where one has to choose between the limited temporal resolution in case of living cells or the disadvantages of chemical fixation.
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To cite this abstract:Rainer Kaufmann, Christoph Hagen, Kay Grünewald; Super-resolution fluorescence microscopy of cryo-immobilized samples. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/super-resolution-fluorescence-microscopy-of-cryo-immobilized-samples/. Accessed: May 29, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/super-resolution-fluorescence-microscopy-of-cryo-immobilized-samples/