Microfluidic cryofixation for time-correlated live-imaging cryo-fluorescence microscopy and electron microscopy of Caenorhabditis elegans

by Giovanni Marco Nocera
Doctoral thesis
Date of Examination:2018-10-15
Date of issue:2018-11-01
Advisor:Dr. Thomas Burg
Referee:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:Dr. Henrik Bringmann
Referee:Prof. Dr. Jörg Enderlein
Persistent Address: http://dx.doi.org/10.53846/goediss-7105

 

 

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Description:Title: Microfluidic cryofixation for time-correlated live-imaging cryo-fluorescence microscopy and electron microscopy of Caenorhabditis elegans __ Keywords: microfluidics, cryofixation, correlative microscopy, CLEM, C. elegans

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Abstract

English

Light and electron microscopy are complementary methods to study biological systems at the cellular and sub-cellular scale. Light microscopy is compatible with live cells, allowing features of interest to be selectively marked with fluorescent molecules and followed in real time. Electron microscopy, in contrast, requires cells to be fixed. Cryofixation is a preferred method of fixation, since rapid freezing can preserve hydrated samples in a near-native state. However, state-of-the- art cryofixation systems require a transfer step that imposes a time lapse between the images acquired in light microscopy and the images acquired in electron microscopy. This transfer step prevents the correlation of light and electron cryo-microscopy of sub-second phenomena. Microfluidics can eliminate the need of the transfer step. The cryofixation event is imaged without interruption from the top of a microfluidic channel that is heated while placed on top of a cryostage. When the heater is turned off, the small thermal mass of the microfluidic channel allows rapid cooling of the channel content. In this thesis, microfluidic technology for cryofixation was shown for the first time to enable millisecond time-correlation between live imaging, cryofluorescence microscopy with immersion optics, and electron microscopy. An important benefit of fluorescence microscopy at cryogenic temperature resides in the arrest of photobleaching at very low temperature. However, the stability of fluorescent molecules at cryogenic temperature is still a field vastly unexplored. The microfluidic cryofixation system was used here to investigate the stability of the fluorescent calcium indicator GCaMP in live imaged roundworms (Caenorhabditis elegans). In another part of the thesis, the transfer of cryofixed samples was achieved for the first time with the intent to demonstrate the compatibility of the cryofixation system with correlative microscopy workflows. To acquire higher resolution images, the samples were transferred to a new immersion cryo- confocal microscopy setup developed by Faoro et al. [Faoro et al., 2018]. Compared to the imaging on the cryofixation system, a 20-fold contrast gain was achieved. In the second workflow, the preservation quality of cryofixed samples was investigated via electron microscopy. Electron microscopy revealed an overall good preservation of the samples with minor ice damage within the nuclei. The results of this work constitute a foundation to enable new experimental paradigms for studying relationships between structure and function during rapid cellular processes such as cell signaling and membrane trafficking.
Keywords: cryofixation; correlative microscopy; CLEM; C. elegans; microfluidics
 
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