Connecting live-cell imaging, cryofluorescence, and electron microscopy through microtechnology
Thomas P. Burg
Technische Universität Darmstadt, Electrical Engineering and Information Technology, Integrated Micro- and Nanosystems (IMNS), Marckstr. 25, 64283 Darmstadt, Germany, Email: firstname.lastname@example.org
In this talk, I will present recent advances of our group towards connecting live-cell imaging and electron microscopy (EM) through microtechnology-based methods for cryofixation and cryofluorescence imaging. Before entering the high vacuum of an electron microscope, biological samples generally need to be fixed chemically or frozen. Due to the time required for this preparation, it is difficult to correlate high-resolution EM images of cell structure with millisecond dynamics previously observed in the light microscope. We have been able to overcome this limitation by cryofixing cells and small model organisms directly in the light microscope through ultra-rapid in situ cooling. Ice crystallization is strongly suppressed by freezing at more than ~10^4 °C/s. Next, in order to image rapidly frozen samples by high numerical aperture cryofluorescence microscopy, we designed a new type of light microscope that allows immersion imaging below the glass transition of water (-135 °C). These technologies are compatible with conventional correlative light and electron microscopy workflows. We therefore expect that our platform will be of interest for studying a wide range of questions involving temporal relationships between cell stimulation, dynamic cell function, and structural alterations at the nanometer scale.