Periodic Reporting for period 1 - umCryofix (A microfluidic cryofixation method for time-resolved correlative microscopy)
Reporting period: 2017-06-01 to 2019-05-31
Correlative light and electron microscopy (CLEM) addresses this problem by combining the advantages of two imaging techniques. First, the well-known light microscope is used to map cellular functions. The biological sample must then be fixated to stop the biological process. With the process stopped, a higher-resolution electron microscope is used to “zoom-in” on the ultra-fine structures that form the building blocks of the cell.
Current CLEM methods, however, suffer from limited time resolution, meaning the light microscope and electron microscope images are not taken at the same timepoint of the biological process. The goal of this project is to develop a new cryofixation, or ultra-fast freezing, method that will enable time-resolved CLEM studies of dynamic cellular processes.
Cryofixation, or cooling the sample at a rate of 10,000°C/s or higher, is widely accepted as the gold-standard fixation method for high-resolution imaging of biological samples. The key to attaining high-quality sample preservation with cryofixation is to avoid ice crystallization, so as to leave the sample in a near-native, glass-like frozen state.
However, none of the current methods lend themselves to the cryofixation of dynamic cellular processes on a time scale of seconds and faster. After a region and time of interest is located in the light microscope, the sample must be transferred to a dedicated cryofixation instrument, or ultra-fast freezer. With state-of-the-art robotic systems the transfer step requires at least one second, though transfer times up to a few minutes are still common. During this transfer time the biological process continues to progress.
Through this action, we developed a new microfluidic cryofixation method for time-resolved correlative live imaging and electron microscopy. Our method eliminates the need to transfer the sample from the light microscope to a dedicated machine for rapid freezing. The key idea is to combine the light microscope and a new freezing technology into just one instrument.
First, a biological sample is loaded into a microfluidic device, which is mounted onto a microscale heater. The heater itself is mounted onto a -196°C copper block. The self-contained microfluidc chip, heater, copper block assembly is mounted directly in the viewing path of the light microscope.
During live imaging, the heater maintains the sample in the microchannel at 25°C. At the desired moment in the biological process, the heater is switched off, and the sample cools rapidly through the contact with the -196°C copper block. The newly developed system allows, for the first time, continuous live-imaged videos of the sample before, during, and after the freezing event. Since the biological process is stopped at a known time, the time resolution is decreased from seconds to milliseconds, which is the time it takes for the sample to freeze. Therefore, once the last image of the dynamic biological process is taken with the light microscope, the process progresses just a few milliseconds before it is fully frozen for high-resolution electron microscopy.
The objectives which we achieved through this action are:
1. We developed a self-contained microfluidic cryofixation system with a cooling rate greater than 10,000ºC/s.
2. We developed procedures to move the already frozen sample from the light microscope system to the electron microscope system. This is a challenge because the sample had to remain below -140°C during this process to avoid the formation of ice crystals that would destroy the sample.
3. We imaged the ultrastructure of the sample and characterized the quality of the cryofixation using electron microscopy.
To achieve the action objectives, a specialized microfluidic device design and device fabrication procedures were developed to achieve an average cooling rate of 24,000ºC/s. The overall cryofixation system was designed as a self-contained, portable unit that could be transferred from the light microscope experimental setup to a deep liquid nitrogen bath. Removing a single access screw allowed the microfluidic chip housing the sample to be recovered at -196°C under liquid nitrogen. Cryo-electron tomography of microfluidic cryofixed C. elegans showed that the ultrastructure of the sample was well preserved with this cryofixation method. Following live imaging and microfluidic-based cryofixation, the sample ultrastructure was imaged using either state-of-the-art electron tomography or more well-established freeze substitution, resin embedding, and electron microscopy procedures.
The detailed results of this work are openly available in the following publications:
1. Fuest, M., Schaffer, M., Nocera, G.M. et al. In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography. Sci Rep 9, 19133 (2019). https://doi.org/10.1038/s41598-019-55413-2
2. Fuest M, Nocera GM, Modena MM, Riedel D, Mejia YX, Burg TP. Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy. J Microsc. 2018;272(2):87-95. doi:10.1111/jmi.12747
The results have been presented to the national and international scientific community at three conferences:
1. M. Fuest, M. Schaffer, G.M. Nocera, R. Galilea-Kleinsteuber, J.E. Messling, J. M. Plitzko, T. P. Burg. “Cryo-Microfluidic Devices Enable Millisecond Time Correlation Between Live-Imaging and Cryo-Electron Microscopy in C. elegans”. MicroTas 2019, Basel, Switzerland, October 30, 2019.
2. M. Fuest, G.M. Nocera, M. Modena, D. Riedel, T.P. Burg. Invited. “Advances in microfluidic cryofixation for correlative light and electron microscopy”. Microscopy Conference 2017, Lausanne, Switzerland, Aug. 22, 2017. As an invited speaker
3. M. Fuest, G.M. Nocera, M. Modena, T.P. Burg. “Fabrication Advances for Microfluidic Cryofixation”. Mikrosystem Technik Kongress 2017, Munich, Germany, October 25, 2017.