A microfluidic cryofixation method for time-resolved correlative microscopy

Informazioni relative al progetto

umCryofix

ID dell’accordo di sovvenzione: 749830

DOI

10.3030/749830

Progetto concluso il 31 Luglio 2020

Data della firma CE 15 Marzo 2017

Data di avvio 1 Giugno 2017

Data di completamento 5 Settembre 2020

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 171 460,80

Contributo UE

€ 171 460,80

Coordinato da

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - umCryofix (A microfluidic cryofixation method for time-resolved correlative microscopy)

Periodo di rendicontazione: 2017-06-01 al 2019-05-31

Sintesi del contesto e degli obiettivi generali del progetto

Detailed characterization of the function of various cellular components provides researchers with a fundamental understanding of normal cellular function. Further, linking changes in cellular function to alterations in the cells smallest structures provides a pathway to determine the underlying cause of disease when abnormalities arise. This is not only important for fundamental science but also to prevent, diagnose, and develop targeted treatments for virtually any affliction, ranging from Alzheimer’s disease, to HIV, to cancer. Yet, no single measurement technique is able to relate functional information from live imaging of whole cells (~microscale) with the smallest structures, or building blocks, of the relevant cellular components (~nanoscale).

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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

Through this action we developed a new technology that allows millisecond time correlation between live imaging and electron microscopy. A roundworm (Caenorhabditis elegans) was used as a test sample to establish compatibility of the new microfluidic cryofixation method with subsequent transmission 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.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

We developed an enabling technology for millisecond time correlated live imaging and electron microscopy. This represents an up to 1000x improvement in time resolution compared to previously reported methods. The new technique developed through this action could potentially be used to arrest, locate, and identify rare or highly dynamic biological processes in a known state and relate the process to the relevant ultra-fine cellular structures.