Technology Development

A key part of our research program is the development of novel technologies for high resolution, 3D imaging of biological materials. We are actively engaged in technology development in both the Cellular Imaging and Cryo-EM sections of our research program.

 

New Technologies for Cellular Imaging

Graphic showing process of correlation between light and electron microscopic images


Light and electron microscopic data can be correlated in the X, Y, and Z planes to create 3D information. Image credit: Kedar Narayan, Center for Molecular Microscopy

Our primary technology for cellular imaging is focused ion beam scanning electron microscopy (FIB-SEM), a technique that allows serial imaging of fixed, resin-embedded biological materials. Our recent development work with this technology has included several key features that allow for more stable, targeted imaging of areas of interest, as well as techniques for correlating light microscopy with the 3D data acquired with FIB-SEM.

The correlation of FIB-SEM volumes with light microscopy of labeled protein, allowing one to localize these proteins to specific features of interest, continues to be a focus of the group. Current areas of interest for technology development for cellular imaging include new, enhanced algorithms for correlation between FIB-SEM and light or super resolution microscopy, as well as novel methods for FIB-SEM compatible tags and labels. Additionally, we are also exploring new methods for sample preparation that will minimize artifacts and expand applications for FIB-SEM in biological imaging. Finally, we continue to pursue solutions to the challenge of efficient computational extraction and segmentation of specific features from FIB-SEM image volumes.

 

 

New Technologies for Cryo-EM

Single particle cryo-electron microscopy (cryo-EM) can be divided in three different steps: (1) Specimen preparation, which consists of applying a small volume of sample to a grid with the goal of obtaining a mono-disperse thin layer of particles in vitreous ice. (2) Screening and data collection, which includes assessing the quality of the sample (screening) and obtaining a good set of micrographs (collection). (3) Data processing, which entails generating a high-resolution 3D reconstruction of the sample.

Our work at the CMM in the cryo-EM field is highly focused on finding new ways to improve all these three steps, from sample preparation to data processing. Almost every aspect of cryo-electron microscopy has been automated over the last few decades. However, one of the challenges that remains elusive is the robust and reliable preparation of vitrified specimens of suitable ice thickness. For that reason, at the CMM, we are developing several methods and new supports that will help with the automation of the sample preparation. We are planning on purchasing a Spotiton V1.0. The Spotiton is a piezo-electric dispensing robot that was developed a few years ago by Bridget Carragher and Clint Potter currently working as directors of the National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, located in New York. We are planning on working together with the goal to improve the sample preparation step.

We are also actively pursuing methods to better streamline and improve image collection in our Titan Krios cryo-electron microscope. We are in the process of generating a very efficient pipeline from data collection to data transfer and processing that will allow us to obtain high resolution 3D reconstructions.

Finally, we are interested in discovering ways to directly image proteins and structures within the cellular context.