Introduction

In the rapidly evolving field of cryo-electron tomography (cryo-ET), the ability to efficiently target and image regions of interest (ROIs) within lamellae is paramount. Delmic's integrated fluorescence microscopes, METEOR and ENZEL, have emerged as game-changers, enhancing workflow efficiency and enabling researchers to achieve unprecedented sensitivity and precision in their studies. With over 25 customers worldwide choosing METEOR, and a growing number of publications showcasing its capabilities, Delmic is at the forefront of advancing cryo-ET technology.

The Power of METEOR in Cryo-ET Workflows

METEOR's unparalleled sensitivity and ease of use have made it a favored choice among cryo-ET labs. Its ability to maximize valuable TEM time by ensuring precise fluorescence targeting and correlation has led to a significant increase in the number of viable lamellae containing desired ROIs. Researchers like Sabrina Berkamp and co-authors (FZ Juelich, Juelich, Germany) have demonstrated METEOR's effectiveness, achieving a more than 2x increase in the number of viable lamellae containing hard-to-target ROIs, such as autophagic p62 droplets. This high sensitivity allows for the detection of even the smallest structures, guiding researchers to optimal sites for tomogram acquisition.

A key element in building a sensitive integrated fluorescence microscope is choosing the right objective lens.

Targeting Sub-Diffraction-Limited Particles with ENZEL

The challenge of targeting sub-diffraction-limited particles in lamellae is one that Phyllis Wang and co-authors (SLAC, San Francisco, US) successfully overcame using Delmic's ENZEL integrated microscope. Her work with the tri-coincident cryo-FIB-SEM-fluorescence instrument allowed her to   uncover and preserve structures like centrioles and nascent inflammasomes in lamellae. The high sensitivity of ENZEL's fluorescence microscopy provided sufficient resolution to correlate with specific clusters of vesicles, offering strong evidence for the presence of NLRP3 within them. This level of precision and reliability is crucial for studying complex biological phenomena at the nanoscale.

The camera efficiency is another key design criterion - the more sensitive the camera, the more photons are collected.

Optimizing Fluorescence Imaging in Cryo-ET

In-situ cryo fluorescence relies heavily on the sensitivity of the microscope used, as the fluorescence signal diminishes with thinning lamellae. Delmic's METEOR addresses this challenge with high-NA objectives, an optimized optical path, and a high-sensitivity camera. This setup allows researchers to study real biology, including endogenously expressed proteins that may be sparsely distributed. By optimizing the optical pathway and choosing components with high photon collection efficiencies, Delmic ensures that even rare events, such as early viral infection time points and low-copy-number proteins, can be captured with clarity.

The combination of the right optical path, the right objective lens and the right camera gives the best performance - saving samples from devtirification and imaging for long times under gentle conditions

Conclusion

Delmic's METEOR and ENZEL integrated fluorescence microscopes are transforming the landscape of cryo-ET, enabling researchers to achieve higher sensitivity, precision, and efficiency in their workflows. With a growing number of labs adopting these technologies and an increasing body of publications highlighting their success, Delmic continues to push the boundaries of what's possible in cryo-ET. As we look to the future, the innovations brought by METEOR and ENZEL promise to unlock even greater insights into the nanoscale world, driving forward our understanding of complex biological systems.

Image courtesy of Honkit Ng (Rockefeller University, NYC, USA). Samples courtesy of Francis Ashwanth (Florida State University, Tallahassee, USA)