Platform for cryo-fluorescence with high stability and improved ease of use

Session

Poster Session

Authors

Martijn van Nugteren (1), Tiemen Smit (1), Arnold Kamp (1), Andrew Davies (1), Peter Grocutt (1), Clara Ko (1), Michael Schwertner (1)

Affiliations

1. Linkam Scientific Instruments Ltd.

Keywords

Cryo-fluorescence, cryo-CLEM, cryo-SXT, correlative microscopy, cryo-sample screening, cryo-Super-Resolution, cryo-sample screening for single particle tomography (SPT)

Abstract text

Cryo-light microscopy (cryo-LM) and in particular cryo-fluorescence are techniques that can be combined with typically electron microscopy (EM), electron tomography (ET) or X-ray tomography to form a correlative workflow, combining data from complementing imaging techniques.

Small biological samples such as cells or protein particles can be frozen rapidly into a vitrified, glass-like state. This allows to obtain nearly perfect preservation down to the ultra-structural level. Vitrified samples are also naturally compatible with the vacuum conditions typically required for high-resolution EM.

Fluorescence imaging and the very versatile fluorescence labelling techniques can help to identify regions of interest and provide biological context. Because of the resolution gap towards EM and X-ray, cryo-Super-Resolution [1,2] is gaining popularity. This is also aided by the typically very low photo-bleaching under cryo-conditions compared to room temperature. Other applications of cryo-LM are cryo-spectroscopy, the preparation of cryo-lamellas for tomography guided by fluorescence [3], the pre-screening of samples for cryo-ET and the measurement of ice-thickness for single particle tomography (SPT) workflows [4]. A key parameter for the ever more demanding high-resolution cryo-fluorescence applications is the drift stability under cryo-conditions.

In response we developed the CMS196V4, our updated cryo-platform for fluorescence imaging which uses a novel continuous liquid nitrogen filling principle for the sample environment and the internal dewar of the system. An additional gain in stability is achieved by an integrated lens-heater system. We will discuss typical data from fluorescence beads measurements obtained with the system.

The user interface with touch-screen joystick is intuitive and we introduced a modular sample interface, allowing to configure the stage for multiple applications or adopting future sample-mounting standards. The CRYOARM cartridge system from JEOL has been added to the family of supported sample mounts as shown below, streamlined quick-release mechanisms for the cryo-stage have also been added and support more convenient sample loading.

Applications, workflow options as well as principles behind operation and design will be covered in our presentation.

References

[1] Danielle L. Sexton, Steffen Burgold, Andreas Schertel, Elitza I. Tocheva, Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans, Current Research in Structural Biology, Volume 4, 2022, p.1-9, https://doi.org/10.1016/j.crstbi.2021.12.001

[2] Okolo CA, Jadhav A, Phillips P, et al. Correlative imaging using super-resolution fluorescence microscopy and soft X-ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development. Journal of Microscopy. 2021; 284: 214–232. https://doi.org/10.1111/jmi.13054

[3]  Alexander Rigort, Jürgen M. Plitzko, Cryo-focused-ion-beam applications in structural biology Archives of Biochemistry and Biophysics, Volume 581, 2015, Pages 122-130

[4] Mart G.F. Last, Lenard M. Voortman, Thomas H. Sharp, Measuring cryo-TEM sample thickness using reflected light microscopy and machine learning, Journal of Structural Biology, Volume 215, Issue 2, 2023, 107965