US 12,553,812 B2 · Vacuum ultraviolet cryo-EM grid screening tool

Abstract

A tool and method for screening cryogenic electron microscopy (cryo-EM) sample grids using vacuum ultraviolet (VUV) illumination in two configurations. First configuration directly images cryo-EM grids using bright-field optical microscopy employing VUV wavelengths and specialized VUV optics. Second configuration converts transmitted VUV radiation from the cryo-EM grid to visible or near-UV light with a scintillator positioned by the grid. The resultant luminescent high-resolution shadow image is viewed using more conventional microscope optics. In both configurations, individual micron-scale grid holes are imaged to determine ice thickness and quality from the optical absorption of ultrathin vitrified water layers with a precision of a few nanometers. Longer wavelengths can be used to independently view protein concentration and distribution within the ice layer. This tool greatly increases yield of high-quality grids before cryo-EM analysis and is compatible with Single Particle Analysis (SPA) and other cryo-EM methods including cryo-electron Tomography and microcrystal electron diffraction.

Claims20

Dependent claims

A screening tool for a cryogenic electron microscopy grid that holds a sample in an ultrathin vitrified water layer, said screening tool comprising:

a) a source for producing an inspection light beam at a vacuum-ultra-violet (VUV) wavelength;
b) an evacuated optical dewar vessel having a VUV input window for admitting said inspection light beam along an optical path defined within said evacuated optical dewar vessel;
c) a cryogen source for maintaining a predetermined temperature within said evacuated optical dewar vessel;
d) a holding structure for holding said cryogenic electron microscopy grid in said optical path to expose said sample in said ultrathin vitrified water layer to said inspection light beam such as to produce a response light;
e) a light-detector for detecting said response light;
wherein said inspection light beam experiences attenuation when passing through said sample in said ultrathin vitrified water layer such that said response light contains information about a physical parameter of at least one of said ultrathin vitrified water layer and of said sample thereby permitting said screening tool to rapidly determine suitability of said cryogenic electron microscopy grid for further electron microscopy analysis.

3 more elements

Dependencies are inferred from the claim text.

Vacuum ultraviolet cryo-EM grid screening tool

Life of this patent

Vacuum ultraviolet cryo-EM grid screening tool

Life of this patent

Abstract

Claims20