Metallic support films reduce optical heating in cryogenic correlative light and electron tomography

Abstract

Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20× the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.

Keywords: CLEM; Cryogenic electron tomography; Fluorescence microscopy; Single-molecule; Super-resolution.

Figure 1:

Low magnification optical image and cartoon top and side views of the three electron microscopy grid types used in this study. (a) R 2/2 holey carbon support film on a 200 mesh copper G200F1 finder grid geometry (Quantifoil N1-C16nCuG1-01). (b) Custom ordered grid with gold ultrAuFoil support film on a 200 mesh gold H2 finder grid geometry (Quantifoil N1-A16nAuH2-01). (c) The same support film and grid as in a with a 50 nm layer of silver deposited on the top surface.

Figure 2:

Thresholds for optical excitation that lead to sample devitrification. a) Top row shows samples excited with intensities just below the threshold. Bottom row shows samples excited with intensities just above the threshold. Listed intensities are the peak intensity of the 561 nm Gaussian beam profile. b) Analysis of vitrification state of all grid squares analyzed post-illumination to determine devitrification threshold ranges found in this study. Devitrification was assessed at the center of each grid square adjacent to the peak optical intensities. Each mark indicates a grid square illuminated for five minutes with the peak intensity listed on the x-axis. Dashed lines indicate the intensity regime in which grids of each support film type are near the devitrification threshold.

Figure 3:

Simulation of optically driven sample heating. a) Simulation geometry intended to represent a single grid square. The boundaries of the grid square were fixed to be 77 K and sample heating was due to the absorption of a 561 nm laser with a Gaussian beam profile. The amount of energy absorbed and how it was dissipated depended on the various support film material properties. b) Representative isotherms from solving equation 1 for the standard carbon grid illuminated by a Gaussian beam with a peak intensity of 50 W/cm². The red star marks the peak temperature, in this case ~120 K c) Plot of the peak temperature as a function of excitation intensity for the three different support films. The red star is the same as that shown in b.

Figure 4:

Grid autofluorescence due to 561 nm optical excitation at room temperature for a) carbon grid, b) gold grid, and c) silver grid displayed with the same intensity scaling. d) Mean background measured for each of the three different grid types. Error bars show the standard deviation of background measured from six different grid squares for each grid type.

Figure 5:

Improved single-molecule imaging and srCryoCLEM using silver grids demonstrated by imaging of PAmKate-PopZ fusion in C. crescentus. a) Background subtracted fluorescence brightness trace taken from an integrated cell pole imaged on a carbon grid with 50 W/cm². Green highlights the contributions from a single emitter and gray denotes the estimated background. b) The same as panel a except imaged on silver support film with 1000 W/cm². Note the difference in time the emitters spend in an emissive state. c) Histogram of lateral localization precision for emitters imaged on silver grids. Precision is determined experimentally from the standard error of the mean from localizations of the same emitter across multiple frames. Localization data comes from 10 cells and 303 emitters. d). Central slice of a representative tomographic reconstruction of a C. crescentus cell. e) Overlay of single-molecule fluorescent localizations (green circles) and central slice of cryoET reconstruction. Each circle is centered on the estimated location of an individual emitter with the radius of the circle being that emitter’s localization precision. There is an additional registration error, i.e. an error in the alignment of fluorescence localizations and CryoET data, of ~30 nm that is not shown, see Materials and Methods section. This registration error would be the same for all localizations and would serve to shift the cloud of localizations slightly. The zoom shows the polar region with the ribosome excluded region manually annotated in red.