Biology subjects

Boltje, D.

Publications and source records attributed to Boltje, D..

3 recordsLinked to original sources

bioRxiv2024

Thickness and quality controlled fabrication of fluorescence-targeted frozen-hydrated lamellae

Cryogenic focused ion beam (FIB) milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 and 200 nm. While the lamella fabrication workflow has undergone significant improvements since its conception, quantitative, live feedback on lamella thickness and quality is still lacking. Taking advantage of a coincident light microscopy integrated into the FIB-SEM, we present three different strategies that together allow accurate, live control during lamella fabrication. First, we combine 4D-STEM with fluorescence microscope (FM) targeting to determine the lamella thickness. Second, with reflected light microscopy (RLM) we screen target sites for ice contamination and monitor lamella thickness and integrity of the protective Pt coating during FIB milling. Third, we exploit thin-film interference to obtain fine-grained feedback on thickness uniformity below 500 nm. We finally present a full workflow for fluorescence-targeted and quality controlled fabrication of frozen-hydrated lamellae, benchmarked with excellent agreement to energy filtered transmision electron microscopy (EFTEM) measurements and reconstructed tomograms obtained with electron cryo-tomography.

biophysics↗

bioRxiv2024

Depth-dependent scaling of axial distances in light microscopy

In volume fluorescence microscopy, refractive index matching is essential to minimize aberrations. There are however, common imaging scenarios, where a refractive index mismatch (RIM) between immersion and sample medium cannot be avoided. This RIM leads to an axial deformation in the acquired image data. Over the years, different axial scaling factors have been proposed to correct for this deformation. While some reports have suggested a depth-dependent axial deformation, so far none of the scaling theories has accounted for a depth-dependent, non-linear scaling. Here, we derive an analytical theory based on determining the leading constructive interference band in the objective lens pupil under RIM. We then use this to calculate a depth-dependent re-scaling factor as a function of the numerical aperture (NA), the refractive indices n1 and n2, and the wavelength{lambda} . We compare our theoretical results with wave-optics calculations and experimental results obtained using a novel measurement scheme for different values of NA and RIM. As a benchmark, we recorded multiple datasets in different RIM conditions, and corrected these using our depth-dependent axial scaling theory. Finally, we present an online web applet that visualizes the depth-dependent axial re-scaling for specific optical setups. In addition, we provide software which will help microscopists to correctly re-scale the axial dimension in their imaging data when working under RIM.

biophysics↗

bioRxiv2022

A cryogenic, coincident fluorescence, electron and ion beam microscope

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

molecular biology↗