Precise light and fluorescent microscopy guided sequential cryo FIB lift-out

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F24%3A00617805" target="_blank" >RIV/68378050:_____/24:00617805 - isvavai.cz</a>

Výsledek na webu

—

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

Precise light and fluorescent microscopy guided sequential cryo FIB lift-out

Popis výsledku v původním jazyce

Background incl. aims Cryo-TEM tomography is a well-established imaging technique for revealing intracellular ultrastructure at the level of individual molecules. However, the requirement for electron transparency limited what could be imaged. Cryo-FIB lamella made deep intracellular structures accessible by removing the excess material. This approach works very well for objects and structures that are abundant in target cells. When the region of interest (ROI) is not found in every single cell, but rather in one or a few cells within a relatively large multicellular organism, the thinking changes significantly. It is no longer effective to rely on a stochastic approach or ,,luck,,. An effective way of target identification and localizations can be provided by cryo-fluorescence imaging and image correlation. Thin samples up to a few micrometers of thickness are vitrifiable by plunge freezing into liquid ethane, directly on TEM grids, making the workflow relatively straightforward. Thicker ones require HPF to achieve vitrification, which results in bulk sample inside a metal carrier and requires cryo lift-out to get the ROI to the TEM. That not only increases the amount of effort required to manufacture every lamella, but also complicates the navigation and ROI localization. We aim to develop and demonstrate a workflow enabling cryo-TEM visualization of a specific subcellular ROI from a multicellular organism such as C. elegans. Methods In a nutshell, the proposed method consists of high-pressure freezing (HPF, Leica EM ICE) in transparent cryoprotectant that results in a vitrified frozen-hydrated sample attached to a suitable support, typically a TEM grid or a HPF carrier, preserved in a life-like condition. The sample is then imaged in cryo light and fluorescence microscope (Leica EM Cryo CLEM) to screen for ROIs and identifiable features and transferred to the cryo FIB-SEM machine (Tescan Amber Cryo). The ROIs are then marked and material around the intended lamella is removed with FIB. Lamella is lifted out using a nanomanipulator (Tescan cryo-nanomanipulator) and welded to a receptor grid with pre-milled slot of appropriate width and thinned down to <200nm through 2 or 3 steps of consequent fluorescent microscopy check and FIB milling. Results We were able to acquire a TEM tilt series on a selected part of cell nucleus containing a fluorescently labeled feature of interest. Since there were single digit nuclei expressing desired phenotype close to each other, multiple consecutive lamellae were lifted out from each processed worm and polished with a reasonable success rate. Conclusion The method demonstrated here enables cryo TEM processing of a fluorescently labeled feature from within large multicellular biological samples. It consists of targeted cryo FIB lift-out using Tescan cryo-nanomanipulator mounted in Tescan Amber Cryo FIB-SEM with multiple fluorescence checks performed using Leica EM Cryo CLEM during the preparation. This will provide researches with multiple high-resolution windows within context of larger volume mapped using cryo fluorescence microscopy. As a result, we can offset the miniscule volume of space visualizable by the cryo TEM tomography which, until now, was mostly limited to abundant objects of interest. While the workflow by itself is relatively labor intensive, for rare objects and phenomena, it is much more practical than any untargeted approach.n

Název v anglickém jazyce

Precise light and fluorescent microscopy guided sequential cryo FIB lift-out

Popis výsledku anglicky

Background incl. aims Cryo-TEM tomography is a well-established imaging technique for revealing intracellular ultrastructure at the level of individual molecules. However, the requirement for electron transparency limited what could be imaged. Cryo-FIB lamella made deep intracellular structures accessible by removing the excess material. This approach works very well for objects and structures that are abundant in target cells. When the region of interest (ROI) is not found in every single cell, but rather in one or a few cells within a relatively large multicellular organism, the thinking changes significantly. It is no longer effective to rely on a stochastic approach or ,,luck,,. An effective way of target identification and localizations can be provided by cryo-fluorescence imaging and image correlation. Thin samples up to a few micrometers of thickness are vitrifiable by plunge freezing into liquid ethane, directly on TEM grids, making the workflow relatively straightforward. Thicker ones require HPF to achieve vitrification, which results in bulk sample inside a metal carrier and requires cryo lift-out to get the ROI to the TEM. That not only increases the amount of effort required to manufacture every lamella, but also complicates the navigation and ROI localization. We aim to develop and demonstrate a workflow enabling cryo-TEM visualization of a specific subcellular ROI from a multicellular organism such as C. elegans. Methods In a nutshell, the proposed method consists of high-pressure freezing (HPF, Leica EM ICE) in transparent cryoprotectant that results in a vitrified frozen-hydrated sample attached to a suitable support, typically a TEM grid or a HPF carrier, preserved in a life-like condition. The sample is then imaged in cryo light and fluorescence microscope (Leica EM Cryo CLEM) to screen for ROIs and identifiable features and transferred to the cryo FIB-SEM machine (Tescan Amber Cryo). The ROIs are then marked and material around the intended lamella is removed with FIB. Lamella is lifted out using a nanomanipulator (Tescan cryo-nanomanipulator) and welded to a receptor grid with pre-milled slot of appropriate width and thinned down to <200nm through 2 or 3 steps of consequent fluorescent microscopy check and FIB milling. Results We were able to acquire a TEM tilt series on a selected part of cell nucleus containing a fluorescently labeled feature of interest. Since there were single digit nuclei expressing desired phenotype close to each other, multiple consecutive lamellae were lifted out from each processed worm and polished with a reasonable success rate. Conclusion The method demonstrated here enables cryo TEM processing of a fluorescently labeled feature from within large multicellular biological samples. It consists of targeted cryo FIB lift-out using Tescan cryo-nanomanipulator mounted in Tescan Amber Cryo FIB-SEM with multiple fluorescence checks performed using Leica EM Cryo CLEM during the preparation. This will provide researches with multiple high-resolution windows within context of larger volume mapped using cryo fluorescence microscopy. As a result, we can offset the miniscule volume of space visualizable by the cryo TEM tomography which, until now, was mostly limited to abundant objects of interest. While the workflow by itself is relatively labor intensive, for rare objects and phenomena, it is much more practical than any untargeted approach.n

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

10609 - Biochemical research methods

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů