Providing a high resolution window into a larger multicellular organism through precise light and fluorescent microscopy guided cryo-FIB lamella preparation

Kód výsledku v IS VaVaI

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Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Providing a high resolution window into a larger multicellular organism through precise light and fluorescent microscopy guided cryo-FIB lamella preparation

Popis výsledku v původním jazyce

The cryo-FIB lamella coupled with Cryo-TEM tomography is a well-established method able to reveal deep intracellular structures at almost molecular level of detail. Many potential objects of interest are abundant enough to be practically found in any randomly selected cell. Targeting rare objects present in only a fraction of cells or a few cells within a larger multicellular organism such as SUN-1 foci in meiotic nuclei of C. elegans examined in this project is, however, quite challenging and a stochastic approach is no longer efficient or even possible in such cases. nInstead, we propose and demonstrate a workflow based on multiple rounds of post-vitrification fluorescence imaging using cryo fluorescence (currently a standalone Leica EM Cryo CLEM) before and between FIB milling steps to screen for, localize and precisely target subcellular objects of interest. To freeze and vitrify multicellular samples, we use high-pressure freezing (HPF, Leica EM ICE) and localize the region of interest based on specific fluorescence patterns under the surface of vitrified solution inside a HPF carrier. We then use the light and fluorescence images to navigate to a correct site, mill and lift-out a cryo FIB lamella [2] using a high-precision TESCAN Cryo Nanomanipulator. The process continues with further imaging in Cryo CLEM, determining the position and depth of the object of interest within the lifted-out volume, and thinning the lamella as needed. This is made easier by an adapter that enables preservation of position and orientation during transfers between Cryo CLEM and FIB-SEM but would benefit greatly from a fluorescence microscope integrated directly inside the FIB-SEM chamber.nThe resulting lamella, approximately 200nm thick, is then used for cryo-electron tomography and volume reconstruction, providing us with a high-resolution window into the context of a larger multicellular structure visualized by light microscopy.n

Název v anglickém jazyce

Providing a high resolution window into a larger multicellular organism through precise light and fluorescent microscopy guided cryo-FIB lamella preparation

Popis výsledku anglicky

The cryo-FIB lamella coupled with Cryo-TEM tomography is a well-established method able to reveal deep intracellular structures at almost molecular level of detail. Many potential objects of interest are abundant enough to be practically found in any randomly selected cell. Targeting rare objects present in only a fraction of cells or a few cells within a larger multicellular organism such as SUN-1 foci in meiotic nuclei of C. elegans examined in this project is, however, quite challenging and a stochastic approach is no longer efficient or even possible in such cases. nInstead, we propose and demonstrate a workflow based on multiple rounds of post-vitrification fluorescence imaging using cryo fluorescence (currently a standalone Leica EM Cryo CLEM) before and between FIB milling steps to screen for, localize and precisely target subcellular objects of interest. To freeze and vitrify multicellular samples, we use high-pressure freezing (HPF, Leica EM ICE) and localize the region of interest based on specific fluorescence patterns under the surface of vitrified solution inside a HPF carrier. We then use the light and fluorescence images to navigate to a correct site, mill and lift-out a cryo FIB lamella [2] using a high-precision TESCAN Cryo Nanomanipulator. The process continues with further imaging in Cryo CLEM, determining the position and depth of the object of interest within the lifted-out volume, and thinning the lamella as needed. This is made easier by an adapter that enables preservation of position and orientation during transfers between Cryo CLEM and FIB-SEM but would benefit greatly from a fluorescence microscope integrated directly inside the FIB-SEM chamber.nThe resulting lamella, approximately 200nm thick, is then used for cryo-electron tomography and volume reconstruction, providing us with a high-resolution window into the context of a larger multicellular structure visualized by light microscopy.n

Druh

O - Ostatní výsledky

CEP obor

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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