Cryo-FIB/SEM of Cultured Cells

William Rice, NYSBC

Zach Freyberg, Columbia University

Cheri Hampton, Emory University

Cultured Pancreatic Cells

• Grow cells on carbon-coated gold finder grid

• Vitrify cells:

– Blot most of water from grid

– Rapidly plunge into liquid ethane held near freezing temperature (-180 C) in lN2 bath

• Image frozen grid in TEM (300 kV)

Cryo-CLEM to identify organelles (Grant Jensen, Caltech)

RAG

• Can only image thinnest parts of cell

• Areas much thicker than 500nm are too thick for tomography

Limitations

Solutions

• Cryo sectioning

– Difficult:

• Need to be able to section well with cryo ultramicrotome

• Need to be able to make sections stick well to EM grid

• Crevasses

• Compression

Solutions

• Cryo FIB milling

– Equipment needed: FIB/SEM with cryo stage

– Shuttle to hold grids

– Ability to transfer from SEM to TEM

Equipment at NYSBC

FEI Helios Nanolab 650 With Quorum Cryostage JEOL 3200FSC

Cryo Tools

Shuttle (Quorum), autogrid (FEI) Tools from Jurgen Plitzko

From Rigort et al, Fig. S1

Challenges

• Autogrid needed to protect grids during handling

• Correct orientation may be hard to determine

• Frost must be avoided at all steps, particularly after lamellae are milled

Grids Easily Damaged

Grid must be right side up

Avoid broken regions

Temperature must be kept cold

(and cells like the grid bars!)

Focused Ion Beam Milling

Milling Sequence

Milling Sequence

Several Cells Milled

TEM Imaging

• Need a cryoholder which can hold autogrid cartridges

– Titan Krios ($$$)

– JEOL 3200: cartridge still under development

– FEI Polara at Columbia University (Joachim Frank): custom cartridge made

Insertion into TEM: Ideal Orientation

Tilt Axis ROI

F/T

Whole Grid Montage

Locate Thinned Areas

Lamella

Scale bar: 1 µm

Closer View

Scale bar: 500 nm

Tilt Series

Tilt series: 5.27 Å/pixel -38° to +55° Increment 3° Align using IMOD 3D reconstruction by weighted back projection + SIRT

Tomogram

Issues / Future

• Surface spots: Gallium deposition?

• Need for cryo rotation stage (Pt deposition)?

• Easier workflow for loading autogrid

• Cartridge for Jeol 3200 (in-house imaging)

• Cryo CLEM FIB/SEM

Acknowledgements

Frank Group Joachim Frank Cheri Hampton Robert Langlois

Robert Grassucci

Freyberg Group Zachary Farino

Travis Morgenstern Jenny Aguilar

William Mitchell Edward Yi Elana Levy

Jensen Group (Caltech) Grant Jensen

Stephen Carter

New York Structural Biology Center

Edward Eng Ashleigh Razcowski Bridget Carragher

Clint Potter

Elizabeth Villa (UCSD)