Bioimaging FAQ

Card access for MB26

From now on, we will be doing the MB26-specific induction every year and keep a digital record. To this end we have developed a web form that also includes a code of conduct and a few safety questions covering the oxygen depletion system.

When you fill in the form (correctly), you will get access to MB26 until October 2023, after which it will automatically lapse and you will have to fill in the form again. This ensures we have a proper record of everyone’s inductions and that all users get any important updates to the induction when they renew.

Fill in the form here.

Please not we can only approve access to people who are trained on at least one instrument and are either based in SLS or registered as SLS visitors for other Warwick departments (including WMS). To get a visitor form, contact Saskia or Ian. Once the form has been processed, you will need to:

* Have an induction in Life Sciences (book hereLink opens in a new window)

* Complete the Moodle SLS/WMS Waste Module (find here)

* Follow the Lab forum (found here)

Scale bars, measuring things, and numbers for Relion

In ImageJ/Fiji, use Analyze > Set Scale to put the scale in the software. The pixel size from the table below goes in the Known Distance box, Distance in Pixels should be set to 1. Then use Analyze > Tools > Scale Bar... to put a scale bar in the image. Other numbers you may require (same for both machines): Cs = 2, Acceleration voltage = 200 kV.

	JEOL 2200/FEG - K2	JEOL 2100/Lab6
80000x	0.0427 nm/pix	0.138 nm/pix
60000x	0.052 nm/pix	0.184
50000x	0.0716 nm/pix	0.22
40000x	0.0907 nm/pix	0.275
30000x	0.12	0.363
25000x	0.15	0.43
20000x	0.19	0.54
15000x	0.25	0.73
12000x	0.32	0.92
10000x	0.39	1.11
8000x	0.46	1.36
6000x	0.64	1.82
5000x	0.69	2.18
4000x	0.88	2.73
3000x	1.187	3.04

Making a figure of class averages

For a quick and dirty way, just use a screenshot of the display.

For a publication or thesis, you'll want the full-size images. To turn them into a nice montage, load the mrcs image stack into Fiji/ImageJ. Use the Image > Stack > Delete slice to remove any empty classes, then Image > Stack > Make Montage to make a montage. Play with the settings until you're happy.

The microscope sees many more greyscales than our eyes, and all the microscope values are in the middle. This means you should adjust the contrast. Open the .tif file in ImageJ (or FIJI) and press ctrl+shift+C. This will open a new little window with a histogram. Press the "auto" button and it should automatically adjust the contrast so you can see things. Now press "apply" and save the image.

Note you should only do this for display purposes. If you are doing class averaging and/or reconstruction, please leave the images as they are - the EM software will cope.

Using and borrowing consumables

If you are staining or freezing grids during your booked microscope session, we can provide grids. However, if you want to prepare grids in advance, you should buy your own. I recommend grids from EM Resolutions (on Opera). For negative staining, you want Formvar/Carbon grids such as FC300Cu here

Please do not remove any tweezers from MB026. You're welcome to use them in MB026 or you can buy some from Scientific Laboratory Supplies.

Our stains are hazardous so again, please do not remove them from MB026 but you're welcome to use them at any time once you have been trained. Do not use them without training and do not show others how to use them, ask Saskia or Ian for training.

Acknowledging the facility

If you publish a paper with data from the facility, please give us credit! Acknowledgements on papers, posters and presentations are important to us, as they help us show that we are important to people's research.

People

If the staff of the Advanced Bioimaging RTP have significantly contributed to your research, for instance in experimental design and data processing, please consider them for authorship on your publications.

Jeol 2200FS/DiSPIM/Cryo-ultramicrotome
"We acknowledge the University of Warwick Advanced Bioimaging Research Technology Platform supported by BBSRC ALERT14 award BB/M01228X/1"

Jeol 2100Plus TEM/Leica GP2 (plunge-freezer)
"We acknowledge the Midlands Regional Cryo-EM Facility, hosted at the Warwick Advanced Bioimaging Research Technology Platform, for use of the JEOL 2100Plus, supported by MRC award reference MC_PC_17136"

What kind of samples do you image?

We focus on biological or organic samples, either at room temperature (negative stain or resin sections) or frozen in vitreous ice (cryo). This includes purified proteins, virus particles, thin slices (sections) of cells, vesicles and latex beads, but if you have something else you want to use our equipment for please ask!

What microscopes do you have?

You can see our equipment here.

I want to find out more about the theory of electron microscopes, where can I find a course?

A good introduction to cryo-EM is Getting Started in Cryo-EM by Professor Grant Jensen from Caltech. It is available for free.

What does an experiment cost?

For academic users (internal and external) costs are currently (2022) as follows:

Instrument	Cost
JEOL2200FS	£157.68/hr
JEOL2100Plus/LaB6	£65.97/hr
Plunge-freezing	£14.89/hr
(Cryo)sectioning/ultramicrotome	£32.16/hr

This works out as under £500 for up to 5 samples for embedding and sectioning (including embedding, sectioning and imaging). A cryo-sample takes about an hour per sample.

DO NOT USE THESE as grant costings, please discuss any grant costings with R&IS

What courses and training are available?

We can train you to use our microscopes independently, the training process is outlined here. If you are based at the University of Warwick, you can then book the microscopes through our booking page. If you are based elsewhere, booking the microscope will be through Warwick Shared Services