bioimaging and optics platform BIOP

FREQUENTLY ASKED QUESTIONS AT THE BIOP


GeneraL

I have a project and/or a nice sample but I never did any microscopy in my life, how can I access the BIOp microscopes?

For a first contact, you will need an appointment, through the request form, with the head of the BIOp (Dr. Arne Seitz) and speak about the best way to take your images and get trained depending on your experimental question.

For advanced users, the use and training on another machine, for a different scientific question, can be done directly with the concerned BIOP staff depending on the microscope. But a a request form is still needed.

How can I thank/quote the BIOP in a publication?

Talk to us, as this is dependent on the type of work and collaboration. At the very least something in the lines of:

We would like to thank [person(s)] at the EPFL BioImaging & Optics Core Facility (EPFL-BIOP) for their assistance in [imaging / image processing].


Working at the BIOp

I have questions on how to get the best images for my experiment.

You can ask any question to the BIOp team concerning microscopy and image processing between 8am and 18pm.

I want to do image processing on a high number of images.

The best way is to take few images, and ask the image processing team if there are any changes to do on the acquisitions before taking all the images.


MICROSCOPY

Where can I start learning basics about resolution, numerical aperture, objectives, saturation, aberrations?

"Seeing is believing?" is an excellent paper for an understanding of the main pitfalls on microscopy.

The other excellent Davidson et al. paper about microscopy and different types of contrast.

Try the FAQ of the "MICRO-MAGNET" web site, on of the references in microscopy

I need advice about stainings for my experiment!
Each BIOp staff has a good knowledge about stainings and dyes. For special cases in histology and colorations you could directly contact the Histology Core Facility through Jessica Dessimoz.
What is a good recipe for DABCO?

The DABCO is widely used as a mounting medium for its anti-fading capacities, price and ease of use.
This mounting medium stays liquid, so you need to seal the coverslip with nail polish.

Recipe for 50ml DABCO

AmountProduct
5.0ml1x PBS ph 7.5 from 10x stock = 5ml
28.7ml50% glycerol from 80% stock
1.2grdabco diazabicyclo-222-octane (final = 24 mg/ml)

Adjust the pH at 7.5 (very hard and important) with HCl, then adjust to 50ml final volume with H2O.

After preparing the mounting medium, don't forget to freeze at -80°C in 50 ul aliquots.

When using a tube, keep it at 4°C and never use it for longer than one week to avoid contaminations.

We provide a PDF version of the protocol

I need to clear my sample, are there any protocols you recommend?

Some clearing reagents for thick tissues as embryos and brains are used inhouse such as: The BABB
Used in full mouse brain imaging to about 1.8mm deep with our 2-photon microscope

Another clearing reagent, TDE seems to have the same specifications with less toxicity. (Technical sheet)

For special cases in histology and colorations you could directly contact the Histology Core Facility through Jessica Dessimoz.

My tissue slice is thick and gets squeezed when I mount it!

You can find now some frames to avoid the squeezing of thick tissues between the slide and coverslip.

This will preserve the geometry of the tissue during acquisition. Datasheet Available

Something isn't working (screen, computer, lasers, mercury lamp, XY-stage, microscope controller, etc...).

Verify that you've switched on each of the needed devices, and in the right order.

I don't have an image on the ocular and/or on the screen...

Is there any light coming from the objective on your sample? If not, verify whether light or laser is on and if the shutter is open. If so, is the light path free to go to ocular or camera? Just check it.

After finishing my session, what do I do?

If somebody is working after you on the scope, just log off and set Argon Lasers to minimum.

If it's free till next day, switch everything off.

Where can I store my data?

The best and safest is in your group's server.

In case you have data you would like to share with us, you can use the BIOP Public Folder at \\svfas6.epfl.ch\biop\public

Data on the microscope's PCs are safe for only 3 days.


Image Processing

I cannot find the pixel size of my image!

If you use Fiji, try importing your data using the Loci Bio-Formats plugin explicitely (Plugins--LOCI--Bio-Formats Importer)

If the metadata is wrong, you can calculate it easily using the following formula:


Pixel Size=  (CCDpx Size x Binning) / (Mag Obj * Mag c-mount)

If you have lost the calibration data on a confocal or need to be sure, you'll have to use a slide with a micro-ruler.

What advantages does deconvolution bring?

You should give the Landmann paper and the Boltes paper a read. In short, you can gain in resolution (particularly in z), SNR and signal-to-background ratio.

Which computer fits my needs?

Your needs will depend on your data and what you want to do with it: Large datasets visualization and analysis need lots of RAM, so choose a PC with over 10GB.

Take a Graphic Tablet PC if you need to do precise manual segmentation.

Any machine will do for visualization

I can't find the PC!

Some of our machines are pretty noisy, so we store them in the small room AI-0141 inside the main office.

How many software licences are available?
  • 2 floating "IMARIS" licences (mainly used with PC64 "White Bear" and PC64 "Graphic Tablet")
  • 4 METAMORPH licences (all the journals are saved on the BIOp server, meaning you can continue a job on any computer of the BIOp)
  • 1 server licence for deconvolution by HUYGENS, piloted through web-interface (Hygens Remote Manager- HRM)
  • Open-source software as: ImageJ, FIJI, Readers, etc...


Contact

BioImaging and Optics platform (PTBIOP)

EPFL-SV-PTECH-BIOP
Station 15
CH-1015 Lausanne
Show on campus map


Dr. Arne Seitz

Office AI-0240
Tel: +41 (0) 21 693 96 18
Fax: +41 (0) 21 693 95 85