The formula used calculating the depth of field is:

D = ((w * n)/(NA^2))*(1 + 1/(2*M))

where

D = depth of field
w = wavelength
n = index of refraction
NA = NA of objective
M = objective magnification

This page is to be used to explain how to go about using the super computing institutes's software package named Huygens. Huygens will allow confocal images to be taken from a non-confocal machine, such as the Zeiss microscope currently set up.

First of all capture all your needed images. All files should be saved as uncompressed .tiff images. All filenames should have a numerical ending, meaning that each name should end in a numerical digit based on the order that they were taken starting from zero. This applies even if there is only a single image.

All files should be placed in a directory that matches the file name, with the exception of the numeric ending.

For example, if the images are named test00, test01, test02, ... textxx where xx is the ending number, then the directory that they all reside in should be named just test.

Now connect to the super computers server. There should be an icon located on the desktop with the name of 'huygens'. By clicking on this icon, you will open a secure shell connection to the server in a pre-configured way.

Once connected, open the file transfer screen (located on the tool bar of the shell window) and copy your directory of images to the server and place them in the incoming folder.

Return to the shell terminal and now type in 'scripts/huygens.pl'. The script should return almost instantly and indicate if the batch job was accepted or not. If so, you may now disconnect and leave as the job will take at least 10-15 minutes, perhaps much more if there are a large number of images to deal with. The result of the huygens script will be located in outgoing//_out.

If the script returns an error indicating that the batch job was denied, first check name and location of the files to make sure that they are correct and resubmit. If this fails, please see a BIPL member to assist you.

FYI: Backprojected pinhole radius for the Olympus Flouview 1000 at 60x is 24.33 nm

This guide will go over how to do a reconstruction in Volocity and how to make a movie with your reconstructed image. You will need access to the Supercomputing Institute (BSCL) to use this software.

This guide will go through how to do 3D and 4D reconstructions in Imaris. You will need access to to the Supercomputing Institute (BSCL) to use this software.

This guide will walk you through doing deconvolution in Huygens. You will need access to the Supercomputing Institute (BSCL) to use this software. The first part shows how to generate a PSF (if desired) and the second walks through deconvolution.

Note: Use Classic MLE instead of Quick MLE you will get better results.

The following PDF contains a basic recipe for a glycerin jelly mount recipe. This may take a little time to make (around 2 hours), but is highly customizible. It has an R.I. of 1.47.

This is a quick guide to the 1024 single photon confocal. A page on Nyquist Sampling rates and a page with ideal Airy Disk diameters are included in the attachments.

This is a quick guide to the Olympus with separate directions for brightfield and fluorescence. There is also a chart of default settings for the camera and a chart of excitation and emission regions for the cubes.

This is a quick guide to the Zeiss Microscope with seperate directions for brightfield and fluorescence. A chart of default cubes and a chart of objectives are included in the attachments

This is a quick guide to the Inverted Microscope with directions to correct for uneven illumination. Included in the attachments are a chart of the default and optional cubes and a chart of the objectives.