Thanks for using iScopeCalc. This calculator was created by Talley Lambert at Washington State Univesity. It is designed to help you configure your microscope to achieve nyquist sampling, optimal field of view, and optimal confocality. It is particularly intended for use with a spinning disc confocal microscope, but may also be used for widefield imaging.
The calculator defaults to a widefield configuration. To change modalities, switch "modality" to confocal and select your Yokogawa CSU model. If you have a Spectral Borealis systems, check the "Borealis" checkbox and a new option will appear on the calculator that lets you change the Spectral CSU coupler between the disk and the scope. This with affect your confocality (Airy units) as well as your sampling rate and field of view. If you chose W1, a new option will appear on the calculator to change the disk and the size of the pinhole.
When you select a camera, the specifications for that particular camera, such as pixel size and chip dimensions, are loaded into the calculator. You may also change the magnification of your camera relay. For spinning disk setups, this relay is placed between the CSU head and the camera, as opposed to the "CSU relay" in a Borealis system which is placed between the CSU and the microscope frame.
Next, enter your objective magnification and NA, select the fluorophore you are imaging and laser emission wavelength (lasers will default to optimal wavelength for your given fluorophore), and chose whether you want to bin your camera pixels. You can then verify that your imaging parameters are appropriate. The "sampling rate" represents the number of pixels per Airy disk radius and should be greater than 2 for "optimal" (Nyquist) sampling. For optimal confocality, when using the Spectral CSU coupler, Airy Units should be around 1. Unless you have a Borealis system or the CSU-W1, it is not possible to change your confocality.
The graphic represents the image of a diffraction limited spot (the Airy disk) as imaged by the camera. The grid represents the size of the camera pixels in the image plane. When in confocal modality, the size of the pinhole in the image plane is represented by a white circle and one can then compare the size of the Airy disk to the size of the pinhole for assessing confocality.
For more detailed stats and a list of equations used, click on "equations" in the menu at the top of the page. These numbers are theoretical and will likely vary slightly depending on your optical configuration.
If you find an error or if you have a suggestion for improving the calculator, I would love to hear from you!
iScopeCalc ©2013 Talley Lambert