Determine optical slice thickness in a CLSM

[luri-fax]

I am trying to figure out the optical slice thickness on a confocal laser scanning microscope with the following:

Lambda exitation: 514
Lambda emmision: 548nm
Pinhole: 2 AU / 222,9 um
NA: 1,2
n (refraction index): 1,33 (W)

I have tried a few equations, but they give quite different results. One of the equations I have used is in this article from Zeiss in the following link:
http://www.zeiss.com/C1256D18002CC306/0/F99A7F3E8944EEE3C1256E5C0045F68B/$file/45-0029_e.pdf"
(page 11, equation 4)



I am going to use this in my Master thesis due soon. Can someone please help me determine the correct way of measuring this?
I must stress that I am a biologist so "be gentle" with the complex formulas

 

[Valerie Park]

The equation you are using is a valid way to calculate the optical slice thickness. However, it is important to note that this equation is an approximation and does not take into account all of the factors that can affect the actual optical slice thickness. To get an accurate measurement, you will need to measure the optical slice thickness directly with a microscope or other imaging system.

 

[astrokreng]

I understand your struggle with determining the optical slice thickness on a confocal laser scanning microscope. The optical slice thickness is an important parameter in confocal microscopy as it determines the resolution and depth of field of the images obtained. After reviewing the information you provided, I believe the best approach to determine the optical slice thickness would be to use the equation provided in the Zeiss article you referenced. This equation takes into account the pinhole size, numerical aperture, and refractive index, which are all factors that influence the optical slice thickness.

However, it is important to note that this equation assumes a perfect Gaussian beam profile and may not give accurate results for non-ideal situations. In that case, it may be necessary to use a more complex equation or to measure the optical slice thickness experimentally by imaging a fluorescent sample with known dimensions.

Additionally, make sure to double-check your units to ensure they are consistent with the units used in the equation. This will ensure accurate results. If you are still unsure, I suggest consulting with a colleague or mentor who has experience with confocal microscopy or reaching out to the authors of the Zeiss article for further clarification. I wish you the best of luck with your Master thesis and I am confident that with careful consideration and attention to detail, you will be able to accurately determine the optical slice thickness on your confocal laser scanning microscope.