A raw 1 micron bead looks very elongated along the optical axis. Why?
A measured 1 mu latex bead was sampled at 100nm in all directions.
In the original image (assuming typical microscope conditions, 1.3NA, 500nm excitation wavelength) the bead should have an axial size of roughly 1.8 mu axially and 1 mu laterally, so 18 samples axially and 10 samples laterally. <br>
A factor which can increase this elongation considerably is refractive index mismatch of embedding medium and coverslip. If this is the case and you can't increase the refractive index of the medium you might consider purchasing a water or glycerin immersion objective. To get a more accurate estimate of what the bead should look like in the microscope, you can simulate the imaging process with the Huygens Professional:
- start Huygens Pro <br>
- generate a sphere + do an FFT on the result <br>
- generate a PSF + do an FFT on the result <br>
- multiply the two and put the result in say image 'c' <br>
- do a reverse FFT on 'c' (with the FFT tool in auto mode this will go automatically) <br>
- do a 'to/from optical representation' (restoration menu) to shift the origin of the image to the center. <br>
- to make things realistic you can throw in some Poisson noise.
The result should not be too different from the measured image.
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Keywords: latex bead axial elongation<br>
Categories: Faq Microscopy, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux Windows Mac AIX<br>
Related products: Hu Ess Hu Pro<br>
