Centre Optical Instrumentation Laboratory

Point Spread Function

1. View slide using objective of choice (usually high NA lens). Use the correct immersion oil for your specimen mounting medium and for any correction collar settings
2. Find a bright bead 0.1µm diameter which will appear as a bright point of light with no discernable structure
3. Set camera gain/exposure so as not to over/undersaturate the image
4. Capture a Z-series through the entire bead, capture 40 z-images using a Z-step increment of 0.1µm
5. Collecting a Z-series using different refractive index immersion oils will allow you to find the best immersion oil for your application.
6. Using your image manipulation software produce a 3D stack of your z-series. its important that your software can rotate the 3D image in the xz plane as well as xy

Using the PSF

Astigmatism (xy view)
Good PSF
Moderate Astigmatism
Very Bad
Moderate Astig
Bad Astig
Axial Skew (xz view) Note on a confocal system the PSF appears as more of a focussed point without the haze above and below
Good PSF
Skewed to the top of PSF
Very badly skewed to the left
No skew
Top skew
Left Skew
Images by Applied Precision
Uses for PSF
1. Deconvolution
2. Objective Resolution
3. Objective Check