Point Spread Function

• 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.

• Find a bright bead 0.1µm diameter which will appear as a bright point of light with no discernable structure.

• Set camera gain/exposure so as not to over/undersaturate the image.

• Capture a Z-series through the entire bead, capture 40 z-images using a Z-step increment of 0.1µm.

• Collecting a Z-series using different refractive index immersion oils will allow you to find the best immersion oil for your application.

• 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
Image	Image	Image

 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
Image	Image	Image

Images by Applied Precision

Uses for PSF

• Deconvolution

• Objective Resolution (FWHM)

• Objective Quality Check