Extended Depth Of Field blurring on high-angle surfaces

Vaughanie

Hi Again,

We are just playing around with the EDF on a shell and we are finding it hard to use the EDF function to focus the entirety of the live image correctly (see attached images: Focal Plane 1, Focal Plane 2, and Composite). Blurring and some pixelation seems to occur when the angle of the shell surface is relatively steep (see image of shell under microscope for an idea of the sort of material we are working with).

Do you know how to remedy this and focus the image correctly where the surface angle of the shell is steep? This is using the colour camera.

Also, the live image appears to micro-virbrate (not just flicker), causing blotches of coloured pixels to vibrate on the live image. This may be due to the heat of the colour camera? Any suggestions would be helpful.



 

YuriG

As I can see on the first image the defocusing diameter is quite large, much more that the default 6 pixels. Try to increase this parameter on the EDF Options page.

I tried your 2 images with Defocusing diameter of 10 and have this result:



I also noticed that the intensity of flat areas is slightly different on 2 images, which might produce artifacts around focus areas, so I used Normalize illumination (Process tab, Combine group, Sub-sample dropdown) to fix this problem.

Increasing "Plane blending" can reduce visibility of illumination differences.

Another way is to keep illumination stable moving focus. Or, if it's not possible and you don't want to increase plane blending, you can acquire images one by one, merge them into a sequence, Normalize illumination and then run Live EDF on normalized sequence.


Yuri 

YuriG

Regarding "pixelated": this effect is caused by intensity fluctuations on different images. As I mentioned in the previous comment, use higher Plane blending (e.g. 20). And if you use manual stacking, also use intensity normalization.

Yuri

Vaughanie

Thanks Yuri!

Vaughanie

Just to follow up with the second part of the question above, do you know how we can resolve the micro-vibration of the image? It is not a flicker but a rapid small-scale vibration that causes blurred pixels to vibrate on the screen when viewing the image live. We will try and send a JPEG of this asap.
It may be due to the heat of the camera itself causing the microscope to vibrate?

Any suggestions would be gratefully received.

Also - when trying to build a focused composite image of a high-angle shell surface we have also tried two different options. The first option involved using live EDF with 'accumulate' off, taking multiple photos focused at various focal fields and stacking them manually (see image manual stacking). The second option involved taking images at various focal fields (not using live EDF) then manually stacking them (see image manual manual stacking).
As you can see, on both composites when the individual images are stacked they become pixelated. Any advice here?

Thanks again for all your help!