A question about microscope "alignment"

[gekko]

I was wondering what the traces produced by focus stacks obtained from a dusty sensor such as those in the thread viewtopic.php?f=27&t=3415&p=31190#p31190 are caused by. In the example in the link (and in focus stacks that I got in the past) the dust speck traces are all parallel to each other. Could it be that the microscope is somehow misaligned, such that the motion of the objective when focusing is not normal to the stage? Or is there a more benign explanation? Many thanks for any suggestions.

[c-krebs]

If you are working on a compound microscope (not a stereo scope) with a 10X or higher objective you should be able to turn off (or set to "0") the adjustments for x/y position (alignment) and magnification. Then, any dust spots you have will not become "trails" but will be simple spots as would be the case in a single image. But... and it is a pretty big but!... if the subject moves at all during the stack acquisition, or if the microscope is quite badly aligned you will not get good alignment without those software corrections. (Also if you happen to touch the camera, or slightly bump the scope base, or even move unconnected objects around on the table during stack acquisition it is not unusual to shift image frames very slightly). For example in the stack you referenced, there is a slight shift in the images between 10/11, 13/14, and 16-20. It is not much, but if you stack with "0" alignment and then again with the software set to do its alignment "thing" you will see a difference if you look very closely at the full resolution image at a high magnification. In this case I suspect that the subject moved ever so slightly during the stack. It is extremely common for natural subjects that are not embedded in a solid material to sag, shift, droop in almost imperceptible amounts while acquiring a stack. Subjects in wet mount slides will also commonly drift or otherwise move slightly. A totally rigid subject or a "permanent" mount is rarely a problem unless there is some significant mis-alignment of the scope. While possible, I don't think it is common at all for the compound microscopes we use to be "off" enough for it to be noticeable.

Once you do turn on alignment and magnification in the software then you will almost always get some dust "lines" instead of spots.

A test would be to carefully acquire a stack from a "permanent" mount and then run it through the software with alignment/magnification "on" and then a second time with it "off". See if there is any noticeable difference in the two versions.

[Johann]

Good question Gekko. I just assumed that the image slightly moves because of an optical result from the different focusing depths.
Seems I was wrong - will take extra care going forward to keep everything still and steady - keep forgetting how the tiniest of movements are having a huge impact at these magnifications.

@Charles, Thanks for the explanation

[Johann]

Oh, and one other thing I learned from this whole exercise:
My camera needs a serious clean!!

[mrsonchus]

Oh, and one other thing I learned from this whole exercise:
My camera needs a serious clean!!

Advice re cleaning your camera's sensor - either don't do it, or be very, very careful. I just 'cleaned' mine with special little swabs the size of the sensor - I was extremely careful and did exactly as instructed - result - clean sensor and a very new (<200 shots) Canon EOS 1200D with two hideous scratched trailing across the middle of it's sensor!

Beware - I'm weeping as I type this message.....

I am a desolate husk and a stupid one at that!

[75RR]

Advice re cleaning your camera's sensor - either don't do it, or be very, very careful. I just 'cleaned' mine with special little swabs the size of the sensor - I was extremely careful and did exactly as instructed - result - clean sensor and a very new (<200 shots) Canon EOS 1200D with two hideous scratched trailing across the middle of it's sensor!

Ouch! No chance those are smear marks I suppose?

[mrsonchus]

Advice re cleaning your camera's sensor - either don't do it, or be very, very careful. I just 'cleaned' mine with special little swabs the size of the sensor - I was extremely careful and did exactly as instructed - result - clean sensor and a very new (<200 shots) Canon EOS 1200D with two hideous scratched trailing across the middle of it's sensor!

Ouch! No chance those are smear marks I suppose?

None - checked, double-checked, wept for 5 minutes, checked again, wailed and wandered wraith-like about the garden, re-checked - the horror is real........
I seem to have forgotten to mention this to my Darling Wife... I wonder if she may believe it was a lightning-strike phenomenon-type thing?

Arggghhhhh.... I'm so stupid sometimes! Ah well, calm.....little 'ching-ching bells' and shaded seat in the garden!

Still, I've just posted a comparison of my 2mp ToupCam and the Canon and can't see much difference - it gets worse!

p.s. the scratches miraculously don't seem quite visible in my pictures - but the camera is now worthless!

If I can bear to do it I'll post some images of the full horror-show - as learning processes go this one is not my favourite!

[gekko]

Charles, thank you so much for taking the time to give such a clear and thorough explanation. This is very good to know.

John, very sorry to hear that. But I would again ask what 75RR asked: it is not that easy to scratch a sensor (it is not the actual sensor, I believe, but a glass filter that covers the sensor), unless you had a hard piece of dust or sand that you dragged across it. I would try to rule out a smear. But whatever you do, try not to make it any worse (rather easy to do when one is faced with such a problem).

[Johann]

Oi sorry John, thats no good
I have a canon approved service shop up the road - gonna take it there.

[billbillt]

Advice re cleaning your camera's sensor - either don't do it, or be very, very careful. I just 'cleaned' mine with special little swabs the size of the sensor - I was extremely careful and did exactly as instructed - result - clean sensor and a very new (<200 shots) Canon EOS 1200D with two hideous scratched trailing across the middle of it's sensor!

Ouch! No chance those are smear marks I suppose?

None - checked, double-checked, wept for 5 minutes, checked again, wailed and wandered wraith-like about the garden, re-checked - the horror is real........
I seem to have forgotten to mention this to my Darling Wife... I wonder if she may believe it was a lightning-strike phenomenon-type thing?

Arggghhhhh.... I'm so stupid sometimes! Ah well, calm.....little 'ching-ching bells' and shaded seat in the garden!

Still, I've just posted a comparison of my 2mp ToupCam and the Canon and can't see much difference - it gets worse!

p.s. the scratches miraculously don't seem quite visible in my pictures - but the camera is now worthless!

If I can bear to do it I'll post some images of the full horror-show - as learning processes go this one is not my favourite!

Hi John,
You just did what anyone else would have done.. It should not be so difficult and sensitive to clean... You did what you thought was right.. Nothing wrong with that!!..

[mrsonchus]

Thanks Bill - at least it doesn't seem to show in my photomicrographs - could be worse I suppose..

[Rodney]

My question is how would Canon, Nikon or any other company clean a microscope or camera optical system when its needed and what are the finished results if you really look close?
Many types of cleaning chemicals can it seems be bound to many types of optical glass and coated lens and may not be easily removed if at all, so you may be left with a light haze across the lens if you look closely, all the cleaning may look really good for the first few minutes but wait a few more minutes and look closely at a possible haze.
Mineral deposits are the biggest problems in cleaners for do it your self, so how does Canon clean?

Rodney