Hi all,
I was helping a coworker to align the Kohler illumination of their microscope. They only had a 20X 0.45NA lens in the microscope and were struggling to place the field diaphragm in focus (it was after a relocation and the alignment was out of whack). I also struggled myself, until I thought I could also close the condenser diaphragm to reduce the illumination NA and therefore increase the depth of field. The condenser diaphragm has a marking that seem to indicate the corresponding illumination NA (0.9 to 0.1). Obviously, from 0.9 to about 0.5 it didnât make a difference (detection NA is 0.45), but then going all the way to 0.1 the field diaphragm started to appear out of focus. From there, we placed the field diaphragm in focus, and fully opened the condenser diaphragm. The field diaphragm was again, slightly out of focus, and we adjusted it one last time.
The reason Iâm posting this here is because my coworker started asking me more details about the explanation above, and I realized Iâm not entirely sure if my theory is correct. The marking on the condenser diaphragm is a strong piece of evidence, but am I correct with my theory above? Is there something else I did not consider? Has someone else used this âtrickâ before with medium to high NA lenses?
Any feedback is appreciated, thank you.
Omni
Reducing the NA of illumination below the NA of acceptance (i.e. the NA of the objective) only increases spatial coherence of illumination and, at the same time, reduces resolution of the microscope as a whole.
Increasing the spatial coherence of illumination will make objects that are not in the focal plane of the objective appear more prominent and contrasted. It can therefore give a kind of âforced planarityâ.
However, I donât quite understand what was the problem you had of focussing the image of the field diaphragm in the first place with the condenser set at the right setting for the objective. It is common with a standard (i.e. non highly corrected) transillumination condenser system for the image of the field diaphragm to be hazy or chromatically slurred showing several shadow images when viewed by the higher power objectives. You just need to chose one of the images to focus on. Trying to get a sharper image by stopping down the condenser shouldnât make a practical difference to the performance of the microscope once you open back up the condenser afterwards since, when you open up the field diaphragm to just larger than the field of view, you shouldnât see any shadow of it in the field of view - and thatâs as good as your microscope is going to get. Slightly focussing on one level or another (because you are chasing different chromatic images at high power) isnât going to help overall - and if it does then using the condenser trick isnât going to make it any better per se. However, the one thing your condenser trick might do is that it might allow you to pick the best focus for your particular scope and, if it does, then thatâs great - but you could probably do exactly as well if you just select the ârightâ shadow / image to focus on with the condenser open.
However, if you really need to stop down the condenser to focus the field stop, this should alert you to the possibility that something in your scope has gone seriously out of alignment.
If you like basic microscope theory, I have a series of videos on YouTube that explain it in detail.
Here are some links to my Köhler videos:
Köhler Illuminator
Köhler 2
Köhler Update 1
PWG, Youngâs slits, Köhlerâs Conjugates (Dominus part 9)
Later, this weekend, Iâll be releasing a new video on âDiffractionâ and, scheduled for December, Iâll be releasing a video on Abbeâs diffraction theory of image formation in the light microscope.
Enjoy.
P.
1 Like
@P_Tadrous
Thank you for taking the time to prepare such an elaborate answer. It is much appreciated.
Some points I want to clarify:
- The microscope was pretty much taken apart and put back together, therefore the alignment was completely wrong to begin with
- We could probably have managed the alignment without this âtrickâ but everyone was stressed and the aligned condenser was surprisingly close to the specimen (we didnât think it should have been this high)
- Iâm not saying this trick improves anything in terms of the performance, I am merely suggesting that it helps finding the field diaphragm more easily to begin the alignment
Here are some pictures I took with a camera to illustrate what we did. Note that the camera is not centered and does not covered nearly the same FOV.
When reviewing the content that you sent, in the first video (Kohler Illuminator) at 3:55 you mention an issue where stray light reflects into the objective lens and creates hotspots. Could it be the issue at play here? To be clear, Iâm not talking about the halo in the âafter alignmentâ picture. Iâm trying to understand why closing the Condenser Diaphragm helps seeing the Field Diaphragm in the second picture (from top).
Thanks again for sharing your experience, it is really valuable for me.
PS: ignore the background in the images, I took the pictures without a sample, just to show what it looks like (I know the alignment should be done with a sample but I wanted quick images this morning).
Take care,
David
Hello @Omnistic
The answer is simply that closing the diaphragm increases the effective focal depth of the condenser (i.e. the range, in depth, over which objects appear in focus). You can explain this in terms of increased spatial coherence or you can model it in terms of a narrower angular aperture - however you model it, the fact is that focal depth is increased with smaller aperture. So your condenser trick helped you to find the edge of the diaphragm by bringing the edge of the diaphragm (that was wildly out of focus - i.e. out of the range of the depth of focus - with a wide condenser aperture) into the (now greatly extended) focal field that you caused to be extended by closing the condenser aperture. You brought the field stop âinto focusâ not by moving the field stop into the focal range of the condenser but by extending the focal range of the condenser till it reached the field stop.
This is also why you should always do your final focussing of the field stop with the condenser aperture opened as far as it can go (to match the objective NA) - because only then will you have the best resolution of focus (i.e. the focal depth will be the narrowest).
The âhotspotsâ I mention in my video refer to localised areas of brightness and lower contrast seen in the image plane due to reflections of stray light off the insides of the objective and other microscope tubes. So, no, it is nothing to do with what is seen here.
Your last picture illustrates what I meant by various âshadow imagesâ in my first reply.
Note: You should always do your final field diaphragm focussing with a sample on the stage and in focus because the glass of the slide and mountant and coverslip will significantly alter the correct focal distance. The idea with focussing the field stop is that the field stop must be confocal with your specimen (as I explained in my first Köhler video).
All the best.
P
@P_Tadrous
Perfect, that confirms my suspicion. Thank a lot for your time and effort. Thank you also for the resources you are putting out for the community.
Take care,
David
