Improving Depth of Field through the microscope.

[georgetsmurf]

As with all photography-to getter better results there is always a trade of to be addressed. I am very new to Photomicrography other than some stuff for uni many years ago. I have however done and published quite a lot of macro work. I have found that to imrove depth of field through the microscope-what works is the same as in macro work. I put a very small hole (2mm) in a piece of copper and placed that between the turret prismn and the microscope. Of course there is dramatic light loss and potential
camera shake but these problems can be tackled some other way. Have a look at the images I have taken of a jumping spider to compare the difference. Taken on a 1970's Nikon microscope with Nikon standard objective. camera is a Nikon D300s. Please try to ignore the poor quality. this was only an experiment in immprovement of depth of field.

[rjlittlefield]

As with all photography-to getter better results there is always a trade of to be addressed.

That's certainly true. In this case the tradeoff is between DOF and sharpness, or more precisely, between geometric blur and diffraction blur.

What you are accomplishing by stopping down is to decrease geometric blur but increase diffraction blur. There is a lot of discussion of this tradeoff in the forum archives. One good place to start is at http://www.photomacrography.net/forum/v ... .php?t=424 and the links therein. Perhaps the most relevant part is this snippet:

You might be amused by the following story...

I started doing photomacrography back in high school, probably '66 or thereabouts. 'Twas all 35mm film at the time, of course. Well, I realized quickly enough that to get large magnification, I had to use short focal length lenses, and all that I had available were microscope objectives. But dang! Every time I tried to use a microscope objective, the DOF got so small it was hopeless. Stopping down a microscope objective is fairly tedious (I machined aperture plates and dropped them in), so I went looking for a short focal length lens with an iris diaphragm. Pretty quick I found a surplus lens from an 8mm movie camera -- a 12.5mm f/1.9-16 Rodenstock-Ronar, serial number 4193938. (How do I know this, you ask? Keep reading.) Cheap, the lens was, and soon delivered.

Well, the lens was a little tricky to mount, but after a couple of hours of lathe work I had a cute little adapter with Pentax M42 threads on one end and some ludicrously fine thread on the other end to fit the lens' filter threads. (21 x 1/3mm, I think it was.)

Mounting the adapter on my bellows, I eagerly proceeded to shoot a test series of frames, and scurried off to the darkroom to develop them. What a disappointment! Even at marked f/16, the DOF was still not enough to be useful, and the resolution was ghastly. I gave up in despair, blaming a cheap lens and looking forward to the day when I could afford something better...a lot better.

Of course, the real problem was that at the time I did not understand about diffraction. And if I had understood about diffraction, I still would not have been able to do anything about it, because at the time computers were Really Big and Really Expensive and still were nowhere near powerful enough to do anything like stacking.

You ask us to

Please try to ignore the poor quality. this was only an experiment in immprovement of depth of field.

If by "poor quality" you are talking about lack of sharpness, then it would be a serious mistake to ignore that. The lack of sharpness is inescapably paired with the increase in DOF when images are formed using ordinary light and lenses. This may be frustrating and/or saddening to hear, but hearing it will save you a lot of time.

Stopping down is not a good path to increased DOF in this regime. Your time will be better spent learning about focus stacking.

--Rik

[georgetsmurf]

Stopping down is not a good path to increased DOF in this regime. Your time will be better spent learning about focus stacking.

--Rik

Thanks for that-please point me in the right direction and as Dick Tracy (i think) would say-"I will get on it right away".
Cheers George

[rjlittlefield]

please point me in the right direction and as Dick Tracy (i think) would say-"I will get on it right away".

Pointer provided in your other thread.

BTW, as Admin I've edited a couple of your posts so that the

tag works as intended. It's important to get the start tag, end tag, and quote marks exactly right. You can see what I've done by clicking the Edit button on those posts. You can check your own new posts by clicking the Preview button, to be sure that they look right before hitting Submit.

--Rik

[georgetsmurf]

You say

Stopping down is not a good path to increased DOF in this regime. Your time will be better spent learning about focus stacking.

--Rik

I have downloaded a trial version of Zerene but it seems that one cannot save output images on the trial version. Is that the case ?
I have put a trial stack together based on 9 images just to get an idea of how to do it. Interesting but obviously a few important aspects must be considered to get it giving good results.
George.

[rjlittlefield]

I have downloaded a trial version of Zerene but it seems that one cannot save output images on the trial version. Is that the case ?

The 30-day trial period is fully functional, no restrictions. To save an output image, be sure the image you want is selected (highlighted), then in the menu system use File > Save Output Image(s).

--Rik

[Charles Krebs]

George,

Welcome to the group!

I'll echo much of what Rik has mentioned. I jumped (back) into microscopy in summer of 2004 and initially set up with a DSLR, figuring I could get "up-to-speed" much quicker with the instant digital feedback, and then put my film cameras on the scope. It didn't take long to realize that one of the biggest advantages of digital was the possibility of dealing with the ridiculously shallow DOF experienced with a light microscope.

With nearly all microscopes, you are immediately into a situation where resolution losses because of diffraction are already occurring. There is still the compromise you mention... DOF versus resolution, and that is a constant decision with mobile subjects, where you may only be able to get a "single shot". But with stationary subjects, or subjects that will sit still long enough to take a number of shots, the technique of "stacking" can produce results that were only a dream a decade ago.

There's still place for an "inserted" aperture in a compound microscope. But I think it's biggest value would be in situations where you are trying to establish darkfield illumination with an objective whose numerical aperture is a little too large for the darkfield condenser arrangement. And even in this situation the reduction in aperture size should ideally be very slight. I'm not quite sure where, in the microscope, you have placed the 2mm aperture you mention here... could you describe the location again?

[georgetsmurf]

Hi Charles. Thanks for the welcome.

You said

There's still place for an "inserted" aperture in a compound microscope. But I think it's biggest value would be in situations where you are trying to establish darkfield illumination with an objective whose numerical aperture is a little too large for the darkfield condenser arrangement. And even in this situation the reduction in aperture size should ideally be very slight. I'm not quite sure where, in the microscope, you have placed the 2mm aperture you mention here... could you describe the location again?

I had placed the copper shim with the 2mm dia hole directly under the ocular tube that I had attached my camera to. Unfortunately it seems that the second image with improved depth of field, albeit increased blurr and obvious light loss didn't make it to to the post. I will try to attach it to this reply.
George.

[georgetsmurf]

I have downloaded a trial version of Zerene but it seems that one cannot save output images on the trial version. Is that the case ?

The 30-day trial period is fully functional, no restrictions. To save an output image, be sure the image you want is selected (highlighted), then in the menu system use File > Save Output Image(s).

--Rik

My mistake. I now find I can save output files. I have attached one of the nine imput files for my first try with the program and the output file. A bit rough but at least I can see it getting somewhere. This test example is just a few threads of a gal screw. Improved DOF is apparent however I need to now refine the process. Thanks for helping me along with that.