Investment and quality

Linden.g · Post by **Linden.g** » Wed Mar 23, 2011 5:25 pm

I've now added reflective lighting on my Olympus BH-2. The following butterfly and moth wings were taken with the Olympus 20x S Plan Achromat. Focus stacked in Zerene Stacker. My question, how much would I gain if I invested in an Olympus 20x Apo.

Linden

Charles Krebs · Post by **Charles Krebs** » Wed Mar 23, 2011 5:54 pm

Linden,

A couple things going on here in regards to your question. You don't mention whether or not you used a cover slip for these reflected light shots. It's possible to get by pretty well without one using the 20/0.46 S Plan Ach. This would not be the case with the 20/0.70 Apo. It does very poorly without a cover slip.

When used with the proper coverslip my 20/0.70 Apo clearly outperforms my 20/0.46. As would be expected from the NA the resolution is higher, but equally noticeable is the much higher degree of color correction. It is a very sweet objective. Not without a few minor headaches.... the working distance is 0.55mm versus 1.5mm so you really need to be careful if you have oiled a slide, or you try to swing it into place on a subject that is some distance from the cover slip ("crunch"... and squished subjects). Optically the 20/0.70 does not like subjects that are too far away from the cover slip. So you'll find more occasions where you pass up a subject because it is just a little too "deep".

Linden.g · Post by **Linden.g** » Wed Mar 23, 2011 6:31 pm

Hi Charlie, thanks for the reply. I didn't use a coverslip, I assume this would be used dry? If you don't mind would you be willing to provide specific critical analysis on what you see above. Please provide examples from your own work if you think that would be helpful. Side by side comparisons would be ideal. For such close working distance on the Apo have you always used light projected through the objective or have you managed to light the subject from the sides.
All the best
Linden

Charles Krebs · Post by **Charles Krebs** » Wed Mar 23, 2011 9:51 pm

For such close working distance on the Apo have you always used light projected through the objective or have you managed to light the subject from the sides.

Never tried. The 20/0.70 is always used with transmitted light and a cover slipped subject.

For opaque subjects like these there are only a few "biological" objectives I'll use on a "biological" microscope... 2.5X, 4X and 10X. But since I set up a microscope with the BH2-UMA I more typically use the "M" type objectives (5x to 100x) if I will be using a microscope. These are designed for no cover slip usage. (Once you start going above a NA of about the 0.46 of your 20X you want to start abiding by the design parameters as far as cover slip thickness and usage is concerned).

When you say that you have added "reflective lighting", what do you mean? Have you put a vertical illuminator on the scope or just added external light sources than can be directed from the side? What was the magnification on sensor? Remember that the higher the NA used for any given magnification, the better the resolution (assuming naturally that the objective quality is about equally good). So if you are using a 20/0.46 with a 2.5X NFK you are getting 50X with a NA of 0.46. If you had a stable set-up where you could "direct project" with a higher power objective with a higher NA you would get results that showed higher resolution.

Mitch640 · Post by **Mitch640** » Thu Mar 24, 2011 4:04 am

Charles, I was looking at your scope setup yesterday, the one with the light pipe illuminators. Will those work for slides with coverslips? I couldn't tell from the images, if your slide had a coverslip on it. I was wondering if they would work on a scope for toplighting of opaque subjects, like Gammarus Shrimp and other subjects that are small, but too thick to see through.

I have managed to get my Fluophot to push light through my lenses, but the filters that are also involved in doing that, filter all but one specific wavelength of light, so useless for what I do. And the clear filter hole has no mirror in it.

Linden.g · Post by **Linden.g** » Thu Mar 24, 2011 7:27 am

Charlie

I'm using the BH-2 optics including the tube 2.5x tube lens. I'm top lighting the subject with two flash units. See below. Its difficult to see from the photo but I'm also using a plastic ring which clips on to the obective and extends beyond the front of the objective lens. This is what is acting as a diffuser for the flash units. The LED lamp on the right is for focusing and a stackshot drive is attached to the focusing knob so I can automatically collect the images for stacking.

twebster · Post by **twebster** » Thu Mar 24, 2011 10:27 am

I love macro images of butterfly/moth wing scales. I especially like number 1. It reminds me of the skeins of colored ribbons you find in a fabric store. This is a fine series of images.

Charles Krebs · Post by **Charles Krebs** » Thu Mar 24, 2011 10:46 am

You can squeeze light into a surprisingly small space as done here. With some subjects, like these scales, it works well. With other subjects it will be too strong from the sides. So if you can get at least a few mm of working distance it is much more versatile. This old link shows how I did quite a few of this type of subject on a microscope:
http://www.photomacrography1.net/forum/ ... php?t=4869

Cover slips are needed for nearly all biological objectives over about NA 0.40. (But with most good "biological" objectives over 0.40 the working distances are too small to do this on a regular basis). If you have a bit of working distance it is not impossible to use external lights with a some cover slipped subjects but it is a hassle and you really need to pay attention to reflections.

Vertical illuminators can direct light through the objective, so working distances are much less of a problem. But epi-brightfield done this way is a very "stark", frontal type of light. Not really attractive for most subjects. And you really need to use crossed polarization to get around the flare issues.

Linden.g · Post by **Linden.g** » Thu Mar 24, 2011 1:30 pm

Thanks for the advice. I did try sending light down one of the eye pieces and it worked somewhat but I got flare from the internal optical surfaces. Do you think its worth trying this again with polarizing filters.
Linden