Discriminating Illumination

pwnell · Post by **pwnell** » Mon Sep 01, 2014 12:00 pm

If there is one thing I love most about photomicrography, it is to observe different structures using different illumination techniques. Reminds me of the elephant and the blind people feeling the tail, leg and trunk. I think this describes pretty much most of science - we see only aspects of reality and project from there what we define as real. The holographic universe is a good theory based on this principle.

Coming back down to earth, most general photography is done by means of white light reflecting off of subjects, and recording that reflection. However, it is but one tiny way we can look at the world. Using different wavelengths (UV), polarized light, light wave interference (DIC), refracted light (darkfield), light wave retardation (lambda/quarter wave plates), and various combinations of these we can see so much more.

Here is the same amphipod specimen I found in my reef aquarium, but interrogated with most of the means to my disposal. I would have loved to have painted it by electron, but that is outside my reach (for now).

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, DF+POL

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, DF+POL+LP, HF A+B

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, POL+LP, HF B

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, POL

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, FLUO-C4

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x, FLUO-C6

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x,BF

Amphipod by pwnell, on Flickr
Reef aquarium amphipod, 10x,DF

Amphipods by pwnell, on Flickr
Amphipods from Reef aquarium, Canon 1DX + MP-E65 @ 5:1, Reflected Brightfield (not microscope).

Jacek · Post by **Jacek** » Mon Sep 01, 2014 12:09 pm

There are no words. Great pictures

carlos.uruguay · Post by **carlos.uruguay** » Mon Sep 01, 2014 3:46 pm

Very interesting!

RogelioMoreno · Post by **RogelioMoreno** » Tue Sep 02, 2014 2:54 am

Nice set showing details with several illuminations.

Rogelio

75RR · Post by **75RR** » Tue Sep 02, 2014 11:53 am

Very nice images.
Always interested in different illumination techniques, unfortunately there are a couple I am unfamiliar with. Could you tell me what they stand for? Thanks
DF = DarkField
POL = Polarized
FLOU = Fluorescent
BF = BrightField
LP = ?
HF = ?

pwnell · Post by **pwnell** » Tue Sep 02, 2014 11:56 am

75RR wrote:Very nice images.
Always interested in different illumination techniques, unfortunately there are a couple I am unfamiliar with. Could you tell me what they stand for? Thanks
DF = DarkField [CORRECT]
POL = Polarized [CORRECT]
FLOU = Fluorescent [CORRECT, C4 = has less UV and more blue, higher emission cutoff wavelength, C6 = has more UV, lower emission cutoff wavelength]
BF = BrightField [CORRECT]
LP = [Lambda Plate]
HF = [Helicon Focus stacking]

hoarybat · Post by **hoarybat** » Tue Sep 02, 2014 2:35 pm

These are stunning, and wonderful examples of differing techniques.

Anatoly · Post by **Anatoly** » Tue Sep 02, 2014 10:58 pm

Very interesting! Thank you!
With the MP-65 which flash? MT14EX or MT24EX?

pwnell · Post by **pwnell** » Tue Sep 02, 2014 11:06 pm

Anatoly wrote:Very interesting! Thank you!
With the MP-65 which flash? MT14EX or MT24EX?

Neither. I used the new MR-14EX II Macro Ring Lite Flash.

Anatoly · Post by **Anatoly** » Tue Sep 02, 2014 11:21 pm

Thanks for the answer!

arturoag75 · Post by **arturoag75** » Wed Sep 03, 2014 4:04 am

Excellent job...last one with no microscope is really impressive

Ecki · Post by **Ecki** » Wed Sep 03, 2014 4:09 am

Very Nice! The second image is my favorite.

Lothar-Gutjahr · Post by **Lothar-Gutjahr** » Wed Sep 03, 2014 4:28 am

Hi Waldo,

EXCELLENT ! Thanks for sharing!

Lothar

piermicro · Post by **piermicro** » Wed Sep 03, 2014 10:02 am

I do not understand how you manage to get the effects in FL. I assume the subjects are not autofluorescent. You are using cubic C4 and C6, but you do not specify the wavelength. Are the final photos in fluorescence the result of multiple photographs linked with photoshop?
Regards, Pier

pwnell · Post by **pwnell** » Wed Sep 03, 2014 10:19 am

piermicro wrote:I do not understand how you manage to get the effects in FL. I assume the subjects are not autofluorescent. You are using cubic C4 and C6, but you do not specify the wavelength. Are the final photos in fluorescence the result of multiple photographs linked with photoshop?
Regards, Pier

Thanks for the comments. This crustacean is auto fluorescent. The red is the ingested algae in its digestive track, the green / blue is the autofluorescence of its carapace. For each of those photos, the image is as seen through the eyepiece. No combining of different colours in post.

FLUO-C4:
Excitation Filter: 330 – 480 nm single band bandpass filter
Dichroic Mirror:495nm
Emission Filter: 500nm long pass edge filter

FLUO-C6:
Excitation Filter: 335 – 379 nm single band bandpass filter
Dichroic Mirror:409nm
Emission Filter: 437nm long pass edge filter