Yet Another First Stack (Sugar crystals polarisation)

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squiggles
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Yet Another First Stack (Sugar crystals polarisation)

Post by squiggles »

Both images taken with a Canon 50D through a Leica DM750 and NPlan 10x objective via direct projection. Shutter time is 1/10s and lighting via built-in LED lamp. Shutter is controlled by PC using EOS Utilities.

Stacking done via Zerene using TIFFs. Minor touch-up was done in Zerene then Photoshop for exposure, contrast and sharpening tweaks.

First up is a 24 image stack using PMAX. There is some flaring/ghosting which is present throughout the stack. Any suggestions how to eliminate this? Is it due to excessive lighting?
Image

Second is a 54 image stack also using PMAX. Still some minor ghosting in the white crystals - think this one is largely due to poor exposure control?
Image

The sharpness is much better than what I could achieve with the Olympus OMD EM5 Mk1. There is still much to learn and thank you in advance for your comments!

rjlittlefield
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Post by rjlittlefield »

These look good, given that I'm looking at just the final images at web resolution.

You ask about "flaring/ghosting which is present throughout the stack". Do you mean that all of the individual images show this effect, or that it appears in only some of them and you had hoped the stacking process would eliminate it from the combined image?

You also mention "using PMAX". The PMax stacking method can sometimes make flare and ghosting worse. PMax also increases contrast and noise, and it can alter colors a little. The other method, DMap, is complementary in that it does not have those problems, but DMap is not as good as PMax at handling complicated geometry. As you do more of this, I recommend to make friends with DMap, then see if you can get a better result from either DMap by itself or by combining DMap and PMax. There's a tutorial on "How To Use DMap", linked on the Zerene Stacker tutorials index page. The tutorial is written in terms of a closeup/macro application with an opaque subject, but hopefully it will give enough basic understanding to help working with transparent subjects also.

--Rik

squiggles
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Post by squiggles »

Rik, thanks for the link to the DMap tutorial - I missed it while looking through the site.

For my case, I think the ghosting/flaring is coming from the phototube. The effect is not so clear in the polarisation photos but very apparent in brightfield.

Here is a diatom taken in brightfield through a NPlan 20x PH1 objective. Shutter 1/100s and flash at 1/16 power.
Image

I'm going to try flocking the inside of the phototube to see if it improves.

Pau
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Post by Pau »

Yes, the light rings at your BF image seems reflection inside a tube.
In that image I can see other issues:
- The background shows some lightly colored banding likely due to the flash head not enough defocused. If your scope allows for Köhler illumination move the condenser to focus the field diaphragm at the slide plane. If not, move a bit upwards or downwards the condenser to have a more even illumination close to its maximum intensity point. Or put a diffuser over the flash head or inside the illumination path.
- There is a light spot at the center and a decentered diaphragm reflection. This will be more difficult to avoid.
- Of course vignette, but this is not that bad. Your system doesn't cover APSC for direct projection but seems close.

Would be nice to see pictures and details of your camera and flash adaptations
Pau

squiggles
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Post by squiggles »

Pau, thank you for the detailed critique! The flaws become readily apparent after you pointed them out and several light bulbs went off in my head...

1. Phototube finishing is poor - circular rings to cut down reflection only extend 1/3 way up the tube. It was an eBay China buy so not surprising. Length is also too long to achieve parfocal

2. Light banding - yes you are right. I neglected to attach the defuser head.

3. Decentered diaphram reflection - I think this is due to my 45-degree slide used to bounce the flash into the condenser. Clearance between condenser and field diaphragm is only 2cm which isn't much space to work with

4. Light spot - probably due to lack of defuser head on flash?

5. Vignetting - yes the trinocular head appears to be just a little small for APSC. Are there other issues that I need to watch for?

Here are some pictures of the setup. It's very crude as I'm still learning the appropriate manual power settings and proper distance to place the flash from the condenser. As it is a LED system, there isn't a simple way of putting the flash below the field diaphragm to achieve Kohler. Any suggestions there?

Image

Image

Image

I've ordered another phototube adapter, this time from Samuel Roa, Spain, that should allow me to achieve parfocal with direct projection. Hoping this will also help with sharper focus at higher magnification. The current setup doesn't deliver good sharpness for small objects at 40x and beyond.

squiggles
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Post by squiggles »

Here are some additional photos - some taken using only the onboard LED and others with flash. All single shot, no stacking.

Algae? 40x brightfield (onboard LED, no flash, shutter 1/10s) edited in Lightroom to accentuate light rings & other defects. Still a hint of the central light spot! Is this a drawback of direct projection?
Image

Aelosoma sp. 20x brightfield (onboard LED, no flash, shutter 1/125s) enhanced in Lightroom. Light rings etc less apparent after processing to enhance detail of subject
Image

Light ring & central light spot visible
Image

Winged Rotifer? 40x darkfield (flash 1/16 with diffuser, shutter 1/160s) enhanced in Lightroom
Image

And in brightfield...
Image

Nematode 40x darkfield (onboard LED, no flash, shutter 1/10s) enhanced in Lightroom
Image

Debris, algae, rotifer? 20x darkfield (flash 1/16 with diffuser, shutter 1/160s) enhanced in Lightroom
Image

zzffnn
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Post by zzffnn »

You can stick a frosted glass filter right under condenser (say with mounting putty or double sided tape). That should save flash power and also provide diffusion for LED, when you use long exposure.

Also make sure your reflector slide is exactly centered under condenser and exactly 45 degrees.

Pau
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Post by Pau »

Now I can have a more precise idea of your system :)

As you say, it would be difficult to put the flash at Köhler position with an integrated illumination scope like yours (you will need an old stile external illuminator with focusable lens and field diaphragm modified to put the flash tube at the incandescent lamp position) but with a diffuser over the flash head and playing a bit with the flash distance you can achieve nice even critical illumination. Because the flash+diffuser light source is big this must not be an issue.
If you can achieve good uniform illumination with your LED I think that the diffuser is better placed over the flash head than under the condenser

You can take a look at our FAQ about flash at the microscope:
http://www.photomacrography.net/forum/v ... hp?t=26185

You could benefit of more flash power for light hungry applications like DF or cross pol changing the slide acting as 45º reflector for a half mirror (beamsplitter) or a cube prism beamsplitter, I use the second option and you can buy it it for little money at SurplusSed:
http://www.surplusshed.com/pages/item/l2047d.html

Others points:
- The central bright spot could also be due to the tube reflections, is so it will disappear when flocking or changing the adapter.
- Try opening each diaphragm to see if the diaphragm reflection disappears and to locate the cause
Pau

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