Diverse views of Pleurosigma on a strew slide

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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Hobbyst46
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Diverse views of Pleurosigma on a strew slide

#1 Post by Hobbyst46 » Sun Oct 13, 2019 6:17 pm

Hello,
I was thinking of oblique-relief-phase contrast attempts so dug a strew slide of benthic marine diatoms, and searched for a large one among the many 20-30um frustules. These diatoms
I had collected about 1.5 year ago, gently cleaned with SDS+EDTA +3% H2O2 and mounted in NOA61 (not the best mountant but that is what I used then).
MicroBob wrote:It is always astonishing, how long one can look at a strew slide and still found new diatom forms sponge needles and other interesting oblects.
And for the first time, I noticed a Pleurosigma (tentative ID!). A whole undamaged 110um-long frustule. Lying among the smaller diatoms and organic remnants (those would have been destroyed had I used corrosive chemicals).

I was very happy with it, so threw away the original purpose of the inspection and just acquired images under diverse illuminations, using mostly the 100X1.3 phase contrast (condenser not oiled). And the simple 100X1.25 achromat for semi-dark field. This objective and the darkfield Ultracondenser were both oiled.
The brightfield image creates a "shadow" along the top rim of the diatom. This artifact occurs in the specific plane of focus that I chose to sharpen as many dots as possible.
Some of the images obtained with a green interference filter, peak 543nm, bad width (at half height) 25nm. Those images required ~4-fold long exposures.
Images are mostly untouched except for resizing and stacking.

Comments are welcome.
Attachments
(1) 100X1.3 Planapo Ph3 oil, brightfield.JPG
(1) 100X1.3 Planapo Ph3 oil, brightfield.JPG (63.11 KiB) Viewed 1179 times
(2) 100X1.3 Planapo Ph3 oil, oblique.JPG
(2) 100X1.3 Planapo Ph3 oil, oblique.JPG (43.87 KiB) Viewed 1179 times
(3) 100X1.25 Achromat oil, Ultracondenser oil, single image.JPG
(3) 100X1.25 Achromat oil, Ultracondenser oil, single image.JPG (63.54 KiB) Viewed 1179 times
(4) 100X1.25 Achromat oil, Ultracondenser oil, 543nm filter, single image.JPG
(4) 100X1.25 Achromat oil, Ultracondenser oil, 543nm filter, single image.JPG (59.56 KiB) Viewed 1179 times
Last edited by Hobbyst46 on Sun Oct 13, 2019 6:22 pm, edited 1 time in total.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Hobbyst46
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Re: Diverse views of Pleurosigma on a strew slide

#2 Post by Hobbyst46 » Sun Oct 13, 2019 6:20 pm

(continued)
Attachments
(5) 100X1.3 Pl.Apo oil phase c., single image.JPG
(5) 100X1.3 Pl.Apo oil phase c., single image.JPG (75.02 KiB) Viewed 1177 times
(6) 100X1.3 Pl.Apo oil phase c., stack of 7 spaced 1um.jpg
(6) 100X1.3 Pl.Apo oil phase c., stack of 7 spaced 1um.jpg (57.11 KiB) Viewed 1177 times
(7) 100X1.3 Pl.Apo oil phase c., 546nm filter, single image.JPG
(7) 100X1.3 Pl.Apo oil phase c., 546nm filter, single image.JPG (74.18 KiB) Viewed 1177 times
(8) 100X1.3 Pl.Apo oil phase c., 543nm filter, stack of 10 spaced ~0.3um.jpg
(8) 100X1.3 Pl.Apo oil phase c., 543nm filter, stack of 10 spaced ~0.3um.jpg (56.55 KiB) Viewed 1177 times
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
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Re: Diverse views of Pleurosigma on a strew slide

#3 Post by MicroBob » Sun Oct 13, 2019 7:14 pm

Hi Doron,
nice find and image series! When looking for the bigger diatoms I have sometimes washed the material in a 50µ stainless sieve. The very best resolution I was able to acheive was with a decentered high power dark field condenser. The image was not beautiful but the pores of Amphipleura pellucida were resolved. The strange setup of the lighting had more impact than the choice of objective. At this resolution absoulte freeness of shock is important. In my lab/office room I have an uneven wood floor and it is a good idea to sit very still and start the exposure shock free.
My Nikon 1j5 has no ir remote function and when controlled via smartphone app it is too slow to use for stacking. So I will have to use my (a bit unwieldy) Sony A6000 in future.

Bob

Hobbyst46
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Re: Diverse views of Pleurosigma on a strew slide

#4 Post by Hobbyst46 » Sun Oct 13, 2019 7:33 pm

Thanks Bob,

Do you positively identify vibration-noise in the images ?

The microscope is small and lightweight (about 7 Kg, at the most) and sits on five 2cm x 2cm x 0.4cm squares of Sorbothane; those are laid on a 20cm x 30cm x 0.4cm ceramic tile, that in turns rests on 4 small low Teflon-coated hard rubber supports, that rest on a desk. The desk is just a 130cm wide plywood plate that is affixed to the back of the open library cabinet, against the wall.
(the description reminds of the support of the flat earth disk as envisaged by the ancient Greek). I am thinking of a new lab bench but cannot afford it in the near future.

The camera is operated by "pressing" the software shutter on the LCD screen, but this is a VERY sensitive touch screen, the mere air pressure when I place my finger tip above it without actually touching the screen fires the shutter.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
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Re: Diverse views of Pleurosigma on a strew slide

#5 Post by MicroBob » Sun Oct 13, 2019 10:30 pm

Hobbyst46 wrote:
Sun Oct 13, 2019 7:33 pm

Do you positively identify vibration-noise in the images ?
No, not at all, but this would be difficult to identify at this performance level. You would just get varying or sub optimal results without knowing it. So it makes sense to be careful about vibration as far as possible. I think a wobbly desk is not too much of a problem as long as you are aware of it's limitations. Your GFL is actually on the sturdy side and not not more vibration prone than e.g. the Phomi. What I'm a bit worried about is vibration when stacking. The shutter shock of the previous image could influence the next image.
In my room the wooden floor boards and the wooden beams they rest upon can transmit my every movement onto the desk - I have to live with it. :roll:

Bob

Wes
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Re: Diverse views of Pleurosigma on a strew slide

#6 Post by Wes » Mon Oct 14, 2019 8:04 am

Thank you for posting, the single phase shot is by far the best in my opinion.

To deal with vibrations I usually set a shutter release timer of a few seconds to provide enough time for residual vibrations to subside. Alternatively I connect the camera to my laptop, when I'm ready to take the shot I lift the mouse off the table, wait a few seconds for everything to stabilize and then click it.

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75RR
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Re: Diverse views of Pleurosigma on a strew slide

#7 Post by 75RR » Tue Oct 15, 2019 3:48 am

Agree #5 i.e. 100X1.3 Pl.Apo oil phase c., single image.JPG is best

Are you familiar with David Walker's article: http://www.microscopy-uk.org.uk/mag/ind ... -test.html

A must read for everyone.

Would have expected #2 i.e. 100X1.3 Planapo Ph3 oil, oblique.JPG to resolve best. How did you achieve oblique?
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MicroBob
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Re: Diverse views of Pleurosigma on a strew slide

#8 Post by MicroBob » Tue Oct 15, 2019 4:43 am

Hi Doron,
I would have expected the oblique image to show more detail. I have made it a bit brighter, bit more contrasty and sharpened abit. Sometimes such editing leads to a gritty look and enhances the remaining dirt but in this case I like the result.

Bob
Attachments
(2) 100X1.3 Planapo Ph3 oil  oblique-01.jpeg
(2) 100X1.3 Planapo Ph3 oil oblique-01.jpeg (66.01 KiB) Viewed 931 times

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75RR
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Re: Diverse views of Pleurosigma on a strew slide

#9 Post by 75RR » Tue Oct 15, 2019 4:52 am

Converted (7) 100X1.3 Pl.Apo oil phase c., 546nm filter, single image.JPG to Black and White
Attachments
(7) 100X1.3 Pl.Apo oil phase c., 546nm filter, single image.JPG.jpg
(7) 100X1.3 Pl.Apo oil phase c., 546nm filter, single image.JPG.jpg (71.39 KiB) Viewed 929 times
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
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