Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

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MicroBob
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Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#1 Post by MicroBob » Sat Feb 06, 2021 6:22 pm

Hi together,
here a photo of the center area of a centric diatom from Wakura Beds, Japan. This is from a slide Hobbyst46-Doron has made with individually mounted frustules. And here the story begins: The letter with his slides took a full 104 days to get here! At a distance of about 3850km this results in an average speed of 1,5 km/h, so the slides would have been here quicker if Doron had walked here, 8 hours a day. :lol: I'm very happy the letter did arrived in the end and has survived all opportunities to get lost on a 104 day trip. Thank you very much, Doron!

Bob
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Zentrum G-Kanal DSC_1382-RGB 1024 klein2.jpg
Zentrum G-Kanal DSC_1382-RGB 1024 klein2.jpg (195.68 KiB) Viewed 7181 times

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75RR
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#2 Post by 75RR » Sat Feb 06, 2021 6:55 pm

.
Nice detail you managed to tease out. Well done

So that it why they call it snail mail ;)

Glad they arrived safely
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)

MicroBob
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#3 Post by MicroBob » Sat Feb 06, 2021 7:05 pm

These centric or triagular diatoms seem to have an inner layer with even finer structure that can just about be resolved with the light microscope. This was with Zeiss DIC old (limiting condenser aperture a bit) and a Leitz NPL Fluotar 100:1 /1.32. I think I will give it another try with a darkfield condenser or n.a 1,4 bright filed condenser and oblique lighting. There should be a bit more to extract.

Bob

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#4 Post by Hobbyst46 » Sat Feb 06, 2021 10:30 pm

Glad to see this, Bob, thanks. Fine resolution. Enhances the appetite to improve my own images of these centric beauties.

With my phase contrast 63X1.4 I did not achieve such resolution. Neither with the 40X0.8 darkfield.
I must try it with the 100X1.3 phase contrast. Or oblique, either with the 63X or 100X.
Phase contrast is supposed to give less resolution.
What is the apparent resolution (from the dots, of course !) in the image you posted ?

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#5 Post by Charles » Sat Feb 06, 2021 10:56 pm

Can you post an image of the whole diatom?

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#6 Post by MicroBob » Sun Feb 07, 2021 8:38 am

Hi together,
the photo was made with the Optovar in 2x setting and a green interference filter in the light path. I tried my Planapo 63 /1,4 with delaminations in multiple layers, but from what I was able to see with my eyes, it resolved less than the 100:1. The camera might have seen more resolution which might have been visible after raising the contrast.
I add two photos of the whole diatom, a stack (which I don't like) and a single image which i prefer. For stacking images with less contrast would probably be better. When trying to resolve Amphipleura pellucida I had the most success with a decentered darkfield condenser. A simle 100:1 Achromat was enough with this condenser when the right illumination setting was found.

Bob
Attachments
Stack 2 komplett-RGB1024.JPG
Stack 2 komplett-RGB1024.JPG (285.74 KiB) Viewed 7113 times
Bearbeitet DSC_1316-RGB 1024.JPG
Bearbeitet DSC_1316-RGB 1024.JPG (226.56 KiB) Viewed 7113 times

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#7 Post by MicroBob » Mon Feb 08, 2021 9:58 am

Hi together,
I did some more technical tests:
-The delaminated 63 Planapo really doesn't resolve as well as the NPN Fluotar 100
-With phase contrast I didn't get a good image of the fine structure. Perhaps the halos superimpose and cover fine detail
-Bright field best showed the real structure
-The slide has little contrast, perhaps the Pleurax is not fully heat-cured
-Olbique (attatched) with the n.a.1,4 condenser shows lots of resolution and contrast, but like DIC is misleading when it comes to show the real structure

Bob
Attachments
Oblique DSC_1403-RGB 1024.jpg
Oblique DSC_1403-RGB 1024.jpg (196.46 KiB) Viewed 7075 times

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#8 Post by Hobbyst46 » Mon Feb 08, 2021 11:46 am

MicroBob wrote:
Mon Feb 08, 2021 9:58 am
Hi together,
I did some more technical tests:
-The delaminated 63 Planapo really doesn't resolve as well as the NPN Fluotar 100
-With phase contrast I didn't get a good image of the fine structure. Perhaps the halos superimpose and cover fine detail
-Bright field best showed the real structure
-The slide has little contrast, perhaps the Pleurax is not fully heat-cured
-Olbique (attatched) with the n.a.1,4 condenser shows lots of resolution and contrast, but like DIC is misleading when it comes to show the real structure

Bob
1. I like this image and resolution !

2. Could be that Pleurax was not fully cured. The Pleurax protocol from the site diatoms.nl states that heating the slide for 15 min at a temperature of 180C is sufficient.
However, I have often observed that the resin was not fully cured. Remained slightly tacky. So did not solidify.
How can we solve this issue ?

Heating at higher temp than 180C is risky. The resin shows discoloration, becomes brown and the coverslip explodes or just detaches and flies away.

On the other hand, I remember that Rod (rnabholz) incubated his slides at 130C for hours after the short higher temp step (hope I remember correctly). It definitely makes sense,
but, curing at 130C (say) for hours requires a hotplate (impractical for me) or constant-temp oven.

When heated, Pleurax gives off smelly vapor - not the IPA smell, more like phenol and/or sulfur, which is logical, since those are the building blocks of the resin.
For this reason, I would hesitate to incubate a Pleurax slide in the kitchen oven.

3. Bob, do you find Leitz Fluotar objectives better than Zeiss objectives of the same mag, on a Zeiss stand ?

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#9 Post by MicroBob » Mon Feb 08, 2021 7:13 pm

2. Could be that Pleurax was not fully cured. The Pleurax protocol from the site diatoms.nl states that heating the slide for 15 min at a temperature of 180C is sufficient.
However, I have often observed that the resin was not fully cured. Remained slightly tacky. So did not solidify.
How can we solve this issue ?

My impression was that you once have to reach this temperature for a couple of seconds and the the Pleurax is good. When curing over the flame of the alcohol burner I see the isopropanol evaporation, then a calm period, then another finer, stinky bubbling and then the Pleurax was good for me. For our last pre-Corona-meeting I built a special cover slip hot-plate to allow the people to make a couple of slides each in the limited time. This was set to 184°, but later I learned that these thermo couples are not especially precise. Here I put the very clean dry material onto the cover slip, add a drop of isopropanol, add a drop of Plaurax, give time to mix, put on hot plate and have a ready slide very quickly. I have never heated Pleurax for longer and would expect yours to be fully cured after your curing method. I only didn't see much in straigt brightfield, which looked like too little refractive index difference to me.

When heated, Pleurax gives off smelly vapor - not the IPA smell, more like phenol and/or sulfur, which is logical, since those are the building blocks of the resin.
For this reason, I would hesitate to incubate a Pleurax slide in the kitchen oven.


I stop right after this special stench rises and wouldn't do this in the kitschen oven too

3. Bob, do you find Leitz Fluotar objectives better than Zeiss objectives of the same mag, on a Zeiss stand ?
No. The performance should be about the same but I got them for prices for which I don't get the Zeiss objectives and they don't delaminate. The CVD correction of Leitz and Zeiss West is very close so I use them together and take Leitz eyepieces for observation and photography for the same reasons.

I always wanted to show the Pleurax cooker - here it is. It is powered by a 3D-printer hot end heater and reaches 180°C quickly. It consumes less energy than a big hot plate. A heat resistant and insulating platform to stand on is necessary, over time the heat goes through a tile and burn the desk surface! :evil: It was very time consuming to build, so not a first class recommendation.

BTW: I sent you a PM yesterday, deleted it and sent it again with additions - did you get it? I can't see it in my "sent" folder.

Bob
Attachments
Pleurax-Kocher 2.jpg
Pleurax-Kocher 2.jpg (185.99 KiB) Viewed 7039 times
Pleurax-Kocher 1.jpg
Pleurax-Kocher 1.jpg (239.58 KiB) Viewed 7039 times

LouiseScot
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#10 Post by LouiseScot » Mon Feb 08, 2021 7:54 pm

MicroBob wrote:
Mon Feb 08, 2021 7:13 pm
Hobbyst46 wrote:
Mon Feb 08, 2021 11:46 am



On the other hand, I remember that Rod (rnabholz) incubated his slides at 130C for hours after the short higher temp step (hope I remember correctly). It definitely makes sense,
but, curing at 130C (say) for hours requires a hotplate (impractical for me) or constant-temp oven.

When heated, Pleurax gives off smelly vapor - not the IPA smell, more like phenol and/or sulfur, which is logical, since those are the building blocks of the resin.
For this reason, I would hesitate to incubate a Pleurax slide in the kitchen oven.

3. Bob, do you find Leitz Fluotar objectives better than Zeiss objectives of the same mag, on a Zeiss stand ?
It's easy to make a simple, cheap hotplate powered by a 12V 5A supply. Just connect a 50W aluminium-clad 3.3 Ohm panel-mount power resistor in series with a 12-24V 8A dimmer switch. Bolt the resistor to the bottom of a small aluminium plate (about 125 x 125 x 3mm) with some heatsink compound. Fit 4 x long thin bolts to each corner as legs. Paint the surface matt black. Adjust with the dimmer switch and use an IR thermometer to monitor the temperature. :) The max theoretical surface temp of the resistor is 150 deg at 50W. I certainly measured the above plate at 120 deg. I can't remember whether it could go higher but if you really need 130 deg, maybe a smaller, thinner plate would be better - say 100 x 100 x 2mm. I'll check mine out in a minute and upload a photo of it, if anyone is interested?

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#11 Post by MicroBob » Mon Feb 08, 2021 8:39 pm

Hi Louise,
good idea, the panel mount resistor. What increased the build time of my micro hot plate was the PID controller with additional wiring, milling, thread cutting...
Recently I made lots of botanic slides for a test series for a new stain. My usual hot plate was too small and got on my nerves due the the clicking of the relais. So I build a hotplate from a 3mm aluminium plate, fat aluminium bar to spread the heat better, two 24V 40W 3D-printer hot end heaters connected in row and powered with 12V. With insulation this heats up to 45° and no more, just what I need, and the controller rarely has to step in, so practically silent and low enery consumption.

Bob

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#12 Post by LouiseScot » Mon Feb 08, 2021 8:48 pm

Here are a couple of pics:
Bottom view:
Bottom_20pc - Copy.jpg
Bottom_20pc - Copy.jpg (71.05 KiB) Viewed 7032 times

Top view and temp:
IR_temp - Copy.jpg
IR_temp - Copy.jpg (85.38 KiB) Viewed 7032 times


Only got to ~115 deg but my living room isn't very warm! Probably would be better (slightly higher temp) with a 3 ohm resistor rather than the 3.3 ohm one I used ->
I = V/I = 12/3 = 4Amps, P= IxV = 12 x 4 = 48W which should give 48/50 x 150 (Max temp) = theoretical 144 deg resistor case temperature. That will drop a bit as heat lost to hot plate and overall plate temperature will also depend on ambient. Can still pick up the hot plate by the legs at 115 deg :) One could make the plate a fair bit smaller to save heat loss and also use an insulating material for the legs rather than metal bolts.

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#13 Post by LouiseScot » Mon Feb 08, 2021 8:57 pm

MicroBob wrote:
Mon Feb 08, 2021 8:39 pm
Hi Louise,
good idea, the panel mount resistor. What increased the build time of my micro hot plate was the PID controller with additional wiring, milling, thread cutting...
Recently I made lots of botanic slides for a test series for a new stain. My usual hot plate was too small and got on my nerves due the the clicking of the relais. So I build a hotplate from a 3mm aluminium plate, fat aluminium bar to spread the heat better, two 24V 40W 3D-printer hot end heaters connected in row and powered with 12V. With insulation this heats up to 45° and no more, just what I need, and the controller rarely has to step in, so practically silent and low enery consumption.

Bob
Hi Bob

Mine uses the 8A 12-24V dimmer controller which controls the power - and the temperature indirectly. The 50W resistor wont go above 150 deg at 50W = 50/12 = 4.12A @ 12V, and the plate temperature will always be lower. Temperature rise from start is quite slow - takes about 25 min to get up to 115 deg at my room temp - which is currently under 15 deg. So it's fairly easy to control/set the temp via the dimmer. Simples!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#14 Post by MicroBob » Mon Feb 08, 2021 9:21 pm

Nice design and much quicker to build! In old times they would have used a bulb to heat the thing - try that with todays LEDs! :lol:
Your room temperature will probably drop by another degree but you could increase the surface temperature by applying a layer of Armaflex insulation to the underside. This is adhesive light foam rubber used for technical insulations. Perhaps a small piece is available as 3D-printer heat bed insulation or you could get a little as a sample from a vendor.

In old times they used alcohol and petroleum lamps and candles for heating stuff for microscopy recipes. Here electicity is just great to have.

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#15 Post by LouiseScot » Mon Feb 08, 2021 9:43 pm

MicroBob wrote:
Mon Feb 08, 2021 9:21 pm
Nice design and much quicker to build! In old times they would have used a bulb to heat the thing - try that with todays LEDs! :lol:
Your room temperature will probably drop by another degree but you could increase the surface temperature by applying a layer of Armaflex insulation to the underside. This is adhesive light foam rubber used for technical insulations. Perhaps a small piece is available as 3D-printer heat bed insulation or you could get a little as a sample from a vendor.

In old times they used alcohol and petroleum lamps and candles for heating stuff for microscopy recipes. Here electicity is just great to have.
The hot plate isn't my room heater! Just making the plate smaller would do if someone needed a slightly higher temperature. It probably doesn't lose much heat from underneath since heated air will heat the plate (assuming no draughts). I don't really need it above ~120 deg C. myself but, if I did, I'd substitute a 3, or possibly a 2.7, ohm resistor. It would be too much effort for me to try and saw the plate to make it smaller (I'm just a little, old lady - literally!). I tend to go for simple solutions that will likely work :)

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#16 Post by LouiseScot » Mon Feb 08, 2021 9:48 pm

ps my plate needs a better coat of black paint! It's only there for the IR thermometer but the first time I used it, some of the paint stuck to the slide! :shock:

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#17 Post by Hobbyst46 » Mon Feb 08, 2021 9:50 pm

Bob, Louise,

I like your designs and comments. May I add though, that a heating finger - inserted into the plate via a bore - is more efficient, since all heat loss from the heater element is to the plate.

I have constructed heating plates in the past. Point is, that I do not dare to work with Pleurax in a non-ventilated space, and in that space where I do there is no electrical power outlet, so my heating plate - aluminum, roughly 100x200x8 mm - is heated by means of a small alcohol burner. When ambient temperature is 20-30C (normal, here) the plate reaches 180C within 20-30 minutes. The slow process is fine, according the instructions by Michel Haak. The plate takes four slides simultaneously, BTW.

Bob, if the temperature sensor in your plate as shown is a thermocouple, it is probably accurate. However, I believe that temperature varies significantly between different points on the surface of the plate - a 10C variance would be common. Still, for the Pleurax slide it should be OK.

I received a deleted PM; will re-check.

Louise, the surface temperature monitor is a good idea. It is being used in the millions nowadays to verify that people's forehead or hand or arm temperature is ~36 (corona). Funny indeed.

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#18 Post by LouiseScot » Mon Feb 08, 2021 9:55 pm

Hobbyst46 wrote:
Mon Feb 08, 2021 9:50 pm
Bob, Louise,



Louise, the surface temperature monitor is a good idea. It is being used in the millions nowadays to verify that people's forehead or hand or arm temperature is ~36 (corona). Funny indeed.
Yes, I've seen them! It does the job for me :D

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#19 Post by LouiseScot » Mon Feb 08, 2021 10:18 pm

Hobbyst46 wrote:
Mon Feb 08, 2021 9:50 pm
Bob, Louise,



I have constructed heating plates in the past. Point is, that I do not dare to work with Pleurax in a non-ventilated space,
Isn't pleurax just dissolved in isopropanol? Safe enough? The naphrax I have is in toluene but the amount given off by a single slide is tiny, so not overly dangerous.

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#20 Post by Hobbyst46 » Mon Feb 08, 2021 10:52 pm

LouiseScot wrote:
Mon Feb 08, 2021 10:18 pm
Hobbyst46 wrote:
Mon Feb 08, 2021 9:50 pm
Bob, Louise,



I have constructed heating plates in the past. Point is, that I do not dare to work with Pleurax in a non-ventilated space,
Isn't pleurax just dissolved in isopropanol? Safe enough? The naphrax I have is in toluene but the amount given off by a single slide is tiny, so not overly dangerous.

Louise
Pleurax is indeed just dissolved in IPA. I would not worry about the IPA. However, when I open the Pleurax vial, I always smell phenol, and even more so when I heat it. That is not a surprise, since Pleurax is produced by reacting phenol with sulfur compound. So possibly there is some phenol residue in the resin. Since I prepare 4-8 slides each session, I do not like anyone in the house to breath the vapors. Perhaps I am being overly cautious.
The prepared slide does not smell of anything; I seal it anyway, now with Hammerite direct-on-rust paint, which resists immersion oil.
It is also possible that Naphrax does not give off vapors (besides toluene).

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#21 Post by Sabatini » Mon Feb 08, 2021 11:07 pm

MicroBob.
What a lovely work.
Beautiful diatom.
Thanks you

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#22 Post by LouiseScot » Mon Feb 08, 2021 11:33 pm

Hobbyst46 wrote:
Mon Feb 08, 2021 10:52 pm
LouiseScot wrote:
Mon Feb 08, 2021 10:18 pm
Hobbyst46 wrote:
Mon Feb 08, 2021 9:50 pm
Bob, Louise,



I have constructed heating plates in the past. Point is, that I do not dare to work with Pleurax in a non-ventilated space,
Isn't pleurax just dissolved in isopropanol? Safe enough? The naphrax I have is in toluene but the amount given off by a single slide is tiny, so not overly dangerous.

Louise
Pleurax is indeed just dissolved in IPA. I would not worry about the IPA. However, when I open the Pleurax vial, I always smell phenol, and even more so when I heat it. That is not a surprise, since Pleurax is produced by reacting phenol with sulfur compound. So possibly there is some phenol residue in the resin. Since I prepare 4-8 slides each session, I do not like anyone in the house to breath the vapors. Perhaps I am being overly cautious.
The prepared slide does not smell of anything; I seal it anyway, now with Hammerite direct-on-rust paint, which resists immersion oil.
It is also possible that Naphrax does not give off vapors (besides toluene).
Phenol isn't especially toxic - at least, not in the amounts we're talking about here. It's used in throat sprays as an antiseptic e.g. Chloraseptic. I wouldn't purposely stand over a heated slide and inhale the vapours, though! I'm on my own so don't have to worry about affecting other people. Obviously if you have others around you have to think about them.

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#23 Post by MicroBob » Thu Feb 11, 2021 7:01 am

Hi together,
these images raised an interesting question: Hobbyst46-Doron didn't quite get these results though he is using a first class instrument and a slide that should be virtually identical. What is the difference? Doron made both slides so the number of possible differences is limited.

Here some of my thougths to begin with:
- My diatom seems to be optimally close to the cover slip
- My slide seems to be an optimal thin preparation, good for the illumination
- I had mentioned that the slide had little contrast in bright field and concluded the Pleurax may have not been cured fully. This will not be the case, it probably is just right.
- The Zeiss DIC old limits condenser aperture, so it is not optimized for the very finest detail
- Oblique with the apl-achr. n.a. 1,4 condenser gave at least the resolution of the DIC image, no advantage for the expensive DIC, this was what I would have expected
- A multi-delaminated Zeiss 63 1,4 and a Leitz Fluorescence 63 1,3 didn't give quite as good an image in the eyepieces, maybe they would have in the photo
- A phase Neofluar 1,3 didn't give a useable image (attatched), the objective itself is good for other uses
- A multi delaminated Zeiss Planapo 100 1,32 didn't give a good image
- A nice Planachromat gave a nice image (attatched), especially when only using the green channel
- The images turned out exceptionally well, I don't alwayse get this performance

So where might further optimization take place? What might be the difference

- Orientation of the diatom frustule upside down?
- cover slip thickness?
- Thickness of mountant layer
Less likely as my images fit to the eyepiece view:
- shutter shock avoided? (My Nikon 1J5 has no shutter, so no shock, it performs about as good as an APS-C camera from 2010, good but not great)
- image editing

@Doron: Please add your photos and procedure, so we can look for a hint for further optimization

Bob
Attachments
Phase green light Neofluar 100 1,3 grenn channel usm DSC_1398-RGB.jpg
Phase green light Neofluar 100 1,3 grenn channel usm DSC_1398-RGB.jpg (120.71 KiB) Viewed 6940 times
DIC white light planachromat 100 1,25 green channel contrast usm DSC_1396-RGB.jpg
DIC white light planachromat 100 1,25 green channel contrast usm DSC_1396-RGB.jpg (155.13 KiB) Viewed 6940 times

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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#24 Post by Hobbyst46 » Fri Feb 12, 2021 9:35 pm

Slide preparation procedure:
1. My coverslips are 18mm in diameter, #1 nominal thickness, inexpensively bought from china. They are cleaned by 15min boiling in tap water, rinsing with distilled water and leaving to dry out on a Kimwipe.
2. A clean, coverslip (inexpensive, from china) is coated with dilute filtered gelatine-based or filtered polyacrylamide-based adhesive. I prefer gelatine.
3. A drop of suspension of cleaned diatom frustules is placed on a matt-black surface and left to dry out.
4. Cleaned diatoms are picked and laid on the coated coverslip, using a mechanical finger and a stereoscope at 60X magnification. The diatoms were cleaned in the past, most of them are
centric, and they appear to be snow-white and very clean indeed. The diatoms are placed at the center of the coverslip, by eye.
5. Cautious, slow exhalation on the coverslip is supposed to fix the frustules in place.
6. 1-2 drops of Pleurax are added, a slide is laid inverted onto the coverslip, and slight pressure is applied if needed to spread the liquid.
7. On each of the two short rims of the slide, a paper clip is fitted, such that one side of the clip lies flat, in contact with the coverslip side of the slide.
8. Four slides are laid side by side, coverslip side down, on a thick 200x100x8 mm aluminum block. The block is placed on a tripod over an alcohol burner. Two thermocouple sensors are fixed onto the top surface of the block at two remote locations.
9. The block is heated; it reaches a temperature of 175-185C within 30 min and is kept as much as possible at this temperature level for 20-30 min, by removing and returning the burner under the block.
10. The prepared slide is ringed with Hammerite Direct-On-Rust paint.
11. Admittedly, the diatoms in the above slide are not "arranged". Rather, they are randomly spread at various distances from the center. Still, the virtue of the adhesive is that it prevents
them from being swept to the rim of the coverslip. So, they are in a more or less disperse group.
12. These Japanese frustules are especially contrasty against the background even under brightfield, compared with various other diatoms that I have cleaned and mounted.
13. Pleurax fills the frustules very well, there are no air bubbles.

(to be continued)

Hobbyst46
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#25 Post by Hobbyst46 » Fri Feb 12, 2021 10:40 pm

(cont'd) the following are various single images of frustules - cousins of the diatom documented by MicroBob above. Diameters 74 and 110um, respectively. They are the sharpest images that I could see - view through the eyepieces did not yield better resolution. As-is JPG photos are shown, as well as green channels of them. Contrast was enhanced somewhat. Two objectives were used - a plain achromat 100x1.25 and a planapo 100x1.3 Ph3.
Attachments
(1) Slide WBJ-7, 74um diameter, 100X1.25 achromat.JPG
(1) Slide WBJ-7, 74um diameter, 100X1.25 achromat.JPG (117.07 KiB) Viewed 6863 times
(2) Slide WBJ-7, 74um diameter, 100X1.25 achromat, green channel.jpg
(2) Slide WBJ-7, 74um diameter, 100X1.25 achromat, green channel.jpg (106.63 KiB) Viewed 6863 times
Last edited by Hobbyst46 on Fri Feb 12, 2021 10:48 pm, edited 1 time in total.

Hobbyst46
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#26 Post by Hobbyst46 » Fri Feb 12, 2021 10:44 pm

(cont'd) notice that different focus depths show different ornamental structures.

Although all these diatoms are "cousins", of course I cannot be sure that two microscopists at separate locations study similarly structured surfaces. Maybe these are different faces of the valves, and the valves are not identical. It is still tempting to think that one objective is better than the other... ;)
Attachments
(3) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield.JPG
(3) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield.JPG (126.26 KiB) Viewed 6861 times
(4) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield, green channel, focus A.jpg
(4) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield, green channel, focus A.jpg (113.21 KiB) Viewed 6861 times
(5) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield, green channel, focus B.jpg
(5) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, brightfield, green channel, focus B.jpg (114.09 KiB) Viewed 6861 times
(6) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, phase contrast, green channel.jpg
(6) Slide WBJ-2, 110um diameter, 100X1.3 Planapo Ph3, phase contrast, green channel.jpg (106.81 KiB) Viewed 6861 times

Hobbyst46
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#27 Post by Hobbyst46 » Fri Feb 12, 2021 11:19 pm

P.S. My 100X1.3 planapo objective is 160/-. So I believe (perhaps wrongly) that it should not be very sensitive to the coverslip thickness.
The 100X1.25 achromat is also 160/-.

LouiseScot
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#28 Post by LouiseScot » Fri Feb 12, 2021 11:40 pm

Hobbyst46 wrote:
Fri Feb 12, 2021 11:19 pm
P.S. My 100X1.3 planapo objective is 160/-. So I believe (perhaps wrongly) that it should not be very sensitive to the coverslip thickness.
The 100X1.25 achromat is also 160/-.

I think, usually, the larger the aperture, the more sensitive to coverslip thickness? I see a Zeiss 100x/1.3 160 planapo on Ebay. It has an adjustable coverslip thickness collar. The only thing is that it's from the States which puts me off.

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#29 Post by Hobbyst46 » Sat Feb 13, 2021 12:56 pm

LouiseScot wrote:
Fri Feb 12, 2021 11:40 pm
Hobbyst46 wrote:
Fri Feb 12, 2021 11:19 pm
P.S. My 100X1.3 planapo objective is 160/-. So I believe (perhaps wrongly) that it should not be very sensitive to the coverslip thickness.
The 100X1.25 achromat is also 160/-.

I think, usually, the larger the aperture, the more sensitive to coverslip thickness?
Yes. However, these objectives are marked as "-" with respect to coverslip, meaning that the objective is expected to perform well with or without coverslip. Intuitively, I would think that means that it is not very sensitive to coverslip thickness; does it also mean that the resolution and contrast provided by these objectives are inherently the same as of objectives designed to work with 0.17mm coverslip ?
Sure, the 100X1.3 Ph3 phase contrast is expected to provide less resolution than a brightfield 100X1.3 objective. But, setting aside this point, I wonder about the "-" design vs the "0.17" design.
Tried some microscopy sites but did not find the answer so far.
I see a Zeiss 100x/1.3 160 planapo on Ebay. It has an adjustable coverslip thickness collar. The only thing is that it's from the States which puts me off.
I would be cautious about two other aspects of all 160mm TL Zeiss objectives: (a) these objectives need Zeiss correcting eyepieces to provide a low aberration image, (b) delamination might be present.

LouiseScot
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Location: Scotland

Re: Centric diatom from Wakura Beds, Japan or how quick do diatoms travel?

#30 Post by LouiseScot » Sat Feb 13, 2021 1:15 pm

Hobbyst46 wrote:
Sat Feb 13, 2021 12:56 pm
LouiseScot wrote:
Fri Feb 12, 2021 11:40 pm
Hobbyst46 wrote:
Fri Feb 12, 2021 11:19 pm
P.S. My 100X1.3 planapo objective is 160/-. So I believe (perhaps wrongly) that it should not be very sensitive to the coverslip thickness.
The 100X1.25 achromat is also 160/-.

I think, usually, the larger the aperture, the more sensitive to coverslip thickness?
Yes. However, these objectives are marked as "-" with respect to coverslip, meaning that the objective is expected to perform well with or without coverslip. Intuitively, I would think that means that it is not very sensitive to coverslip thickness; does it also mean that the resolution and contrast provided by these objectives are inherently the same as of objectives designed to work with 0.17mm coverslip ?
Sure, the 100X1.3 Ph3 phase contrast is expected to provide less resolution than a brightfield 100X1.3 objective. But, setting aside this point, I wonder about the "-" design vs the "0.17" design.
Tried some microscopy sites but did not find the answer so far.
I see a Zeiss 100x/1.3 160 planapo on Ebay. It has an adjustable coverslip thickness collar. The only thing is that it's from the States which puts me off.
I would be cautious about two other aspects of all 160mm TL Zeiss objectives: (a) these objectives need Zeiss correcting eyepieces to provide a low aberration image, (b) delamination might be present.
Perhaps the '-' means no coverslip? I wasn't about to buy a Zeiss from the US, at least. I just happen to come across it :) The Swift one I have is probably fine for most things anyway.
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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