Stephanodiscus

Smokedaddy · Post by **Smokedaddy** » Fri Feb 03, 2017 10:06 pm

Well this is my first shot at imaging a Diatom (6 stacked images with Zerene). Yea, nothing like the experts post here but I'm just getting started. <g> Cactusdave was kind enough to send me a few Diatom slides and they arrived today. I couldn't wait to give my MM-11 a try. I'm not sure HOW to use the collar on this particular objective? Anyone know? Hopefully down the road I'll buy a couple of oil objectives.

Smokedaddy · Post by **Smokedaddy** » Fri Feb 03, 2017 10:40 pm

How would you go about measuring the diameter of a Diatom (BTW, this image is cropped)?

carlos.uruguay · Post by **carlos.uruguay** » Sat Feb 04, 2017 7:56 am

Beatsy · Post by **Beatsy** » Sat Feb 04, 2017 9:07 am

Smokedaddy wrote:How would you go about measuring the diameter of a Diatom (BTW, this image is cropped)?

Nothing wrong with those images. Looking good.

Ref: measuring. You can roughly work out the image scale by dividing the width of the camera sensor by the magnification onto it. So say you're using a 40x objective with 2x relay lens onto a full-frame 36mm sensor. The field of view will be 36/80 = 0.45mm (or 450 micron). From this you can work out microns per pixel, measure the pixel width of your diatoms and voila - you have a measurement. This is prone to some inaccuracy due to magnifications not being exactly as stated. So...

The best way is to get a stage micrometer which is a slide with a 1mm graticule (usually) divided into 0.01mm intervals. Take pictures of the graticule with each of your objectives and work out the image scale from there.

Here's a cheap chinese stage micrometer. I'm not particularly recommending this one - it was the first cheap one to come up in an ebay search.

http://www.ebay.co.uk/itm/Calibration-S ... SwgQ9Vgi9z

Smokedaddy · Post by **Smokedaddy** » Sat Feb 04, 2017 9:22 am

What is a relay lens? <red-faced> My sensor is 23.7mm x 15.6mm (APS-C). If I had a stage micrometer side I don't understand how you would use it. Surely you don't stack a diatom slide on top of the micrometer slide.

Beatsy · Post by **Beatsy** » Sat Feb 04, 2017 9:33 am

Relay lens is whatever is between the objective and camera to project an image onto the sensor. Never mind that though - the slide is easier.

No, you don't put diatoms on the micrometer slide. You just take pictures of the micrometer slide with each of the objectives you plan to measure with. Looking at the pictures will tell you exactly how wide the field of view is with each objective (you will count the graticule marks in view). Given this info, and the number of pixels across the image (depends on camera mpix), you can calculate the exact size of anything in OTHER pictures taken.

For example, using simple numbers. If your 40x objective covers 0.25mm (250um) of the graticule and your captured image is (say) 3000 pixels wide before cropping, then each pixel covers 250/3000 = 0.083 microns. To get the size of a diatom, just take a picture of it, measure how many pixels long/wide it is in the image, and multiply by 0.083 (in this case). That will be the size of the diatom in microns. You will use a different scale factor for each objective of course.

Smokedaddy · Post by **Smokedaddy** » Sat Feb 04, 2017 1:58 pm

Beatsy wrote:Relay lens is whatever is between the objective and camera to project an image onto the sensor. Never mind that though - the slide is easier.

Thanks for explaining, BTW all I had were extension tubes, no lens.

-JW:

Smokedaddy · Post by **Smokedaddy** » Sat Feb 04, 2017 6:00 pm

I wonder if anyone here has used this ImageJ plugin?

http://imagej.net/Microscope_Measurement_Tools

-JW:

Smokedaddy · Post by **Smokedaddy** » Mon Feb 06, 2017 3:58 pm

Assuming I did the calculations correct I came up with this. I used a stage micrometer and ImageJ.

Smokedaddy · Post by **Smokedaddy** » Tue Feb 07, 2017 1:31 pm

This is my first image taken with my newly acquired Optiphot. Being a complete novice to microscopes I'm not sure I have Köhler Illumination setup properly. Also, one of the chrome knurled knobs on 'bottom surface' of my Phase Contrast was extremely hard to rotate. Can the PC device be taken apart and cleaned? This image much better than my poor lighting setup on my MM-11 rig. If I knew what I was doing illumination wise I might be able to get a little more resolution out of it. I didn't mess with the collar on the dry
objective either. This is a 100% crop of the overall image. I'm pretty happy with the image.

rjlittlefield · Post by **rjlittlefield** » Tue Feb 07, 2017 3:49 pm

To my eye this latest image appears to be chock full of stacking artifacts -- hard-edged halos of various sorts -- that may be mistaken for fine detail.

Can you show us a single frame, for comparison?

--Rik

Smokedaddy · Post by **Smokedaddy** » Tue Feb 07, 2017 4:46 pm

... and I thought it was a great first image. <red-faced> When I stacked the group the end product was a DMap image before taking it to Photoshop. This is a 100% crop of the 21st .TIF file (.JPG here).

rjlittlefield · Post by **rjlittlefield** » Tue Feb 07, 2017 6:04 pm

Thanks for the single frame.

This confirms my suspicions.

I think if you scroll through the set of source frames, what you'll see is that the colored fringes slide around from one frame to the next. There's no really sharp detail for DMap to lock onto, so, at least with default settings, it ends up making decisions based largely on pixel noise or at least on unreliable light/dark patterns. This results in rapid and frequent changes from one source frame to the next, and in combination with the sliding colors, that produces a plethora of sharp edged colored halos.

I would have to play with the source stack to be sure, but my guess is that to get decent results with this subject, you would need to
1. increase the radius settings quite a bit, and
2. position the contrast threshold slider pretty far to the right so that it's only making decisions based on high contrast actual detail.

To be explicit about one other thing... It's always a good idea to compare stacked output against individual source images, to be sure that the stacking process is doing something plausible. After you've had more practice you'll be able to spot artifacts in isolation, but nothing beats direct comparison for revealing problems.

--Rik

Smokedaddy · Post by **Smokedaddy** » Tue Feb 07, 2017 6:40 pm

Thanks for the suggestions. I figured this was to easy. <g> Here's another shot, radius at ER at 70 and CTS around 90 as well. I tried a few different settings too, ER at 40 and 90 as well. I'm not sure why the center seems to be out of focus unless the stack wasn't deep enough?

Smokedaddy · Post by **Smokedaddy** » Tue Feb 07, 2017 6:43 pm