First picture with the new Universal

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

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Free2Fish
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First picture with the new Universal

Post by Free2Fish »

Hi folks,

I've just taken this picture of what might be some type of Vorticella, a few algae and loads of bacteria. I'll list some of the relevant information.

Canon 40D
Zeiss Universal
Phase contrast Neofluar 40/0.75
Optovar 2X

The condenser has an NA of 0.63 and will likely be replaced by an NA of 0.90 by this evening. Also the camera adapter does not have enough magnification to properly fill the sensor and I'm looking at doing something with that too.

I'm hoping to add DIC capability sometime but this is what I'm working with for now.

What would some logical next steps be to improve my photos besides better objectives? I've got Elements but haven't touched the photo. I wouldn't even know what to do to improve it with Elements.

Any comments or suggestions would be appreciated.

thanks,
Harry Image

Free2Fish
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Post by Free2Fish »

This is quite disappointing.

I purchased a better microscope in large part because of the helpful response to my student microscope photos on this forum. Since getting my Zeiss Universal I've posted twice; once to show my new microscope and the second time to ask for help in improving a photo.

I've had 51 views and no responses.

Harry

rjlittlefield
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Post by rjlittlefield »

Harry,

So, perhaps 51 people have looked at your image and didn't feel that they could help you. It's not necessarily a bad thing that those people remained quiet.

I represent 1 or 2 of those views. For myself the limitation is that I use full Photoshop, not Elements, so it is hard for me to give advice targeted to your situation.

In general, I will offer that your image has an obvious blue cast and is lacking "snap", by which I mean some combination of contrast and sharpness.

To illustrate, I have taken your image as posted, and applied two changes:
1) "Levels Adjustments" to neutralize the color and increase the contrast, and
2) sharpening via an "Unsharp Mask" filter using parameters 60% and 1 pixels.

Here is the result:
Image

Perhaps someone who knows Elements can comment on how to do something similar in that tool.

I hope this is helpful.

--Rik

Free2Fish
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Post by Free2Fish »

Thanks Rik, clearly an improvement. Both those features are available in Elements and I'll now try to duplicate your results using the old photo. Mine still has a redish cast but I'm getting closer. :)





HarryImage

rjlittlefield
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Post by rjlittlefield »

In my case, the levels adjustment values were:
RGB: 0, 0.61, 206
Red: 0, 1.15, 226
Green: 0, 1.00, 255 (default)
Blue: 0, 1.00, 255 (default)

The values are min, gamma, and max. I have no idea whether Elements uses the same parameterization.

The procedure I used was to display both the image and its histogram, while I adjusted the parameters by hand to get a result I liked. A generally neutral appearance happens when the red, green, and blue peaks in the histogram roughly line up with each other, and none of them is notably above the others in the top half of the histogram. Green and blue were already pretty good, so I adjusted the red gamma and max so as to make red line up also, then adjusted the RGB (overall) gamma and max to give good contrast without blowing out too many pixels.

The starting and ending histograms looked like this:

Image Image

--Rik

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Post by rjlittlefield »

Forgot to mention... I was using a levels adjustment layer, so the modifications were non-destructive and I could keep tweaking them until I was happy. Then before I did the sharpening I duplicated the image layer so I could easily get back to the original there also. Layers are your friends for all this stuff.

--Rik

Charles Krebs
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Post by Charles Krebs »

Harry,

With PS and PS Elements there are often a variety of ways to accomplish the same task.
Mine still has a redish cast but I'm getting closer.
Elements has a very useful "adjustment" (that PS only has in its raw conversion utility). Go to "Enhance", then select "Adjust Color", then select "Remove Color Cast". You will get an eyedropper tool. Click the eyedropper on a part of the picture that should have a neutral color (gray, white). That's it! It should "clean up" the color cast on the shot you re-posted.

I would click it on the near-white area surrounding the Vorticella. (If the area you want to select is too small on the screen, zoom in first).

Also, any time you have an eyedropper tool in PS or PSE it can be set to a "sample size". In PSE I believe the choices are "Point", "3x3" and "5x5". With rare exception it is best to use the 3x3 or 5x5 (pixel) sample size.

Most microscope shots will benefit from some contrast adjustment (Typically use levels or curves. Basic "Levels" using the three sliders is a bit more straightforward. Curves is very powerful but takes a bit of practice and study).

Most will also benefit from some color correction. This can be done with that "Remove Color Cast" option. It is fast and very effective for correcting a "global" color cast. (There are many more complex ways using levels, curves, "color balance", "selective color", "replace color" that you can get into as you gain more experience if you find you need unusually high control over certain areas).

Elements has an eclectic mix of controls and features. In some cases the control is exactly the same as in Photoshop and is very complete and can get quite sophisticated in use (if desired). Sometimes a control seems overly simplified (or not available). It has many choices for "Auto" correction of most image parameters. You can play around with these but they are usually hit and miss as far as doing what is needed. Best to work "manually".

Familiarize yourself with the choices under:

Enhance -> Adjust Lighting
Enhance -> Adjust Color

Since nearly 100% of microscope images will benefit from some sharpening, so also familiarize yourself with:

Enhance -> Unsharp Mask
(Typically this is the last step after image has been adjusted and resized for its intended use).

Free2Fish
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Post by Free2Fish »

Thanks Rik and Charles, this is very useful information and will provide a template for how I can improve my photos. I'm in the process of assembling a "toolkit" with which to move forward within the hobby and would benefit enormously from any advice.

Perhaps it is best to describe where I am now in terms of equipment and experience. I'm relatively new to photomicrography but not new to photography. Years ago I tried PS and after some time trying to learn it, took the advice of of an expert in my hobby (bird photography) and purchased PS Elements. So to my first question; Should I get back into PS or is Elements good enough? I understand any answer will be highly subjective but I am interested in the opinion of those using either program.

The microscope I've purchased has the following Zeiss objectives:

Plan 2.5/0.08
Plan 6.3/0.16
PH1 Plan 16/0.35
Neofluar 25/0.60
PH2 Neofluar 40/0.75
PH3 Neofluar 100/1.30 oil

Also an Optovar unit with 1.25, 1.60 and 2.00.

I have two universal condensers; one with a 0.63 NA condenser and the other with a 0.90 NA condenser. The 0.63 takes much better photos and I'm presently trying to figure out why. I've ordered Klaus Kemp's 8 form test plate to help with this.

My camera is a 40D and is attached to the Zeiss via a Diagnostics Instruments Z Clamp and 0.60 camera adapter. Obviously this will not provide efficient coverage of the Canon sensor. I'm looking for a suitable replacement. And as soon as I can acquire the parts for DIC I'll be jumping into that with both feet.

I'm fairly computer literate and have been reading as much as possible on web sites and in books about this subject (photomicrography). Based on my reading I had initially hoped to find a BHS microscope but this Zeiss Universal fell into my hands first.

With the equipment I have, I'm presently researching oblique and COL contrast enhancement given the excellent results these methods seem to give vis-a-vis phase contrast.

To me, the above photo is the best I can presently do given my equipment, the oblique techniques I've tried and the number of specimens I've dug out of the snow.

With the above in mind, is there a natural next or first step for me to improve my photos while waiting for a DIC setup to materialize?

Thanks for reading all this!!!

Harry

soldevilla
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Post by soldevilla »

Hello. You've touched on a sensitive topic. The image processing is usually the best kept secret of the crown ... because often there is no secret. I often hear questions about a standard process to enhance photographs, and that does not exist. Each image, with its color and noise, need a specific processing. In my version of your image, I have two layers to neutralize the dominant two layers of high-pass filter (I like more that the mask unsharpening; gives me better control over the noise). A layer of contrast adjustment levels and finally a layer of very soft unsharp masking.
but:

a / the process to be followed in each image is dictated by experience.

b / I'm no good treating images ...

No more shortcuts to practice and practice.

regards

Image

Charles Krebs
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Post by Charles Krebs »

Should I get back into PS or is Elements good enough?
I would say stick with Elements for now. For the type of processing you need to do to most microscope images it will be fine.

One valuable thing that PSE lacks is a "Color Balance" dialog box. I could never figure this out. Just about every photo editing program has this. (The closest thing in PSE is "Color Variations" under "Adjust Color". There are other ways this can be done (curves and levels) but that is much more complex when starting out.

One other thing that might be a little frustrating is that PSE is more limited as to what adjustments can be applied as "adjustment layers". (At least this is my recollection, and probably goes back a version or two from the current 11). Adjustment Layers are nice to work with but not essential.

It is really hard to make any suggestions based on a single image. The quality of a photomicrograph of a live subject on a wet mount can vary greatly due to a large number of factors. It would only be after seeing more images that a consistent problem can be seen, or any technical suggestions made.

Free2Fish
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Post by Free2Fish »

Thanks again for the advise. I've still got an old version of PS around; Photoshop 7.0 according to the books in my bookshelf. The version of Elements I've got is about 7 or 8 years newer...Elements 8.0.

I still plan to experiment with all the methods of oblique illumination and will now also try to develop more skill with using Elements.

Must say I'm still enjoying this immensely and can hardly wait for the spring landscape to offer up more specimens. :)

Harry

ChrisJones
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Post by ChrisJones »

I have Elements 8.0 at home and Photoshop CS6 at work. I prefer the full version of Photoshop in general, but it's hard to judge what features Elements lacks as Adobe seem to have put a lot of effort into arranging the interfaces differently. I'm pretty sure though that you can't auto blend a stack of images to increase depth of field in Elements though.

However, for single shots like this Elements is fine. I've had a go at processing your image, here's what I did:

- Increased sharpness (1px, 150%)
- Lightened shadows by 10%
- Adjusted levels using an adjustment layer - auto worked nicely

I then applied two Hue/Saturation layers, which may not to be to everyone's taste:

- The first reduces the saturation of every colour except for green and yellow across the whole image. I did this to remove any colour that didn't look like it should be in the image.
- The second boosted the saturation of green and yellow a bit.

Image

Free2Fish
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Post by Free2Fish »

Thanks for your efforts Chris. More improvements, this time with a somewhat different method. To me this illustrates both the strength of these products and the frustration I sometimes feel with them. There seem to be so many different ways of achieving the same effect, a beginner like myself gets somewhat intimidated, even with the "simple" product.

If I could ever locate a local "PS for the Microscope" course, I'd likely invest in Photoshop again but until then I'll stumble along trying to learn Elements.

Harry

ChrisJones
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Post by ChrisJones »

I started off with Elements soon after I got my first DSLR a couple of years ago and use it as a matter of course for "normal" photography. The basics are similar for any type of photography and definitely worth learning - I haven't yet come across an image taken by a DSLR that can't be improved in some way.

I did a short Open University course when I was starting out that helped immensely. Alternatively, there's a lot of stuff on YouTube for both Elements and full PS.

I'd advise learning one at a time though - even though you do the same things, they're often done differently in Elements and PS. I don't really see why they should be set up differently, but they are.

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Post by ChrisR »

The "Elements" I saw last had "Levels" but I couldn't find a Photoshop-style "Curves", which would be a sad omission. Is it included?

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