Ok, here we go.

[oxkarthemighty]

Hey folks,
This is my first photo (and I mean first) of my recently purchased BH-2 trinoc that Mr. Krebs graciously helped me to acquire/advise. I would like to thank him, and as well the user APO for the great deal he gave me on the adapter and the eyepiece for it. Thanks a ton fellas!!

I have some questions for those that stack images.
1. How do you clean your sensor to the point when you stack images its fairly clear?
2. Is this image decent for a first go around?
3. Am I expecting too much out of the objectives I have? (they came with it DPlan's. A 10x 0.25 160/.17, 40x .65 160/.17, and 100x 160/.17 oil)
4. I realize that these are darkfield objectives, however they should give me an equal amount of quality between the darkfield and brightfield (other than the oil objective) am I correct in this statement?
*EDIT* This is roughly a 40 image stack, I cant remember.
Original photo


Here is the photo with HEAVY cloning and slight sharpening, as well as some contrast adjustments. (probably a little much)

[Charles Krebs]

This is my first photo (and I mean first)

I think it is a great start...really! You don't say what objective this is, the 40X? Why not provide some subject info as well. A 40 shot stack at this magnification is pretty ambitious for a first shot.

How do you clean your sensor to the point when you stack images its fairly clear?

Welcome to the world of microscopic dust . Small pieces that do not show at all in "normal" photography really show up in microscope images. Cleaning a sensor is a whole science/craft/art-form. There are a variety of methods that work. For me a key requirement is to be able to see the surface being cleaned. This means some type of magnifier, preferably one that has sufficient working distance to permit working while observing. I use a low power stereo scope (because I have one) but there are a goodly number of magnifiers and loupes that are offered in the new "sensor cleaning industry" designed for this task.

A stacking "tip". For microscope images (of stationary subjects) you might try turning off image scaling/magnification and even x/y alignment in the stacking preferences. If your microscope focus stage is in good shape you may not need these adjustments. This at least keeps the dust spot in the same place and can make retouching a little easier at times. (Rik may offer more info on this).

Am I expecting too much out of the objectives I have? (they came with it DPlan's. A 10x 0.25 160/.17, 40x .65 160/.17, and 100x 160/.17 oil)

Well, I don't know what you are expecting . The thing is when you start taking microscope images you need to understand that you are almost always well "into" diffraction territory. I believe you are using a 2.5X NFK photoeyepiece. So with the 40X you are at 100X on camera sensor. At such a magnification you really can't compare image "sharpness" or "resolution" on sensor with photographs made at normal distances. There is no way to avoid diffraction losses when using full spectrum light to take images like this.

Your objectives are good mid-range achromats. I wouldn't lie to you... Olympus S Plan Apo's would often look better. Olympus S Plan Achromats were the next level up, but I am not so sure you would see a big difference there. One of the advantages of the S Plan Achromats is that they provided a larger image circle so they can be used with super-widefield viewing heads and the appropriate eyepieces.

The numerical aperture (NA) is what will determine how much fine detail can be resolved. So if you have two objectives that are of about the same quality level, the one with the higher NA will offer better resolution. So a 10/0.40 objective will resolve more fine detail on the subject than a 10/0.25. (Don't get too excited about minor differences like 10/0.25 compared to 10/0.28, or 20/0.40 compared to 20/0.42). If you look at a manufacturer's objective lines you will usually see that as you go up the price scale, the numerical apertures for a given power increases.

I realize that these are darkfield objectives, however they should give me an equal amount of quality between the darkfield and brightfield (other than the oil objective) am I correct in this statement?

No, these are not considered "darkfield" objectives, they are entirely suitable for a range of illumination methods... but primarily brightfield and darkfield. ( They will not work for phase contrast because you need specialized phase-contrast objectives and condenser for that type of illumination). They will probably work just fine with polarized light as well. (There are "special" strain-free objectives made specifically for very "serious" polarized light users but for most general uses with polarized light plan achromats work well).

One final thought for now. Unless you are using a color balancing filter somewhere, you want to set your camera white balance to "Tungsten" (or 3200k). Then be sure when you photograph you check the voltmeter on the base to insure that the that the light is turned up to recommended operating voltage. Otherwise you will get a strong red/yellow cast to your pictures. If it is really strong you may not be able to fully correct it, but usually you can correct that in software (and if you also shoot a raw file it is easy to correct). Typically in a brightfield shot the background should be white or a neutral shade. The above shot looks a little red/yellow to me. Easy to fix, but better avoided if possible.

[rjlittlefield]

A stacking "tip". For microscope images (of stationary subjects) you might try turning off image scaling/magnification and even x/y alignment in the stacking preferences. If your microscope focus stage is in good shape you may not need these adjustments. This at least keeps the dust spot in the same place and can make retouching a little easier at times. (Rik may offer more info on this).

Sure. There's a long discussion HERE. The short version is just what Charlie says: when shooting through a microscope it's often better to turn off most or all of the alignment options because they're likely to cause more problems than they solve.

--Rik

[oxkarthemighty]

I think it is a great start...really!

Thanks Charles! Before I had my stackshot I used to have to do all my stacking manually (not the best rail OR tripod either), it was incredibly more tedious than the microscope is. If it was my first round to use a manual rail I imagine that my stack would have probably been smaller.

You don't say what objective this is, the 40X? Why not provide some subject info as well.

It was at a 40x as you presumed, the image was of a worker bees leg on a prepped slide set purchased by my wife....great chick.

One final thought for now. Unless you are using a color balancing filter somewhere, you want to set your camera white balance to "Tungsten" (or 3200k). Then be sure when you photograph you check the voltmeter on the base to insure that the that the light is turned up to recommended operating voltage. Otherwise you will get a strong red/yellow cast to your pictures. If it is really strong you may not be able to fully correct it, but usually you can correct that in software (and if you also shoot a raw file it is easy to correct). Typically in a brightfield shot the background should be white or a neutral shade. The above shot looks a little red/yellow to me.

I was wondering why it was looking like I was shooting through urine! I was thinking there was something in the scope that I would have to replace....Good!

I still need to get a darkfield condenser, I am really itching to get some photos in that area. Might be a little since I just rolled all the cash out for this little hobby. Good to know that my objectives are mid range as well...I was hoping that these might last me a little.

Sure. There's a long discussion HERE. The short version is just what Charlie says: when shooting through a microscope it's often better to turn off most or all of the alignment options because they're likely to cause more problems than they solve.

Hey Rik! Thanks for the link, I will be reading it!

Here is another photo of the section of a Tilia stem, shot with the 40x with the color temperature change, the lamp power change, options for the aligning in Zerene off, and I switched to my 5D finding it had less dust as well. I am sooooo much more happier with this result! Few adjustments made in Photo-chop: sharpening, and a little cloning. Any other suggestions I might take?

[rjlittlefield]

This looks much better. I'm glad to hear it's feeling better too.

Few adjustments made in Photo-chop: sharpening, and a little cloning. Any other suggestions I might take?

I'm glad to see that sharpening is in your list. High magnification shots can and should be sharpened much more heavily than low mag work. That's because at high mag, the optics are always getting hit hard by diffraction -- lots of contrast loss for fine detail due to falloff in the MTF curves. You can often make much more detail visible to the viewer by heavy sharpening, and by "heavy" I mean USM at maybe 100% and 2 pixels. The exact values depend of course on the optics that you're using. The goal for using such a heavy and wide USM filter is to prop up the MTF curves so that after sharpening, the overall system MTF becomes a lot more flat instead of trailing off so bad at the end due to uncompensated diffraction.

--Rik

[oxkarthemighty]

Yeah, when I first started up with fixing my 10x Nikon on my Canon I couldn't figure out why my image quality was so poor. Charles had pointed it out that part of it was that I had too high of an expectation from an objective, and you had pointed out that I had needed to sharpen the poo out of my photo. Previously I had been using the 65mm MPE, and required very little. Ever since then, after all my other adjustments I always made sure to sharpen...and even still I probably don't sharpen my photos enough. I without exception, LOATHE noise or any artifacts in my photos; so I tend to brake a little before I apply too much. I suppose that I should just run with it until I get the hang of this.

[Charles Krebs]

Yeah, when I first started up with fixing my 10x Nikon on my Canon I couldn't figure out why my image quality was so poor. Charles had pointed it out that part of it was that I had too high of an expectation from an objective

Hmmm.... don't really remember that . A "direct-projected" 10X objective can look pretty sharp in a camera at 10X. (It is true that direct projection with a 10/0.25 will give about an "effective" f/20 so you are into diffraction, but not all that much). On the other hand, a 40/0.65 on a microscope used with a 2.5X NFK photoeyepiece gives you 100X on sensor, at an effective aperture of (at best!) of about f/77. So there will be a very big difference in the perceived "sharpness" between the 10X and the 100X shot.

[oxkarthemighty]

F/77!! Intense. All the math that is involved in this makes my head go a little loopy. It was with my ant photo that I was speaking of....a PM maybe. I think the next thing I'm going to look into is the scale bars sticky and learn how to work that out! Thanks guys, you all are invaluable!!!

[oxkarthemighty]

Ok ladies and gents, how is this one? Areas I can improve?

28 image stack of Waterthyme leaf, 40x objective
Sharpening, and some light cloning.

[rjlittlefield]

This looks good to me. I have no further suggestions about the image.

F/77!! Intense. All the math that is involved in this makes my head go a little loopy.

The formula for effective f-number is just magnification/(2*NA), so 100/(2*0.65) = f/77. That's with the condenser wide open. If you stop down to say the 70% setting that's often quoted, then the NA drops and the f-number rises proportionally, say 100/(2*0.7*0.65) = f/110. Most of the time there's no need to actually do these calculations, but sometimes they give some helpful insight.

I think the next thing I'm going to look into is the scale bars sticky and learn how to work that out!

There is much good information in the scale bar stickies. However, there is one important piece of advice that tends to get lost: get yourself a stage micrometer. Those devices make life a lot simpler, and I see that as of this morning they're now available new on eBay for under $16 with free shipping. Once you have a stage micrometer, you can use the photo layers technique shown HERE, no math required. See HERE for an application at 40X NA 0.65.

--Rik

[Charles Krebs]

I think this looks great!

The only thing I might quibble about is that to me it looks there is still a little too much yellow in it (the background measures about R:213, G:213, B:170). But at this point it is in the range of personal preference. I can see how the background plays nicely against the blue stain of the leaf, and think many people will like this rendition. My personal choice would probably be to make the background neutral and slightly brighter. This give a more pure, brighter blue, but you do lose the near complementary color scheme.




(I've added these little edited clips of a section of your shots because you have specifically asked for improvement suggestions, and this is in the "beginners" section.
I may be stretching the forum re-posting rule, so if you would rather not have these in the thread let me know and I'll delete them).

[oxkarthemighty]

However, there is one important piece of advice that tends to get lost: get yourself a stage micrometer.

I will be checking this out Rik. I had used the formula from the spreadsheet Charles had on his website from the link within the posting. I will get the micrometer to double check to see if I did it correctly.

The only thing I might quibble about is that to me it looks there is still a little too much yellow in it

I totally agree Charles, I can't seem to get this correct in my camera. I didn't notice this until I looked at it on my iPad. Something must be off on my laptop.....The final image looks really yellow in my opinion. How did you do this in Photoshop? Contrast adjustment, or by adjusting the color balance??

I may be stretching the forum re-posting rule, so if you would rather not have these in the thread let me know and I'll delete them

As far as I am concerned: Any postings that I have my photos in, you can feel free to edit as desired. I am trying to become a better photographer in this area so I totally appreciate the needed help. You won't catch me being a jerk about things (unless someone tries to rip a photo...then of course I would be upset). I will edit my signature reflecting this decision that way any users that would like to contribute by using my images to reflect or propose an idea or technique will be free to do so if this works with everybody.....