Lighting for macro photography of fern gametophytes

Lou Jost · Post by **Lou Jost** » Tue Dec 08, 2015 4:59 pm

Maybe gelatin would work for you. All I can say is that even solid marble is non-rigid when doing high magnification microscopy, so I am skeptical about gelatin. But what is rigid and what is not rigid depends on the magnification and the frequency of the environmental vibrations, so I guess there's a chance gelatin would work in your situation. I wouldn't bet on it though.

It's definitely worth watching your Live View monitor (at its highest magnification, not at full view) as you touch stuff in your setup. The dancing pixels will give you a feel for what is and what is not rigid there.

ChrisR · Post by **ChrisR** » Tue Dec 08, 2015 6:01 pm

Dentist's wax is a possible. It softens in your fingers enough that you could get a leaf to stand up in it, then when it cools it's still quite soft, but definitely a solid. Cheap on ebay. My dentist gave me some for swapping a tap washer...

Chris S. · Post by **Chris S.** » Tue Dec 08, 2015 7:16 pm

Or perhaps modeling clay?

(I was surprised by the phrase "expensive agar." Some time ago, I used agar at home to culture fungi, and in those days, agar wasn't expensive. But I see, in an interesting Nature article published just today, that there is an agar shortage and the price has skyrocketed.)

--Chris

jsp · Post by **jsp** » Wed Dec 09, 2015 12:43 am

Thanks, I'll look into these options. I was also very surprised by the price of agar. The nutrient mix and fungicide are very expensive too, and I don't have autoclaving facilities, so that is out.

Chris S. · Post by **Chris S.** » Wed Dec 09, 2015 1:16 am

jsp wrote:I don't have autoclaving facilities, so that is out.

I did my autoclaving with my grandmother's pressure cooker. Worked very well! For fungi substrate back then, the recommended protocol was to pressure-cook the substrate, wait a few days without opening the pressure cooker, and then pressure cook again. The idea was that the second pressure cooking killed bacterial spores that survived the first pressure cooking, and then sprouted--at which point they were easily killed by the second round of pressure cooking.

-Chris

jsp · Post by **jsp** » Wed Dec 09, 2015 3:20 am

Hi Chris S. Normally I would consider that, but I'm permanently frazzled, accident prone, and looking after a five year old, so I think pressure cookers and microbe culture are probably best left for another year. :-)

jsp · Post by **jsp** » Sat Dec 26, 2015 9:50 am

Hi,

I have a tiny bit of progress to report. I'm still working on the higher dpi scanner, but it's proving tricky because it has a different kind of stepper motor.

While I'm doing that I decided to retry the coin test on the older, low dpi scanner to see if the steps are small enough to accommodate the tiny depth of field of the MP-E lens on x5 magnification.

For my Christmas I was given two Yongnuo flashes and a polaroid filter, so I used those too, and took the shots on 1/250th exposure to remove motion blur.

The images were stacked using Helicon focus. There were 45 slices.

I wondered if anyone with a more experienced eye than mine might be able to tell from this photo whether the steps of the scanner are small enough? When I look at the slices it seems as if there is a large overlap between the focussed area of one slice and the next.

Thanks for looking, and Merry Christmas!

Jen

jsp · Post by **jsp** » Sat Dec 26, 2015 10:26 am

This is what one slice looks like:

I'm trying to upload the next slice but getting web errors. I'll keep trying.

It seems to me that there is plenty of overlap between the focused areas of two consecutive slices.

I haven't tried with a big base board and rubber feet yet. I set the camera to mirror lock but when I tried to trigger the shot using the EOS software over USB nothing happened, so I went back to using the infra red remote control with the live view turned off, so I don't think I was using mirror lock.

jsp · Post by **jsp** » Sat Dec 26, 2015 10:27 am

Here we are:

This is the next slice along:

jsp · Post by **jsp** » Sat Dec 26, 2015 12:11 pm

I had a go at a fern gametophyte and it comes out at a decent size with the x5 lens.

This is a bit of a mess, but it might be better once I figure out how to trigger the camera remotely with mirror lockup, and get the flash to fire. Currently I can only get the flash to fire if I press the button on the actual camera.

jsp · Post by **jsp** » Sat Dec 26, 2015 12:50 pm

Just out of interest, here is the 5x MP-E lens with some extension tubes added. I was photographing the top of the "n" on my Canon lens cap upside down, so it looks like the bottom of a "u".

the First the MP-E at 5x:

jsp · Post by **jsp** » Sat Dec 26, 2015 12:52 pm

Then the same with the MP-E x5 and 105mm of extension tubes (mix of Canon and 3rd party):

jsp · Post by **jsp** » Sat Dec 26, 2015 12:55 pm

and this is the MP-E at 5x with 40mm of Canon brand extension tubes:

So it seems as if extension tubes are a definite possibility for increasing the magnification of the fern with this lens.

Charles Krebs · Post by **Charles Krebs** » Sat Dec 26, 2015 2:00 pm

Do you recall the aperture used and the number of steps? I can't see any focus banding in the first image just posted, but you should look at it at 100% in Photoshop or some other image program. If, as you previously stated, you can get 42 microns per step, I think you will be OK with one step at 5X with the aperture at f/4

The "contours" in the out-of-focus background could be the result of slightly differing exposures (not that unusual when using flash). Be sure to have the "Brightness" box checked in your "Autoadjustment" tab in the Helicon preferences.

For my Christmas I was given two Yongnuo flashes and a polaroid filter, so I used those too, and took the shots on 1/250th exposure to remove motion blur. ....

This is a bit of a mess, but it might be better once I figure out how to trigger the camera remotely with mirror lockup, and get the flash to fire.....

I set the camera to mirror lock but when I tried to trigger the shot using the EOS software over USB nothing happened, so I went back to using the infra red remote control with the live view turned off, so I don't think I was using mirror lock.....

The fastest flash sync speed with the Canon 5DII is 1/200. I would not try to use a shutter speed faster than that with "regular" flash units. The motion/vibration "freezing" ability comes from the duration of the flash burst, not really the shutter speed used. (But use a fast sync speed and keep the continuous ambient light level relatively subdued to avoid "ghosting" due to the continuous light adding something to the image.). Do you know what power level the flashes were set for? As the manual power settings are reduced, the duration ("on time") of the flash burst get very short and effectively stops motion to a very high degree.

You cannot fire a non-Canon (or non-Canon dedicated) flash from live view when the camera is set to use Mode 1 or Mode 2 "Silent Shoot" in the Live View/Movie func. set. menu. In that menu set "Silent Shoot" to off if you wish to use a non-Canon flash from live view. (However... if you are using continuous lighting from "live-view" you definitely want to set Silent Shoot to "Mode 1 or Mode 2 from that same menu).

If you are using electronic flash for lighting, and you don't need to use live-view during stack acquisition, you should set C.FnIII-6, mirror lock-up to 1: Enable. Then when you a picture you need two shutter "presses"... the first flips the mirror up, the second actually takes the picture (pause briefly after the first press to let vibration dampen out).

Try a little more lighting diffusion for subjects like the lens caps, it will cut back the colored specular highlights significantly.

ChrisR · Post by **ChrisR** » Sat Dec 26, 2015 4:37 pm

Extension tubes will give you more magnification, but they will also give you a smaller effective aperture, so more blur from diffraction.
At M=5, the effective aperture is
(5 + 1 ) x 2.8 = 16.8, which is small enough to affect sensors which have small pixels.
At M=7 you'd get eff/22.4, and so on.
What that means, is that the extra magnification is likely to be "empty".
If you use a close-up lens/dioptre instead, you don't reduce the effective aperture (ie make the number bigger) with more magnification, so that's a route worth trying as well.
Iirc Brian Valentine (LordV) showed an example a while ago.

Both methods will reduce your DOF, but the dioptre more than the extension tube.

The effect of a small source for the illumination also has the effect of reducing the effective aperture. It's worth avoiding, at this scale.