# Proof of concept: UV-reflectance stacking with MP-E 65



## Zeidora (Dec 9, 2016)

This right up front, this is not a great picture, it is a proof of concept, to be refined.

I am interested in UV reflectance photography and to see whether there are insect patterns not visible in "normal" light on some minute orchid flowers. The flower is 1.76 mm (no, not a typo) wide. Shot with full-spectrum modified 5D2, MP-E 65 at f/2.8, manually stacked 10 frames, Baader UV-pass visible opaque filter, ISO 400, about 4 second exposure, illuminated with single UV 365 nm LED flashlight. 

First off, the MP-E 65 DOES transmit some UV. That's already great. I also have a Nikon EL 80/5.6 but still waiting for some parts to arrive to put it all together. Should have much better UV transmission, so will be interested to see the differences.

Further refinements are additional UV flashlights (can't diffuse them because of absorption and fluorescence issues), and stacking with StackShot (have that).

Some single shots on some other orchid flowers do show some different patterns, so that works as well. The dotted highlights, those are the individual cells, with their rounded surfaces (verified by scanning electron microscopy). In visible light, the flower is uniformly green.


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## Maximilian (Dec 9, 2016)

Hey that's great!

Although I am personally not interested in doing something like that myself, I know about this UV insect patterns and I'd like to see more if you continue working on that. 

Thanks for sharing and good luck!


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## tpatana (Dec 9, 2016)

Very interesting. Please keep posting more.


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## LordofTackle (Dec 9, 2016)

Amazing! You are better equipped than my old lab


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## lion rock (Dec 9, 2016)

It is amazing to try unusual techniques on everything. This opens up another avenue of opportunities.
Glad you've started this method. Please keep perfecting the lighting and send us samples of the various "targets."
We do have to innovate!
-r


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## Zeidora (Dec 9, 2016)

Glad this generates some interest. I will post some more as they come along. Loads of fun. And may even get a paper out of it


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## LordofTackle (Dec 9, 2016)

Zeidora said:


> Glad this generates some interest. I will post some more as they come along. Loads of fun. And may even get a paper out of it



May I ask what you do for work? 
Sounds like biologist/scientist.

And keep the good work coming 

-Sebastian


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## Zeidora (Dec 9, 2016)

LordofTackle said:


> Zeidora said:
> 
> 
> > Glad this generates some interest. I will post some more as they come along. Loads of fun. And may even get a paper out of it
> ...



I am a natural history museum curator, officially for marine invertebrates, but lately I've broadened a bit into orchid systematics. I also run the museums scanning electron microscopy facility, and am the go-to person for any odd scientific imaging needs. Apart of the SEM, all the photo stuff is personal. It would be foolish to ask the museum to buy a $400 filter for me. So I just get that myself. I enjoy an imaging challenge, particularly in the macro realm. Like using cross polarization to image harpsichord strings for a multimedia presentation of a baroque orchestra. Or getting z-stacking going on my compound microscope.

So Ph.D. in marine biology, and a bunch of classes in scientific photography in dept phys chem at the University of Basel, Switzerland (Gschwind and Heilbronner were great). Then building on that.


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## LordofTackle (Dec 9, 2016)

Thanks for the answer. Sounds like a really cool job and it must be very nice to be able to combine job and passion that way. 

And I have to ask: is Heilbronner a person? Cause I am from the (german) city Heilbronn....^^


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## Zeidora (Dec 9, 2016)

LordofTackle said:


> Thanks for the answer. Sounds like a really cool job and it must be very nice to be able to combine job and passion that way.
> 
> And I have to ask: is Heilbronner a person? Cause I am from the (german) city Heilbronn....^^



Renée Heilbronner was one of the two instructors of the various classes. Gschwind at some point got a position in Zürich, not sure about Heilbronner. It's been a mere 20+ years! 

I know Heilbronn, but mainly for the traffic jams on the Autobahn ;-) 

And, yes, I'm darn lucky having this job. Curator jobs are pretty impossible to get. Best career advice I got many moons ago was "Mann, vergiss es!" [Dude, forget about it]


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## LordofTackle (Dec 9, 2016)

Zeidora said:


> I know Heilbronn, but mainly for the traffic jams on the Autobahn ;-)
> 
> And, yes, I'm darn lucky having this job. Curator jobs are pretty impossible to get. Best career advice I got many moons ago was "Mann, vergiss es!" [Dude, forget about it]



Ad it's gotten waaaay worse since then 
Seriously, that highway square between Karlsruhe, Heidelberg, Heilbronn and Stuttgart is a major PITA

I'm currently in Konstanz, were I studied...biology 
also not that far Basel


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## Zeidora (Dec 25, 2016)

Here's another installment in my project. Today it's mainly about post-processing. Still not great photographs, but this is about getting technique sorted out.

One problem is that during camera conversion, some of the Bayer filter elements most likely got damaged. This is no big deal for single captures, but if you stack 50 or more, one gets a lot of "creative" color streaks. That leads to a lot of post-processing after stacking. I thought of doing a dust delete data in the 5D2 body, but that does not work, because during conversion, the dust shaker hardware is removed, and without it, the dust delete data cannot be obtained. 

But because these color dots are in fixed positions, they can be screened out with a Gaussian blur mask. Do the following in Affinity Photo:
- Open a dozen images in AP.
- Add a Gaussian Blur live filter on top of the first image.
- Add a mask to the Gaussian Blur live filter. Blur of about 2.5 pixels worked well for me, but that depends a lot on your camera and the type of Bayer damage you have.
- Fill mask layer only with black.
- Paint with a small white brush (4 pixels worked well for me) over any color spots in the mask layer. Do that at 150-300%. You'll spend some quality time at this. Don't bother fix those color spots right at the edge of the frame. The Gaussian Blur from the edge of the image is going to mess it up.
- Once done with first image, copy-paste Gaussian blur filter with Mask into next source image.
- Check for pesky color spots in second image and refine mask.
- Copy-refine for the the rest of the images.

Open one of the CR2 source images, develop it. Now start macro recording and paste the final version of the Gaussian Blur live filter with mask on top of the image layer. Stop macro recording and save it as something like "UV prep". The cool thing in AP is, that the live filter with mask is saved in the macro step. So the macro does not paste from computer memory, but the stored filter-mask. That means even if you quit the application, the Blur-Mask is still stored. Super cool.

In Open-batch, select the Macro, select your output format (e.g., tif for Zerene), add files, and press OK [Another cool thing in AP, saving/export is not part of macros, but is in the batch dialog. No more infuriating confusions about save functions as in PS]. I am extremely impressed with AP batch processing. All 12 cores on my MacPro desktop go continuously full throttle (100%) for several minutes. I have not seen that with any other application. They really program the batch processing well.

I also tried APs focus merge function, and am quite impressed. PS is worse than useless for serious macro stacking, so most people use either ZereneStacker or HeliconFocus. AP is quite impressive, but Zerene still seems to have the edge. I have not found a way to touch up the final stack layer by layer, as you can in ZS or HF. If I stack with the color artifacts still in place, AP has trouble with proper alignment, while Zerene nails it. Last but not least, tone mapping is much better in Zerene than in AP. This could be fixed before feeding the files into AP.

One problem with APs tif export is, that Zerene only reads them as B&W file (or single channel?). Feeding the same files into AP gives me a color image. Not sure yet what is going on there, but am reasonably sure that I can figure that one out.

Attached four images.
- Stacktrail Zerene. Notice the color streaks, but clean edges of flower and good tonality straight out of stacking.
- Stacktrails AP. Notice color streaks, and edge of flower is blurry (alignment issue) and image is overexposed.
- AP stack: stack of images after batch processing with Gaussian blur mask in AP. Flower edges are clean, but still a bit over exposed. It seems that AP-alignment is misled by color artifacts.
- B&W Zerene: Cleaned tifs stacked in Zerene, resulting in B&W image (hmmm ....)

Enjoy!

For the botanists, it is Barbosella duesenii


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## Zeidora (Dec 25, 2016)

Just noticed, the stack trails are not showing well. So here's a 100% crop from the Zerene file.


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## Zeidora (Dec 25, 2016)

Before I forget, I also thought about reflectors for UV photography. The typical paper reflector does not work, because of UV brighteners in most paper that fluoresce the UV back in as blue light. Plastic does not work, because plastic absorbs UV readily. However aluminum foil works very well.

just 2 more c.


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## Zeidora (Dec 25, 2016)

One more thing, these latest images were taken with a Nikon EL 80 mm f/5.6. It has about 10x/3-stop better UV transmission, and the MP-E 65 seems to cut off at a longer wavelength. I would have expected worse transmission by the MP-E 65. With bellows and tubes, I can get to about 4:1 magnification.


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## Zeidora (Dec 26, 2016)

Here's now one that I am reasonably happy with. Oberonia costeriana. Flowers are about 2 mm long.

First image is a visible light stereomicroscope z-stack.
Second is the UV version. No striking patterns, but that is information as well. 
The last small methods adjustments include:
- RAW development in DxO.
- Removing color spots in AffinityPhoto. Note that DxO tifs required a new Gaussian Blur mask. Seems that the different RAW developer engines are putting color spots in slightly different places.
- Run AP cleaned-up tifs through DxO once more, so that Zerene will see them as a color file.
- Run stack through Zerene.

Now that methods have been sorted out, the real fun begins. Off to the greenhouse ...


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## zim (Dec 26, 2016)

Hi Zeidora,
Fascinating stuff really enjoying reading thanks for posting. You should post the same on AP forum I'm sure it would be well received and it would help the devs as I guess there won't be too many beta testers around doing this kind of detailed work!

Regards


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## Zeidora (Dec 26, 2016)

Zim: thanks for the flowers  Posted over in AP forum and also on the Photomacrography forum.


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## Maximilian (Dec 27, 2016)

Zeidora said:


> Here's now one that I am reasonably happy with. Oberonia costeriana. Flowers are about 2 mm long ...


Really fascinating, Zeidora.

If I can recall it right, those UV patterns sometimes look even more geometrical.
I wonder if it's just because these flowers are just so small? 
Or are geometrical patterns not so common?

Do you know more about that?


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## danski0224 (Dec 27, 2016)

Have you considered trying the Coastal Optics UV IR VIS macro lens?


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## Zeidora (Dec 27, 2016)

danski0224 said:


> Have you considered trying the Coastal Optics UV IR VIS macro lens?



The Coastal optics lenses cost >$5K each, while the Nikon EL 80/5.6 was $40. The UV Nikkor is about $10K, if you can find it anywhere. I have not seen any comparison on the optical performance at >>1:1 of the Coastal Optics or the UV Nikkor lenses to enlarger lenses. Given the rather experimental nature of my project, I am not quite ready to drop 5-10K on it.


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## danski0224 (Dec 28, 2016)

Maybe the Coastal lens can be rented in your area. Lensrentals has it in the USA.


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## Zeidora (Dec 28, 2016)

Danki: have you tried/compared the Coastal optics in for that purpose? Lens rental wants $226/week for it; I do live in the US. If I feel the need, I will buy it, but not quite there yet. Some 20 years ago I used the UV Nikkor at University, but for a UV fluorescence class assignment, which is a waste of that glass. Oh, the follies of youth.

Also, solved the Zerene file reading issue. Had to uncheck the "use external tif reader" in the Zerene Preferences. Rik L. kindly suggested that on the Photomicrography forum.


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## danski0224 (Dec 28, 2016)

Zeidora said:


> Danki: have you tried/compared the Coastal optics in for that purpose? Lens rental wants $226/week for it; I do live in the US. If I feel the need, I will buy it, but not quite there yet. Some 20 years ago I used the UV Nikkor at University, but for a UV fluorescence class assignment, which is a waste of that glass. Oh, the follies of youth.
> 
> Also, solved the Zerene file reading issue. Had to uncheck the "use external tif reader" in the Zerene Preferences. Rik L. kindly suggested that on the Photomicrography forum.



Nope, I haven't tried that lens. I have a Sigma camera that will do full spectrum (no mods other than to remove the dust protector), but it's challenging to get Nikon lenses to work with it, much more difficult than the easy adapter for Canon. Right now there is only one Sigma DSLR camera with live view (and EVF), and that is the new sd series.

Having a Canon converted to full spectrum has crossed my mind, but it would have to have live view. An EVF would also be nice, which points at the latest M. I am near a point where I would consider renting one that has been converted to see if I like it.

$226 seems like a bargain rental to see what the Coastal lens can do.

I saw a UV Nikkor on eBay not too long ago for just under $10k. Haven't used one of those, either.


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## scyrene (Dec 28, 2016)

I'll concur: fascinating! I hope you continue to progress on this


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## Zeidora (Dec 29, 2016)

danski0224 said:


> I have a Sigma camera that will do full spectrum (no mods other than to remove the dust protector), but it's challenging to get Nikon lenses to work with it, much more difficult than the easy adapter for Canon. Right now there is only one Sigma DSLR camera with live view (and EVF), and that is the new sd series.
> 
> Having a Canon converted to full spectrum has crossed my mind, but it would have to have live view. An EVF would also be nice, which points at the latest M. I am near a point where I would consider renting one that has been converted to see if I like it.
> 
> ...



There are M42 to Sigma adapters, eg. http://www.adorama.com/kasgpxs.html No problem putting a Nikkor EL on a Sigma. Live view/EVF, though, is almost a must for z-stacking. Allegedly, the EL80 has relatively little focus shift in UV, but screwing on a filter after you adjusted a >>1:1 is asking for trouble.

Re renting a Coastal lens, what I do is ongoing. If I get one, I buy it. Rental would just be paying $226 more in final purchase price.


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## Zeidora (Dec 29, 2016)

Maximilian said:


> Zeidora said:
> 
> 
> > Here's now one that I am reasonably happy with. Oberonia costeriana. Flowers are about 2 mm long ...
> ...



Sorry, missed that. The "why" question is the whole point here, and goes towards my botanical research. Photography is just a tool to investigate the biology of those flowers. Not sure whether there is a size correlation to UV patterns. Yellow sun flowers are famous for their concentric rings in UV. I've seen some lines on a yellow pleurothallid orchid (also relatively small flowers, also many flower on inflorescence). Current working hypothesis is that pollinator is not visually oriented, but rather attracted by odor. That could fit the limited observations on fungus gnats, and one species at least smelling like fungus (Oberonia fungum-olens).

To get a reasonably founded answer will take years. Research is painfully slow.


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## Maximilian (Dec 29, 2016)

Zeidora said:


> Maximilian said:
> 
> 
> > Zeidora said:
> ...


Thanks for your reply.


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## scyrene (Feb 17, 2017)

I'm currently sort-of experimenting with this myself. I don't have a modified camera or proper filter, but I've found the results with an astro light pollution filter quite good even if I'm crossing streams, so to speak  Here's my first direct comparison (few flowers here at this time of year!), it's the rear underwing of a swallow-tailed moth, Ourapteryx sambucaria, in visible vs filtered UV light. Focus stacks using the MP-E and Affinity Photo. Surprisingly few moth specimens I've tried show much in UV but this one was interesting enough to persist with.


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## Zeidora (Feb 17, 2017)

scyrene said:


> I'm currently sort-of experimenting with this myself. I don't have a modified camera or proper filter, but I've found the results with an astro light pollution filter quite good even if I'm crossing streams, so to speak  Here's my first direct comparison (few flowers here at this time of year!), it's the rear underwing of a swallow-tailed moth, Ourapteryx sambucaria, in visible vs filtered UV light. Focus stacks using the MP-E and Affinity Photo. Surprisingly few moth specimens I've tried show much in UV but this one was interesting enough to persist with.



Neat. I think what you are doing is essentially a blue channel image. There are some faint yellow lines visible in the first image, and those are black in the second. Yellow = no blue, so those yellow areas will be dark. Compare the blue channel information from the first image with the second, and I would think they will be very similar.

To image UV light, you cannot get around a modified camera body.


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## scyrene (Feb 17, 2017)

Zeidora said:


> scyrene said:
> 
> 
> > I'm currently sort-of experimenting with this myself. I don't have a modified camera or proper filter, but I've found the results with an astro light pollution filter quite good even if I'm crossing streams, so to speak  Here's my first direct comparison (few flowers here at this time of year!), it's the rear underwing of a swallow-tailed moth, Ourapteryx sambucaria, in visible vs filtered UV light. Focus stacks using the MP-E and Affinity Photo. Surprisingly few moth specimens I've tried show much in UV but this one was interesting enough to persist with.
> ...



If I follow you right, you're saying that desturating the green and red channels in the visible light image should give the same result, is that correct? I don't think that can be quite right. As a comparison, using this technique made green leaves bright red. That would not be the case if I did what I think you are suggesting.

I agree a modified camera must be better, but UV filters are sold for a reason - and not just to protect the lens's front element! The sensor must be sensitive to some UV, or otherwise to fluorescence in UV? I'm no scientist, so apologies if I'm way off


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## Zeidora (Feb 17, 2017)

You're right, something else is going on. So most likely fluorescence, i.e. UV light is shifted into visible light wavelength.

All sensors are UV sensitive to around 320 nm, but there is filter in front of the sensor that cuts off UV at around 400-420 nm. That filter is taken off when a camera is converted, and replaced with a UV transparent filter. In order to take UV images, you need to exclude all visible light, and also the IR. Sensors are less sensitive to near UV than to visible light, so you need to exclude ALL visible light. If you look through your filter against a bright light source and you see anything, then it is not suitable for UV reflectance imaging. Hope that helps a bit.


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## scyrene (Feb 19, 2017)

Zeidora said:


> You're right, something else is going on. So most likely fluorescence, i.e. UV light is shifted into visible light wavelength.
> 
> All sensors are UV sensitive to around 320 nm, but there is filter in front of the sensor that cuts off UV at around 400-420 nm. That filter is taken off when a camera is converted, and replaced with a UV transparent filter. In order to take UV images, you need to exclude all visible light, and also the IR. Sensors are less sensitive to near UV than to visible light, so you need to exclude ALL visible light. If you look through your filter against a bright light source and you see anything, then it is not suitable for UV reflectance imaging. Hope that helps a bit.



Thanks for clarifying  I wonder what the precise cutoff for the in-camera UV filter is? My light source is rated at 395nm so very close, but just under 400. I guess I'll have to get an appropriate filter and see if anything is visible in just that light. I rather like some of the images I've taken with the current setup, however, so maybe it's worth pursuing for its creative merits in any case. Thanks!


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## Zeidora (Feb 19, 2017)

395 nm is very close to visible. It is essentially the "Disco" light for glowing cloths. There are 365 nm flashlight, and I am currently working on modifying a flash for UV photography: remove yellow plastic UV filter, and replace with UG11. If you are interested in this, look at the http://www.ultravioletphotography.com/content/ forum. UV is hazardous, so take appropriate precautions.


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## Zeidora (Dec 31, 2017)

Here's am update on my experiments, with which I am quite happy.

Set-up: Canon 5D2, Lens Nikon EL 80 mm f/5.6, Baader U-Venus filter on lens, cheap Russian M42 bellows in between; various adapters all throughout. Illumination with Metz 45 CT1, removed plastic front shield and replaced with cheap Chinese UG11 equivalent filter. Aluminum foil on opposite side brightens right side of image. Metz is triggered via FlashZebra slave, with Canon MT26 EXRT connected to 5D2, firing single head at 1/512 power. The whole rig on Cognisys Stackshot. Have to put 10s firing delay in program, so that the flash can re-charge between firings. Flash head gets quite warm after 60-100 shots.

Processing through DxO with Premium noise reduction removing all color artifacts. No more messing with gaussian blur masks in AffinityPhoto. Easier, but takes a lot of time on computer. Then in AffinityPhoto, steepened a and b curves in Lab space quite bit (lower corners moved halfway to middle, so doubled slope). Plus a bit of fine tuning so that black background is neutral in color.

Biggest problem is low power of flash. Have to go to ISO 400-1600 at full power of flash to get enough UV through the system. May get two Metz 60 flashes, so that should get me in ISO 100-400 range.

Still no pattern on flowers, but there is some differentiation between flower and rachis. About 2.5:1, some 70 frames at f/5.6 and 100 µm steps.


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## danski0224 (Dec 31, 2017)

Wow, pretty darn impressive.


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## privatebydesign (Dec 31, 2017)

So you are using a $1,000 flash as an optical triggering device to trigger an underpowered $20 flash? I don't see that as making any sense, why not just plug the Metz into the PC port on the camera?

But a simple 'studio' light is going to give you way more bang for the buck anyway and get you to the power output you need rather than mess around with AA batteries.


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## NancyP (Aug 7, 2018)

Very cool. What orchid? The local terrestrial orchids (Spiranthes) tend to be of this size or a little larger (2 to 3 mm at most). https://en.wikipedia.org/wiki/Spiranthes

Also, do you have a recommendation on a 365 nm UV flashlight? 



Zeidora said:


> This right up front, this is not a great picture, it is a proof of concept, to be refined.
> 
> I am interested in UV reflectance photography and to see whether there are insect patterns not visible in "normal" light on some minute orchid flowers. The flower is 1.76 mm (no, not a typo) wide. Shot with full-spectrum modified 5D2, MP-E 65 at f/2.8, manually stacked 10 frames, Baader UV-pass visible opaque filter, ISO 400, about 4 second exposure, illuminated with single UV 365 nm LED flashlight.
> 
> ...


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## Jim Saunders (Aug 7, 2018)

NancyP said:


> Also, do you have a recommendation on a 365 nm UV flashlight?



Spectroline makes good ones. They charge accordingly of course, but they're available filtered pretty tightly around 365nm for use in an industrial field called non-destructive testing.

Jim


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## Zeidora (Aug 8, 2018)

danski0224 said:


> Have you considered trying the Coastal Optics UV IR VIS macro lens?



Bought the 105 Coastal recently and am currently working out some protocols. I also upgraded to a Paul C Buff white light 1600 with uncoated, UV-emitting tube. Now I can keep ISO at 100 with Baader U. The problem is that the heat blast wilts the flowers during the stacking sequence. Have to see whether greater distance but higher power will be better than close distance low power shooting. Still a work in progress ...


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## Zeidora (Aug 8, 2018)

privatebydesign said:


> So you are using a $1,000 flash as an optical triggering device to trigger an underpowered $20 flash? I don't see that as making any sense, why not just plug the Metz into the PC port on the camera?
> 
> But a simple 'studio' light is going to give you way more bang for the buck anyway and get you to the power output you need rather than mess around with AA batteries.



The problem is the sync voltage of the Metz, which will fry the 5D electronics. At any rate, now have a PC Buff white light 1600 so can radio control that.


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## Zeidora (Aug 8, 2018)

NancyP said:


> Very cool. What orchid? The local terrestrial orchids (Spiranthes) tend to be of this size or a little larger (2 to 3 mm at most). https://en.wikipedia.org/wiki/Spiranthes
> 
> Also, do you have a recommendation on a 365 nm UV flashlight?



It is an epiphytic tropical orchid in the genus _Oberonia_. Do some systematic/phylogentic work on that genus, and got interested in the pollination biology because everybody asks me that question.


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## cactuspic (Aug 24, 2018)

Zeidora, you might want to check in with Dr _Klaus Schmitt_ who has done a substantial amount of UV macro work. He knows which macro lenses transmit UV, although I see that you purchased the Coastal. I also had several thoughts. The best lens I know of in the 1x-3x range is a lens taken from the Minolta Dimage 5400 film scanner. Like enlarging lenses, it is not the sort of lens that would come into contact with UV and therefore it may not have any coating that would hinder UV transmission (but I did not test). 

I do a great deal macro work with plants in the studio. The greatest boon to my photostacking has been continuous light LEDs (no heat). I have not checked as to whether they have UV LED panels in the wavelengths you need but they may. I did a brief internet check, and it seems there are a bevy of products in the the various UV wavelengths for a number of industrial uses. That way you won't have to blast the full spectrum Bluffs to get sufficient illumination.

Keep up the good work.


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## Zeidora (Aug 24, 2018)

I've been in contact with Klaus before, know his very useful website. There is also the ultravioletphotography forum. Thanks for the pointers re UV LEDs. As far as i understand UV LEDs, they are very narrow in terms of wavelengths, which result essentially in monochromatic (B&W) UV reflectance images. I am not aware of full spectrum UV LEDs (say 320-400 nm). I think I rather have to be careful in re-calculating DOF and step size, and play with distance vs output of flash unit, or find sweet spot with ISO setting. A UV pass IR block filter may be the holy grail, but not aware of one, and at the size I need it would cost a decent fortune. UG11 is blocking visible, but not IR, and then is also attenuating UV. Tried that with the Metz.

The scanner lens is an interesting idea. But first I'll fiddle a bit more with the Coastal.


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