Some fluorescence shots

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 8:28 am

I'm surprised there aren't more fluorescence photos posted here. It produces very appealing images. Last year, after acquiring an FITC filter cube for next to nothing I made a simple attachment for my microscope out of legos and sheet metal (see last photo). A $5 3w LED flashlight lightsource. It can accept any manufacturer's filter cubes.
House plant leaf showing multiple stomata

fly eye

jade plant chloroplasts

Cactusdave · Post by **Cactusdave** » Thu Mar 15, 2012 8:44 am

Very creative, but be aware that it could be throwing out significant amounts of eye harming UV. Presumably you are using the camera LCD to focus as well as record images? Normally fluorescence microscopes have a UV barrier filter to keep UV away from the eyepieces, and some sort of UV shield around the objectives to stop direct viewing of the short wavelength illumination.

Litonotus · Post by **Litonotus** » Thu Mar 15, 2012 10:36 am

great and very creative (: I used legos (mostly technic) for many things too (:

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 12:57 pm

Thank Dave, yes I'm aware of the dangers of UV light but this is a white LED. Xenon & Mercury (XBO & HBO respectively) short arc bulbs are used in production fluorescence scopes. The light from these lamps is dangerous.

Litonotus, while I was building with the legos I was thinking how useful a material they are for precision building. They have near perfect x y z measurements and click into place. Can't beat it.

Mitch640 · Post by **Mitch640** » Thu Mar 15, 2012 1:26 pm

Dave. I don't think a 3W LED flashlight is going to burn eyes. I have one with 51 bulbs in it and doesn't seem dangerous. It won't work as a light source for the Fluophot either.

Curt, did you need dyes? I would have thought that the dyes would be the hard part to acquire.

The images came out very nice.

Cactusdave · Post by **Cactusdave** » Thu Mar 15, 2012 2:27 pm

LEDs, even 'white' ones can emit significant amounts of potentially eye damaging UV wavelengths, though obviously far less than mercury arcs or Xenon burners. There has been a lengthy discussion of risks from LED illuminators in microscopes on the Yahoo microscopy forum http://tech.groups.yahoo.com/group/Micr ... sage/66359 See also the Cree LED safety datasheet http://www.cree.com/products/pdf/XLamp_eyeSafety.pdf . Good eyes are one thing that microscopists can't do without.

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 2:37 pm

Thanks Mitch, don't underestimate the power of LEDs based on wattage. They are very efficient and mine actually has a focusing mechanism(all for $5 on ebay!). You can achieve a high lux with it fully focused. It would probably blind you for a few minutes if you looked at it. I'm assuming your LEDs are the round type which are not as impressive as the single emitter cree style. In the photo you can make out my transmitted light LED heat sink. Its a 30w array and provides enough light for a 100x 1.25 objective with crossed polars for DIC at 1/30 sec exposures at iso 100 for videos. Chris_M uses a 5w single emitter LED for his 100x DIC videos. Production models generally use a 100w halogen light sources for DIC and I wouldn't be surprised is my 30w LED out performs them.

This is autofluorescence. No dyes were used. Chlorophyll will fluoresce red when excited with blue light. Here is a graph showing the specific wavelengths of excitation and emission.

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 2:54 pm

Cactusdave wrote:LEDs, even 'white' ones can emit significant amounts of potentially eye damaging UV wavelengths, though obviously far less than mercury arcs or Xenon burners.

Unfortunately this is not true Dave. A simple google search will show 100 images of the white led spectrum. I have tried blocking all but the uv wavelengths with a dichroic filter and there was nothing emitted. The link you posted from cree suggests the eye damage risk is from visible blue light and not UV. The only decent uv leds I've seen are made by a company called nichia and the 365nm 3w emitter costs $100!

A white led is actually a blue led with a coating of phosphor that fluoresces yellow to create a balanced white temperature. There is also another type with blue, green, and red emitters (rgb) that appear white. But single emitter white leds have the phosphor coating

Mitch640 · Post by **Mitch640** » Thu Mar 15, 2012 4:02 pm

Curt, do you have a link to the E-bay flashlight?

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 4:59 pm

Sure Mitch, mine is last year's model. This is the same emitter and probably similar driver.

http://www.ebay.com/itm/Adjustable-Zoom ... 5ae4f6f065

I noticed there are some slightly cheaper that have the same emitter but use 1 AA battery. Avoid those. They're probably weak. 3 AAA delivers more current than 1 AA.

The plant photo exposures were between 3 seconds for the 1st, 1 second for the 2nd and the fly eye required 8 second exposures.

Here's the transmitted illumination LED and driver I use. I have it mounted on an old processor heatsink.
http://www.ebay.com/itm/30W-Cool-White- ... 5d31446914

Mitch640 · Post by **Mitch640** » Thu Mar 15, 2012 5:07 pm

Thanks. I'll be getting a couple of these.

curt0909 · Post by **curt0909** » Thu Mar 15, 2012 5:10 pm

Don't pay the asking price. Make him a best offer. I bought 5 and they came down to $5 a piece.

Cactusdave · Post by **Cactusdave** » Fri Mar 16, 2012 2:40 am

Thanks for all the information Curt. You obviously seem to know what you are doing.

René · Post by **René** » Fri Mar 16, 2012 11:09 am

Hi guys, my two cents, I have my 100W halogen replaced by led, type Luminus SST50 single chip 5 mm^2. Colour temp 4500K (I wouldn't go for the 'cool white' ones over 5500K, even though they are the most powerful). When measuring exposure times, this led is about 2-3 more powerful then 100W halogen, and that's at the recommended 1.75A current (pushing to 5A allowed). As a comparison, the Cree equals 100W halogen. At the moment the latest Luminus LED (SST90) is 2-3x more powerful, and at roughly 9mm^2 chip surface would nearly fill the condenser aperture without a need for diffuser. That would push the power to a next level!
So what do I want to do with this eyeblinding amount of light?? I am eagerly awaiting the moment that I can use it as a flash with DIC. But that will take another year or two, I suppose. But not more than that!

Concerning the UV leds, I have played around with good succes with small 20mA leds. Not great visually but adequate for a cmos sensor. Here in the lab I have replaced our HBO lamps with Nichia 365/385 nm leds of about 300mWatt, and they are never pushed to the max, so 3W UV sounds like overkill.

Best wishes, René

Curt, nice work with Lego, will keep that in my mind for experimenting!

curt0909 · Post by **curt0909** » Fri Mar 16, 2012 1:37 pm

Your welcome Dave and thanks for the input Rene.