Using LEDs as a low-cost source to detect GFP and DsRED

Fluorescence detection components are too costly to be installed in every stereoscope. Often, our fluorescent markers are very bright and do not require the full capacity of such equipment. Here we present an LED setup that costs only about $100 and can detect bright fluorescent markers such as myo-2::GFP. The setup (Fig. 1) consists of

  • Xacto helping hands or anything that can position the LED (amazon.com)
  • Lamp cord that has a switch (local hardware store)
  • 17-watt Xitanium LED driver (LED Supply)
  • Xitanium driver connector (LED Supply)
  • Heatsink (LED Supply)
  • Spot lens (LED Supply)
  • Luxeon V Star optic holder (LED Supply)
  • Royal-Blue Luxeon V Star LED for GFP detection (LED Supply), or Green Luxeon V Star LED for dsRED detection (LED Supply)
  • Optional excitation filter: Roscolux #4290 CalColor 90 Blue for GFP, or #389 Chroma Green for dsRED (Rosco.com | Edmund Optics)
  • Emission filter: Roscolux #12 Straw for GFP, or #19 Fire for dsRED (availability same as excitation filters)

When assembling the LED to the driver, make sure that the +/– ends on the LED and the neutral/line ends on the lamp cord match those on the driver. Use a non-conductive glue such as silicone adhesive (Devcon, part No.12045, local hardware store) to put the lens holder on the LED. The excitation filter can be glued to the lens by applying a small amount of glue on the edge. The emission filter can be simply taped under the microscope objective.

This setup has a long operating life, requires no warm-up or cool-down time, and has no radiation. However, it can only detect strong signals. We have used the setup to detect the following markers: myo-2::GFP (Fig. 2), sur-5::GFP, ajm-1::GFP, and myo-2::dsRED (Fig. 3).

More information can be found at http://130.15.90.245/gfp_stereoscope.htm and http://wormlab.rice.edu/LED.

Figures

Fig1_LED_setup
Figure 1: The LED setup.
Fig2_mIn1
Figure 2: Using the LED setup to detect myo-2::GFP in an mIn1 animal.
Fig3_dsRed
Figure 3: Using the LED setup to detect myo-2::dsRED.

Comments

Kathryn Hedges December 22, 2009 at 5:31 am

I used your setup as a starting point, and refined it with some real Chroma filters, to screen for GFP in neurons and the excretory cell. The filters cost more than Roscogels, but make a huge difference! The setup is still cheaper than a commercial fluorescent stereoscope. If you don’t mind, I’ll probably write it up for the next WBG.

We’ll probably use something similar to your version for classroom use visualizing GFP (or red dyes) in bacteria and cell cultures.

Francesco Spinelli May 18, 2010 at 6:19 pm

Hi! your instructions on how to make a stereo-fluorescent-microscope are truly excellent and very, very useful!! I did s you suggested and now I can observe my sample very easily. I used the set-up with the blue laser pointer (405 nm) since I’ve marked with GFPuv the plant pathogenic bacteria I’m working with. The only thing that does not work with me, is that the pointer produces an elongated beam (not circular, and quite concentrated). Therefore I can only illuminate a very little part of the sample and never, the whole field. Do you have any suggestion on how diffuse the pointer beam? Or do you have any idea for an economic light source quite intense an with an emission in the rage of 395-405 nm??
Thank you gain for the brilliant suggestions and looking forward for more tips!
cheers
Francesco

Kathryn Hedges February 2, 2011 at 5:02 am

Francesco,

Do I understand your GFP is optimized for UV? You can get LEDs that emit in the UV range (often used by dentists to cure certain resins), but my concern with DIY stuff using UV is safety. The royal blue LEDs suggested in the original version gave off enough UV or similar wavelengths that they hurt my eyes even when I pointed them at the wall. The 470nm blue LEDs did not. Also, do you need to view the whole plate visually or with a dissecting microscope? If you’re just scanning the plates, you might try a handheld UV lamp and the appropriate safety goggles.

When I was experimenting with LEDs, I also hoped that diffusing the beam would let me see the whole plate at once. Unfortunately, that reduced the intensity. I got my best results by adding an old microscope eyepiece to focus the actual LED array on the plate. (I’m going to write this up for WBG after my PLoS ONE paper is done, but meanwhile, the long version is in Appendix D of my thesis at http://humboldt-dspace.calstate.edu/xmlui/handle/2148/608 ). If you can use a holder with very precise vertical control for adjusting the image on the surface of the agar, this will help a lot. We had a spare micromanipulator (my PI is a neurophysiologist) that was much better than a Helping Hands as shown in this article.

My 5W Luxeons are outdated now; you can get 40W LEDs from LEDEngin for about $40 or 90W for about $75 in February 2011 that have a larger array and will cover a larger spot on the plate. I’d love to try them out, but I’ve graduated and am not working with C. elegans at the moment. Maybe when BioCurious is up and running in Mountain View, I’ll work on it.

Adam Kent Williams February 28, 2011 at 10:59 pm

Well done for getting this info ‘out there’, I have been using a car kit blue LED set with yellow safety glasses to view GFP for a few years now and thought lasers could be an improvement but was unsure how precise the wavelength had to be (I knew from past experience anything bright blue for GFP and rich yellow for the filter would work, but not sure for Texas red/ DsRed). It is indeed awesome to view GFP for less than $15, people think you’re performing witchcraft. So thanks heaps for the links and laser update!!

Adam Williams, Dept of Genetics, University of Melbourne, Australia

Francesco October 21, 2011 at 12:05 am

Hello,
I made some modification to your setup and I optimized it for visualizing the plant/bacteria interaction. If you don’t mind I’ll try to write a short comunication on a plant pathology bullettin… let me know if you have any concern about that. I addition. How can I cite your job?
BR
F.

Farhahna May 21, 2012 at 4:02 am

Hi Kathryn,

I have been looking for an alternative to using the Mercury lamp when visualising GFP in our transgenic tobacco lines. I was very excited when I can across the Worm Breeders Gazette. Should I make any adjustments to the original set-up if I want to view whole leaf tissue?

Francesco, I see you mentioned some changes, Could you kindly assist?

Thank you!

Kindest regards
F

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