GFP Stereoscope

Update:  A company called NIGHTSEA has made a nice LED package.


Green Fluorescent Protein (GFP) is a small protein which forms a chromophore that absorbs blue light and fluoresces in the yellow-green spectrum. GFP as a transgenic reporter  is a very useful tool to view the expression of proteins in living organisms. Unfortunately fluorescence microscopy equipment can be very expensive and most universities or high schools cannot afford to purchase such microscopes for lab  demonstrations. 

I hope you find this resource useful and perhaps we can finally bring GFP demonstrations into the classrooms and labs.

For more information about GFP check out this link.

How it all started:

We routinely use GFP markers in our C. elegans genetic crosses and analyses and  thus our fluorescence stereomicroscope (Leica MZFLIII) was  heavily used. One of our GFP markers on chromosome II (mIn1[dpy-10(e128) mIs14] II) expresses strong GFP in the pharynx. Sometimes we just wanted to check quickly to see if our cross progeny carried the GFP marker. To avoid these bottlenecks at the fluorescence stereoscope we came up with a very inexpensive (under $200) way to convert a Leica MZ6 (or other M series or the Zeiss V series) stereo microscope to view medium to strong GFP expression.  The modification is simple and consists of an  excitation light source and a barrier filter. We use a lumileds  5W Luxeon V star LED (light emitting diode)   450nm blue light source. These are the world's brightest LEDs and fortunately for us come in a 450nm wave length which is required for GFP excitation.  There are also other wavelengths (i.e. 550 nm green)  that may be useful for Red Florescence Protein markers.  The fluorescence is also applicable to  fluorescent dyes such as FITC and Texas Red. A company called Optotech manufactures these LEDs in a standard MR-16 halogen bulb format and also sells the AC adapter to drive this LED.  You can also buy these LEDs  from www.luxeonstar.com.

Benefits of using LEDs over conventional Mercury arc bulb light sources:

Long Operating Life 50,000 hrs+, Cheaper, MR-16 Form Factor,  No UV or IR Radiation,  Range of Colors therefore no need for excitation filters, Cool Beam,  No warm up time required, and Low energy use.

Downside:

Not as bright as mercury bulb light source, Beam spot not as focused (as the the technology develops this will be less of a problem)  

Below are some pictures of the set-up (and a sample picture and movie) and a list of parts that you will need to modify your Leica M series stereomicroscope (you may have to choose a different  size of  barrier filters that suits your scope):

 

 (the light is an intense blue but the digital camera does not get the colour correct. Also the beam spot (inner stronger intensity spot) from this height is about 2cm in diameter--smaller (focused)  the better). 

 Parts Required:

    1 Endura Bright Royal Blue (450nm) LED  Cat # OT16-5100-RB www.optotech.com ($76.00)  The beam angel is 10o . The smaller the beam angle the better it is for exciting your GFP specimen. For those who are good with optics you may want to try putting a focusing lens onto this bulb.  These LEDs are very bright and you should not look directly into the light as it may cause eye damage. 

Update: If you cannot obtain one of these from optotech then you can purchase the LED itself but it must be mounted to a heat sink or it will over heat. I have use a heat sink from an old computer's CPU.  See the following link to buy the LED by itself:

http://www.luxeonstar.com/luxeon-v-star-led-royal-blue-lambertian-700-mw-700ma-p-295.php

Note: We have also tried the 470nm Blue light bulb, but we found that 450nm works better. We use Andy Fire's  GFP vectors with the S65T mutation for most of our work. The 470nm Blue may work better for other variants of GFP.

 1 AC converter Cat# OTMI-0060  www.optotech.com  This is a modified 700mA Xitanium LED driver . For those who are electrically inclined you might want to purchase OEM from the manufacture and add the plugs to the driver.  Note:  You need this AC to DC converter you cannot simply plug these bulbs into any halogen light bulb fixture or you may  blow your LED.

 

 2  Long Pass 515nm 12.5mm filters (You may have to buy larger filters to fit your scope). Edmund Optics Cat #NT54-653 ($12.20 ea)  Note:  These filters are 3mm thick and will fit nicely into the magnification changer once the binocular tube (eye piece) is removed.  If your scope does not accept this configuration another alternative is to use a long pass filter (cut off 470nm i.e. allows wave lengths longer than this to pass through) over the objective or any location where the light passes through. See below.

 

A cheaper (but not as good) alternative to the glass barrier filter is to use flexible filters. Edmund Optics sells a color book of 200 filters  in which #12 straw works well as a barrier filter Cat#NT39-417 ($9.20) You can probably find larger sheets of this filter somewhere. I prefer the glass long pass filters (it blocks out more reflected light), but with these you could probably wrap it around (or tape it in front of) the objective lens.

  

Remove the Binocular tube (eye pieces)  and drop the barrier filters in. Replace the Binocular head.

or cut a piece of filter to cover the two lenses or cover the objective lens.

 Attach the AC converter to the bulb. Plug it into a standard 110-120v outlet.  Note: there is no on or off switch, but RadioShack and other electronic/hardware stores sell wireless switches that plug into the outlet to remotely turn on or off a lamp  or this which can be a very  convenient  way to turn the LED light on and off while you are sitting at the microscope.

The trickiest part of the setup is positioning the light source. Use an  articulating arm to position and hold the light if you have one, or a cheap "articulating arm" can be found at  Radio Shack  (helping hands) tape works just as well too. The easiest way to position the light is to  place the bulb edge  at the edge of the glass plate and change the angle by moving it up or down until the GFP looks the brightest.  As the spot diameter gets smaller (focused spot) and centered it will yield the strongest fluorescence. 

 Samples Pictures:  


mIn1[dpy-10(e128) mIs14] II  AVI movie

   

Good Luck and Have Fun!

Notes:

We have found that we can significantly reduce the glare from the background by putting a plastic translucent blue filter in front of the LED. We use the #4290  CalColor 90 Blue roscolux filter (see above).

C. elegans GFP reporters that can be detected:

mIn1mIs14 (GFP in pharynx)

sur-5:GFP (GFP in ~all nuclei)

mec-4:GFP (very weak but can detect GFP in touch neurons)

F25B3.3:GFP (pan neuronal - detect in the nerve ring and head neurons)

unc-119:GFP (pan neuronal-- detect in the nerve ring and head neurons)


 November  2009 Update!  Using laser pointers as an excitation light source for fluorescent proteins or dyes.

Now that Blue laser pointers are relatively cheap (compared to about  $1000, 2 years ago) they provide us with an easy hand held device to check for GFP or RFP with Green laser pointers and Roscolux #26 Light Red filter.

Blue 405nm (this is also the same wavelength used in Blu-ray DVD players. ) 5mW laser pointer for GFP.  It's not the optimal wavelength for GFP excitation  but it works great! 

 

You can find blue laser pointers on eBay for under $15!  Green ones are even cheaper.

Caution: These are Class IIIa lasers (<5mW) and you should avoid direct eye contact. Minimize the time you use the laser pointer with naked eye.

 These safety glasses work well for blue lasers (GFP) and if you don't have the barrier filters on the microscope these will also block the background blue light and let the GFP fluorescent through.

 The red colored  Laser safety glasses should work with the green laser pointers (eg. RFP) and can be found here.

 

Disclaimer:  I have provided this webpage only as a reference for visualizing GFP.  Reference in this website to any specific commercial products, does not constitute its endorsement. LEDs and laser pointers have potential risk for eye injury. Use at your own risk.

Comments or suggestions are always welcome.

This paper reference our webpage.

You can leave comment at the worm breeder's gazette link:

http://www.wormbook.org/wbg/volume-18-number-1/using-leds-as-a-low-cost-source-to-detect-gfp-and-dsred-2/

See also Weiwei  Zhong's website to find an updated list of suppliers:

http://wormlab.rice.edu/LED/

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