Use of infrared light with microscope

The “12 LED IR InfraRed night vision camera flashlight” arrived yesterday. I purchased it at e-bay for $9.99 plus $4.75 shipping. I removed the top part and attached 2 18 inch wires from the top + and - parts to the + and - parts of the case. The case contains the on/off switch. The flashlight is powered by 3 AAA batteries. I like to use rechargeables as I am able to recharge them with the 5 volt DC available at the computer USB ports. I tried 2 webcams (random selection). Both worked with the IR flashlight(frequency not given). The pictures below were taken with my highest resolution webcam (An inexpensive 5 megapixel webcam - about $14.00 plus shipping from e-bay).

The first link is to a picture taken with a 100 X lens (meant to be used with oil, but used here in air) showing a calibration slide with a 10 micron separation. I was hoping that the use of IR light would negate the need to use an oil between the lens and the slide.

http://picasaweb.google.com/lh/photo/pLpaCJ-VRP15lz8hTLgulQ?feat=directlink

The second link is to the same slide with a regular (visible light) LED.

http://picasaweb.google.com/lh/photo/B_77yE_LMAC5rIuy53LF7Q?feat=directlink

The second set of pictures were taken with a 30 X lens of a William Baffin pollen grain that was stained with acetocarmine. The same 5 megapixel webcam was used. The pollen grains are different.

http://picasaweb.google.com/lh/photo/RL9OoJBpeSLBUU8nPmoTwg?feat=directlink

http://picasaweb.google.com/lh/photo/hxhyKcIfOPUL3ldyae0uoQ?feat=directlink

You’ve got focal problems.

You need to have the grains as close to the cover slip as possible. One way to do that is to put the pollen grains in a couple of drops of water with a speck of solid glycerin and mix that with a toothpick. Then let it dry on a surface heated with a lightbulb underneath it.

That way, all the pollen is at one elevation and you’re not getting diffraction by light going through organic matter in the water between the coverslip and the slide. Then mount the coverslip on a glycerin and water mix and let it air dry. (If it’s good enough and you want to keep it, cut off the glycerin on the sides of the coverslip with a single edged razor blade and seal the edges with nail polish.)

When you’re working with a slide like that, THEN using emersion oil and a 100x ocular will give you great detail.

These photos are interesting, but you’re not seeing the three pores in the pollen grains. (That bothers me, because you should be able to see them.)

I’m a bit fussy about microphotography because I have done a lot of it and it can be done well. But it’s not always easy.

Are the grains fluorescing? Some organic material, when certain wave lengths pass through, will give off colors unlike they have in real life. That the pollen has material inside it, could make epifluorecense even more distracting.

An example: pollen grains that are clear in blue spectrum transmitted light, when hit with certain wavelengths (and viewed through certain filters within the microscope) will appear shades of yellow.

Thank you Ann. In the other post I gave pictures of both John Davis and William Baffin pollen. I thought the quality was world’s apart. I mentioned that the difference in my technique was that I had to crush the anthers of William Baffin to get some pollen. Perhaps the crushed (non pollen) particles of the anthers are keeping the coverslip above (not touching) the pollen grains and therefore producing the effect that you describe.


A private e-mail suggested that for the infrared I should try a blue stain.


Does any have any experience with darkfield microscopy? Today UPS delivered an e-bay purchased 4 lens Bausch and Lomb research microscope that has the darkfield lower unit (costs $13.06 plus $16.67 shipping - there were 5 bids).

The link below makes it sould like it may be a valuable tool with pollen.

Link: en.wikipedia.org/wiki/Dark_field_microscopy

Please remember I’m typing this at 11pm and will probably have more to clarify and expand tomorrow.

The easiest way to get a darkfield is to use two polarized filters at right angles to each other. This cuts out all ‘straight’ light rays. For things like diatoms and radiolaria (generally studied with 40x objectives, this works well.

I also used to use two techniques (expensive lenses) that were called Nomarski and Zernike phase. These were used to give great detail to edge features of fossils but not for internal details.

Incident light…especially useful for freestanding things. The problem with putting the object of your study in a substance to keep it from moving around is that often there is/are so many things in the medium that can diffuse the light. Keeping the light rays parallel is the key to detail, especially as you go to higher magnifications.

How are we going to knock the pollen grains off the anthers and onto the coverslip? A couple of medical probes that can be sterilized with a candle flame is one way. Ideal way would be in a disposable test tube submerged for a couple of minutes in a ultrasonic cleaner (microvibrations).

Thoughts for now, will have more tomorrow.

Ann