Biologists usually need to enhance and differentiate details within living or fixed cells. To do this, special dyes are applied. To increase the sensitivity of observation, dyes (fluorochromes) that fluoresce under invisible or visible radiation are used to stain and delineate the structures of interest. Sometimes, the inherent tendency of some cellular components to fluoresce without additional staining may be usefully employed. This autofluorescence can be seen in cellular components ranging from plant chlorophylls to proteins in eye tissue. Unlike traditional biological staining (e.g., methylene blue and eosin for tissue differentiation), due to the remarkable efficiency and detectability of fluorochromes fluorescing under UV or visible light, much smaller concentrations of these dyes (generally toxic) can be used. This translates into fewer side effects to living cells with minimum disruption to their normal physiology. Also, the biochemical workings of cells, such as calcium ion transport, can be recorded in time-lapse mode using fluorescence microscopy.

Generally, high-pressure mercury or xenon light sources are used for fluorescence microscopy. The lamps are very bright and produce ample light for most work. Sometimes, these sources are too bright and harmful for the living organisms being studied. Excessive brightness will cause rapid photobleaching of the dyes used to stain the tissues in question. Also, very powerful lights can destroy cells or, at least, disrupt their normal behavior. There are substantial costs and some hazards involved in using mercury or xenon lamps. To get around these problems for certain applications, I developed a novel technique employing easily obtainable ultrabright LEDs.

With standard fluorescence microscopy, the light source always requires a narrow-band exciter filter, as the lamp's output wavelengths are too broad. My LED technique differs in that the fairly narrow output wavelength of an LED diode may obviate the need for exciter filters. If an exciting filter is needed, the associated cost is much less than with standard fluorescence microscopy. A blocking filter is required to be placed on the recording camera or in the eye's path to block direct illumination, be it LED, mercury, or xenon. Only the generated fluorescent light should be transmitted. Here again, the cost for blocking filters for LED fluorescence is less than the cost for filters needed for standard techniques. Standard mercury and xenon lamps last several hundred hours in normal operation. When mercury lamps die, they may go out with a bang! LEDs can work satisfactorily for well over 10,000 hours.

Unlike the high-pressure, gas-filled lamps, LEDs are available for use at a moment's notice. As expected, LED brightness is less than that of traditional light sources. However, coupling the LED illumination with low-cost but exquisitely sensitive CCD cameras will open the world of fluorescence microscopy to many more experimenters. Quiet, inexpensive, nice to work with—that's LED fluorescence.

LED Microscopy setup

Seen here is a typical setup using one LED lamp and a light-coupling prism resting on the slide. The glass prism should be optically connected to the microscope slide with a drop of immersion oil or glycerine, depending on the refractive indices of prism and slide. Then, due to total internal reflection, the light beam is efficiently transmitted to the specimen. The technique works well with very thin specimens. In these cases, the microscope objective tends to get in the way of an incident light source, so channeling the light really helps. When the specimen is thick (e.g., a plant leaf), simply directing the LED light at an incident angle to the specimen works best.

Microscope setup with LEDs

In use with a standard transmission microscope, a very sensitive CCD camera (or a cold camera, such as the type used by amateur astronomers) should be coupled to the camera tube. Then, after carefully adjusting illumination and focus by eye, the image can be studied on the video monitor. It is quite amazing how much detail becomes obvious when viewed in this fashion. This is especially true if the fluorescence produced is in the red, far red, or infrared part of the spectrum. Then, the CCD + monitor setup is crucial! Images otherwise totally invisible to the eye pop out in crisp detail. But, most of the time, just peering through the oculars in a darkened room will reveal much.
The following digital slide sheet shows photos taken using my LED fluorescence technique. Various LEDs and filters were employed. A standard transmission microscope was used. For additional information, check out the references for this section.
GREEN FLUORESCENT PROTEIN

Arabidopsis thaliana has become a household name in molecular biology labs everywhere. This weed, sometimes called the mouse-eared cress, has a small genome that has been mapped, has a short life cycle (from seed to seed in under two months), is self-fertilizing, and is easy to grow (once you get the hang of it, that is). Producing mutations of the weed's flowering, growth, etc., is straightforward.

The lines grown for the following photos were obtained from the ABRC in Ohio. Different lines produced fluorescence in different plant organs. The original seeds were donated by Dr. Haseloff's laboratory in England. Green fluorescent protein (GFP) reporter genes were improved for the purpose and employed by Haseloff to transform A. thaliana plants. Selective transformation, in turn, allows identifying the effects of different genes in Arabidopsis by modifying the proteins produced by those genes. When plant genes can be tricked into producing the green fluorescent protein, first found in a jellyfish (Aequorea victoria), along with the normal plant proteins, researchers can then associate those genes with specific activity within specific organs in the plant. The GFP signals its presence (i.e., reports) by its telltale fluorescence when illuminated under UV or blue light. For these photos, a stereoscopic microscope was used with a blue LED lamp and appropriate filters.

Before continuing with LED fluorescence in microscopy, let's take a quick look at how LEDs can be used for infrared and ultraviolet experiments.

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