Types of Fluorescence Microscopesby Richard Gaughan
When microscopes were invented around 1600 C.E., natural philosophers turned their eyes onto a world within a world. When Antony van Leeuwenhoek crafted small, highly-curved lenses and a mechanical holder for adjusting the view, he opened a window onto the microscopic world of bacteria, blood cells, protozoa and the cellular structure of plants. But throughout the history of microscopy, there has always been one question: What are these strange things seen through a lens? Fluorescence microscopy refers to a set of techniques that minimizes that uncertainty -- because in fluorescence microscopy when light is shone on a sample, it shines its own light right back.
By far the most common fluorescence microscope is the epifluorescence configuration. In an epifluorescence microscope, a light source -- typically a mercury or xenon lamp -- shines through a filter that selects a narrow region of wavelengths. The filtered light shines onto the sample through the microscope objective lens. The incoming light is absorbed by fluorophores -- molecular labels that emit light of a long wavelength when they absorb light of a shorter wavelength. Light from the fluorophores, along with scattered light from the illumination source, goes back into the objective lens and to the detector or eye. Along the way, another filter blocks out the illumination light, so all that is left is the fluorescent light from the sample.
An epifluorescence microscope collects light from everywhere within the field of view of the microscope. Some of the excitation light is absorbed before the focal plane of the microscope, some at the focal plane and some beyond the focal plane. Because the microscope collects all that light, the image will contain a sharp picture of light at the focus, but it will also have out-of-focus light from other regions. A confocal microscope fixes that by focusing a laser spot in the same plane as the microscope is focused. Then, a pinhole goes in front of the detector, where it blocks all the light that doesn't come from the microscope focus. By scanning the sample, a clean three-dimensional image of the object can be obtained.
In a confocal microscope the alignment is very sensitive. If the laser spot, the microscope objective, the collecting optics and the pinhole are off even the slightest amount the microscope performance suffers. A multiphoton microscope gets around this problem by using a laser wavelength that's only half as energetic as it needs to be to excite the fluorophores in the sample. The only way the fluorophores will get excited and emit fluorescence is if the laser light is bright enough so that two particles of light -- photons -- strike the fluorophore in a very short time. That happens only when the laser is focused to a very tiny spot. So the only place in the sample that will emit light is where the laser is focused, which keeps the image nice and clean because there's no extra background light to get rid of -- which means no pinhole to align.
Total Internal Reflection Fluorescence (TIRF)
Another way to get very clean images is to make sure the excitation light doesn't get very far into the sample. If a blob of neurons, for example, is placed in a drop of solution on a glass slide, then some of the neurons will adhere to the glass surface. In a total internal reflection fluorescence (TIRF) microscope the light is directed sideways into the glass slide so it doesn't really make it into the solution holding the cells. But some of the light just barely leaks into the solution -- just very close to the surface of the glass. This means the only places that will emit light will be in a very thin region right up against the glass surface. For something like neurons, where so much interesting stuff happens on the surface of the cells, this technique can be very effective.
All microscopes -- including fluorescence microscopes -- are limited by the physics that governs the propagation of light. One of the basic rules is that a focused spot of light can only get so small -- and no smaller. For visible light, that size is about 200 nanometers, or 200 billionths of a meter. But single molecules are only a few nanometers in size, so there are lots of interesting features that are below that size limit, called the diffraction limit. Scientists are developing "super-resolution" techniques to sneak around that limit. Structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy, for example, are both fluorescence microscopy methods that limit the size of the light-emitting spot by shrinking the size of the excitation light spot.
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