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| The essentials for success in fluorescence microscopy |
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Optimum quality of the fluorescence filters
The fluorescence light generated must be treated with care. The filters must transmit the required wavelengths, and block the unrequired ones as completely as possible. This is very difficult to do, since the intensity of the excitation light is many times higher than that of the emission light. The entire exciting light must be kept away from the microscope image, while the emission light, however, must “arrive” in its entirety.
High-intensity excitation
The light source must provide a large amount of excitation energy in very narrow ranges of the spectrum – typically 10 to 50 nm. For this purpose, so-called line emitters, usually high-pressure mercury lamps, are used.
High light transmission of the objectives
The objectives of the microscope – particularly those offering optimum imaging quality – often consist of many individual lenses. Nevertheless, objectives which are suitable for fluorescence feature high light transmission values into the UV range.
No autofluorescence of the microscope optics
If lenses and filters – or immersion liquids – of the microscope exhibit autofluorescence, this disturbing light will be mixed inseparably in the fluorescence image. The result is a brightened background which reduces the contrast available.
High numerical aperture of the objective
As with a radio transmitter, the fluorescence excited in the specimen is radiated in all directions. The objective must gather as much of this radiation as possible. The output will be low with a small numerical aperture (A). A higher aperture angle (B) will be much more effective. If the objective aperture is doubled in size, approximately four times the fluorescence light can be gathered. Immersion, particularly with oil – also eliminates the loss of light caused by light reflection on the surfaces. The image will become even brighter.
continue with: Fluorescence becomes visible
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