Stereo microscopes with TripleBeam Technology

Third illumination path for better signal-to-noise ratio in fluorescence microscopy

April 02, 2013

Especially in fluorescence microscopy, excitation light is friend and foe in one. On the one hand, energy-rich excitation via a specific light wavelength of the fluorochrome resulting in a bright positive fluorochrome signal is highly welcome. On the other hand, "noise" caused by reflections of excitation light passing through the surfaces of optical elements needs to be extremely slight to generate a perfect black background. This relation is described as "signal-to-noise ratio", which is highly relevant for differentiating optically between fluorescence positive and negative cells.

 

Separate beam path for fluorescence illumination light

The crux of this system is that the more excitation light (energy) you introduce to the system, the more reflections appear at optical elements, resulting in increased "noise" and in "bright" instead of "deep dark" background. This makes the differentiation between fluorescence positive and negative cells quite complicated. The best solution for preventing such "noise" is to avoid excitation light coming into contact with optical elements of the observation beam path.

The triple beam principle does exactly that; it consists of two parallel beam paths for stereo observation and a separate third beam path exclusively for fluorescence illumination light. The benefit of this principle is that the observation beam paths are free from any direct excitation light. Compared with systems using the same beam paths for observation and illumination, e.g. systems using a fluorescence filter cube with a dichroic filter to separate excitation and emission light, the triple beam principle has a significantly better signal-to-noise ratio.

Bright fluorescence signal

Moreover, the zoom of all three beam paths is synchronized. The illumination light is only bundled on the field of view of the stereo microscope. By increasing the magnification, the excitation is bundled even more and the amount of excitation light (energy) per area increases.

Another advantage is that the three beam paths are designed to be quite close to each other. The three needed fluorescence filters, one for excitation and two for emission, are mounted in a single small, easily exchangeable slider. Because of its small size, up to four filters can be used in a triple-beam principle stereo microscope setup.

Overview:
1.   Coupling for light guide
2.   Light (full spectrum)
3.   Excitation filter
4.   Excitation light
5.   Zoom system, including 2 observation and 1 emission beam paths
6.   Common main objective
7.   Fluorochrome labeled probe
8.   Emission light
9.  Emission filters
10. Trinoc binocular
11. Eyepieces
12. Camera

In sum the triple beam principle is a very effective system  for illuminating specimens with excitation light and observing the resulting fluorescence with a perfect signal-to-noise ratio. The result is a bright fluorescence signal surrounded by a deep dark background.

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