Friday, 23 September 2016

Some remarks about FLUORESCENCE

In standard light microscopy FLUORESCENCE is clearly one of the most challenging methods. It requires a clear understanding of the scientific background as well as a proper microscope setup: there are many options to miss a satisfying image result. As the costs of a traditional Mercury illumination setup (still the most flexible approach) are remarkable, a beginner in FLUORESCENCE should take his time to evaluate the adequate microscope & filter hardware.

Have you ever driven an old car? In this case you are surely familiar with the phenomenon of resonance in a mechanical system. A piece of hardware, potentially able to vibrate with its “own” resonance frequency, is stimulated by an external energy source. The right front fender of your car may start to vibrate and to create noise at exactly 80 mph speed just by driving on the asphalt covering of a highway. To avoid this noise, just drive faster or slower, and you miss the resonance frequency.

A more impressive example of mechanical resonance is the case of a bridge starting to sway with high amplitudes under the influence of a gale. Watch”Gallopin' Gertie” on November 7th, 1940.

For FLUORESCENCE, specific dyes have been developed to stain specific structures within the specimen: nuclei, microtubules, genes on a chromosome; nearly any component of a cell can be stained selectively. The user has to find the matching excitation frequency (e.g. “speed”) to initiate the resonance of the dye(s) in use. Once found, the resulting resonance of course is not of mechanical/acoustical nature, but a visual effect: All stained structures will emit a defined wavelength (=color). If you miss the excitation frequency, there will be no resonance, no visual effect, in worst case no mage at all. That is why you have to check first the excitation data of your dye (the internet supplies these data abundantly).

From the offered spectrum of your HBO light source, the excitation filter of your filter cube cuts out the excitation “window”. As there is an obvious loss of energy during the FLUORESCENCE process (it is said that the energy relation excitation/emission is 104:1), the emission is always less energetic, means the emitted color has got a longer wavelength. This effect is called “Stokes Shift”:

So the following systematic is comprehensible: 

Excitation UV →
Emission BLUE

Excitation BLUEEmission GREEN

Excitation GREEN Emission RED

Excitation RED Emission IR

To separate excitation and emission, a third filter element, the dichroic mirror, is implemented in the fluorescence filter cube. But finally it is the emission filter (band pass BP or long pass LP) which is responsible for the color impression of the image result. A narrow emission window stands for a selective detection and a pure color impression, a broad window (a LP filter in this case may be regarded as an extremely broad BP filter) causes a brighter image with “dirty” mixed colors. A long pass barrier filter may be chosen to observe simultaneously both stained structure and background.

The complete filter cube looks like this:

The orientation of the cube depends on the microscope type:

Upright microscope

Inverted microscope

Once these technical issues are finished, the preconditions for a satisfying FLUORESCENCE imaging are given. But don’t underestimate the need for a precise alignment of the Mercury lamp house. Take good note of the instructions given by the instruction manual or the tutorials on the website of the microscope manufacturer.

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