Spectral Imaging

The oldest method of (deliberately) separating colors of light was described by Sir Isaak Newton in his book “Opticks” of 1704: the employment of a prism. A copy of his drawing from that book is shown with the preface in this article. Today, our interpretation is that light of shorter wavelength will be diffracted at a boundary of optically different media more strongly than light of longer wavelength (to make a simple conclusion). If a mixture of colors – like the composed emissions of a set of fluorochromes – is fed through a prism, the composed emissions will be decomposed spectrally.

The strength of decomposition depends on many technical parameters, but is independent of the sample or the sensor. This is a very efficient and straightforward solution for the problem of having emissions from a set of fluorochromes pointing in different directions, where they can be subsequently recorded. In the simplest case, one would just place a series of detectors along the spectrum. This concept is realized, but has severe flaws in collection efficiency and flexibility. A better solution is a multiband device which allows individual selection of any fraction of the full spectrum for each sensor to be recorded.

A prism has the advantage of white (flat) transmission, i.e. there is no absorption modulation by the prism (within the specified spectral range). The transmission and the dispersion are independent of the direction of polarization. This is an important fact, as the emission of a fluorochrome is always unpolarized. And last but not least, the dispersion occurs only in one single direction – there are no other “orders” that may reduce the intensity in the selected order.

There is some discussion about the linearity of the dispersed spectrum. A prism-based spectrum is not linear with respect to the wavelength. For technical designs, this is not a problem, as long as one is not bound to use linear detector arrays, such as multi-anode photomutipliers or similar devices.

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