Optoscan - CAIRN RESEARCH - #19

_Principles of Operation

In practice, therefore, there is a very important trade-off to be made between bandwidth and light intensity. This is particularly relevant for biological fluorescence applications, where for fluorescence excitation, high light intensity is generally more useful than narrow bandwidth, but nevertheless some compromise must ultimately be made. Most monochromators make some sort of provision for varying the slit widths, although this may just be as crude as providing (or offering to provide...) a set of interchangeable slits of different widths. This is hardly user-friendly, and since the slits must be both manufactured and located with some degree of precision, it is clearly undesirable for the user to become involved with such things in any case. Those who are somewhat more fortunate with their choice of supplier may get some kind of calibrated mechanism that allows the slit widths to be varied, as provided on the lowest-cost version of our instrument. However, it is not generally appreciated that the relationship between mechanical slit width and optical bandwidth is wavelength-dependent, and is different for the two slits, so setting the correct slit widths for a given optical bandwidth is not as trivial a matter as might have been thought! Furthermore, the whole POINT of using a monochromator is because one wants to switch wavelengths, so even to maintain the SAME bandpass characteristic it would be necessary to readjust both slits.

On the other hand, the best compromise between bandwidth and light intensity for a given application may well DIFFER according to the currently-selected wavelength. Consider the pH indicator BCECF, for example. This is a dual-excitation ratiometric indicator, which is normally excited at 440 and 490nm. The excitation spectrum for this indicator is shown in Fig. 3. The excitation bandwidth at 490nm must be fairly narrow, to avoid overspill into the emission region for this indicator, which starts at around 510nm, but on the other hand the fluorescence excitation at 490nm is relatively efficient.

400 450 500 550 6O0 650 Wavelength (nm)

Excitation at 440nm is much less efficient, but we can improve the situation considerably by using a greater bandwidth here (remembering that the improvement is with the square of the bandwidth as explained above).

The ability to change bandwidth as well as wavelength is thus of great practical value, so if one is interested in changing the wavelength rapidly (i.e. on a millisecond timescale), then one should really be able to change the slit widths with similar rapidity. We believe the Cairn Optoscan to be the first monochromator which allows you to do exactly that. Furthermore, the microprocessor option makes this facility extremely easy to use, since for each wavelength the optical bandwidth can be specified directly, and the microprocessor looks after the chore of calculating and setting the required mechanical slit widths.

However, as for setting the wavelength, users who wish to provide their own electrical signals to set the slit widths will need to perform equivalent calculations, and this information is provided here.

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