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Authors: Prof. Robin Williams and Gigi Williams

Ultraviolet induced fluorescence:

Fluorescence from biological specimens is often both fleeting and weak. It is difficult to measure or meter these weak fluorescences, and exposure tests and "bracketing" are always recommended. When using a continuous source of ultraviolet, such as a hand-held mercury vapour lamp, expect long exposure times - several seconds or even minutes is not unusual.

Allowance must also then be made for reciprocity law failure, which will not only further increase the required exposure time, but may also alter the colour recorded. (Under normal conditions exposure in photography is equal to the product of illuminance and time, but when dealing with very short or very long exposure times this relationship breaks down.) Because colour film is actually three emulsion layers, each sensitive to a different part of the visible spectrum, the reciprocity characteristics of the three layers may be different. The result is, that not only is there an overall speed loss with very long exposures, but the change in speed of each of the three layers will be different - resulting in a shift in colour balance. Colour compensating filters will in turn increase the exposure time. For example, when using Ektachrome P800 at exposures of longer than one second, 05cc of red and 05cc of yellow must be added, along with an increase in exposure of half a stop.

Accurate rendition of the colour of fluorescence is difficult therefore without tests. It is best to avoid the complications of reciprocity law failure by using exposures no longer than one second - this may mean using a wider aperture or keeping the radiation source very close to the subject. With some fluorescence there is also the problem of exhaustion extinction - the longer the subject is irradiated the weaker the fluorescence becomes. Also the fluorescence from some chromatograms and gels is of a temporary nature, and if left for a significant length of time before the photography is done they may no longer exhibit any fluorescence.

Electronic flash avoids both the problems of reciprocity law failure and exhaustion extinction, and its use is recommended despite, the inconvenience of not being able to actually see the fluorescence (a continuous source of radiation for viewing and orientation overcomes this problem). With moderately powered units, it may be found that one flash provides insufficient radiant energy to stimulate fluorescent emission. In normal photography it would be the practice to give several flash exposures, each of which would add to the overall exposure. In fluorescence work, however, this is not true and the practice is not recommended. There is often an energy threshold for stimulating fluorescent emission, and single isolated doses of stimulating radiation will not add up to this threshold. The only solution is the use of a more powerful flashgun or to move the flashgun closer to the subject.

Care needs to be exercised with the selection of background as it may fluoresce much more strongly than the subject at the same exposure level. This is particularly true of white paper, card, or light-coloured materials that are often impregnated with fluorescent dyes. Some experimentation will be necessary to find a neutral background which does not itself fluoresce.

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© 2002 Prof. Robin Williams and Gigi Williams - Disclaimer
Last modified: 3 May 2002