Using optical interference filters to measure autofluorescence in substrates and coatings

In fluorescence microscopy and related applications, fluorescence from optical elements in the system can be a limiting factor in achieving low background levels. Such autofluorescence is considerably weaker than the fluorescence produced by the organic dyes used in fluorescence-based analytical techniques. While spectrally detailed information about autofluorescence is desirable, such information comes at a cost of sensitivity. We present a straightforward approach to making sensitive fluorescence measurements using a simple chopped laser and phase-lock detection scheme. By using high-quality optical filters in the excitation and detection paths, the trade-off between spectral information and ultimate sensitivity is controlled by the choice of filter edge locations and bandwidths. An initial implementation of this approach, using a 405 nm diode laser as an excitation source, achieves a sensitivity of -1 pW optical power of detected fluorescence over a bandwidth of similar to 125 nm centered at 505 nm. Data from measurements on glass substrates are presented, and challenges for evaluating autofluorescence from coatings on glass are discussed.

Automated summary

In fluorescence microscopy, autofluorescence from optical elements in the system can limit background levels. Although weaker than fluorescence from organic dyes, detailed spectral information about autofluorescence comes at the cost of sensitivity. The study presents an approach using a chopped laser and phase-lock detection scheme for sensitive fluorescence measurements, controlling the trade-off between spectral information and sensitivity through filter edge locations and bandwidths.