Fluorescence-based detection methods and apparatus

Abstract

In apparatus for detecting the emission of fluorescent radiation from a sample in response to excitation by two or more different radiation sources (145, 160) over the same time period, each radiation source (145, 160) is imprinted with a modulation regime which can then be separately detected in the emitted fluorescent radiation. For instance in techniques based on fluorescent resonant energy transfer ('FRET'), the drive current to two or more different LED sources (145, 160) may be frequency modulated, or pulse width modulated, according to different modulation regimes. Responses of the sample, for instance of different donor / acceptor probes contained in the sample, to each of the radiation sources (145, 160) can then be separately detected by means of the different modulation regimes, even where the wavelengths or wavelength ranges of the responses are the same or overlapping.

Description

technical field [0001] The present invention relates to fluorescence-based detection methods and devices, such as may be used in assays or imaging. The invention is particularly suitable for use in methods and devices for fluorescence microscopy and in methods and devices based on fluorescence resonance energy transfer. Background technique [0002] In luminescence-based assays or imaging methods, susceptible molecules are physically, chemically or mechanically excited by a stimulus and subsequently emit light, which can be detected. The emitted light can be used to find the position of these molecules and can thus be used in imaging and / or to detect the participation of the luminescent molecule in a process. [0003] A convenient form of stimulus is light that can be directed to a sample including the susceptible molecule, and this form of luminescence is called fluorescence. The emitted light has a different wavelength or range of wavelengths than the stimulating light s...

AI Technical Summary

Problems solved by technology

Even though it is possible to directly distinguish the fluorescence response of the specimen to different excitation wavelengths, e.g. because of the fluorescence of different donor molecules using different wavelengths, the common "quencher" format and the fluorescence of acceptor molecules using different wavelengths, in practice It is also difficult to obtain the multiplexing of a very large number of different probes

For example, when using FRET in real time to monitor the progress of PCR, there is a practical limit of about four

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