Molecules called flurorophores emit light in response to illumination. However, when a flurorophore absorbs light, it becomes susceptible to chemical interactions with its environment. These chemical reactions can irreversibly render the fluorophore insensitive to light, eliminating its light emission capability, a process called photobleaching.
Although photobleaching is usually thought of as being detrimental to fluorescence microscopy, this effect is exploited in the present technology to extract more data from a sample. The technology has the distinctive advantage that it can distinguish between more than one fluorescent species present in a sample in fluorescence microscopy. This technique, is referred to as bleaching-assisted multichannel microscopy (BAMM).
More particularly, the technology relates to novel methods which may be used to enhance multiplexing capability of methods for distinguishing fluorescent species based on their spectral emissions or that can be used independently of their spectral emissions.
The technology developed is a method which makes use of a fundamentally different property of fluorophores than standard spectrally based fluorescence microscopy. Therefore, the developed method can distinguish multiple fluorescent species using only a single emission channel, as well as separating spectrally identical fluorescent species. As an example, it is possible to extract up to five fluorescent channels using only two spectral emission channels, to provide a 2.5x increase beyond what is possible using only spectral information.
The bleaching-assisted microscopy could be integrated into any fluorescence microscope as a software package consist of two parts: custom hardware control instructions, and an image processing module.
Patent protection has been sought for this novel method and system for distinguishing between more than one fluorescent species present in a sample in fluorescence microscopy.
Fluorescence microscopy is widely used in biological and clinical research for high contrast imaging. Fluorescence microscopy enables the study of properties of organic and inorganic substances using fluorescence instead of, or in addition to reflection and absorption properties. Bleaching-assisted microscopy could be integrated into any fluorescence microscope as a software package. The software package would consist of two parts: custom hardware control instructions, and an image processing software module.
Advantageously, the method of the present invention can be implemented using any digital fluorescence microscope, making it an attractive means to enhance the capabilities of existing microscopy suites. A particularly beneficial application of the developed technology is that it works well with laser scanning confocal microscopes.
In conventional fluorescence microscopy, only a single fluorescent species can be separated for each spectral channel observed. Observation of photobleaching characteristics in the developed technology provides an alternative means of distinguishing the fluorescent species present in a sample, or alternatively provides an additional dimension of observation, making it possible to at least double the number of resolvable fluorescent species within a sample when compared with observation of spectral characteristics alone. Moreover, observation of photobleaching behaviour enables distinction between fluorophores having substantially identical emission spectra, which is impossible using spectral information alone. Bleaching-assisted microscopy can also be used to separate autofluorescence from exogenous dyes, a common problem in fluorescence imaging.
A microscope equipped with the developed bleaching-assisted multichannel microscopy software would be capable of performing more complex and informative assays by increased fluorophore multiplexing.
Other benefits include: