A method for distinguishing between more than one fluorescent species present in a sample

A method for distinguishing between more than one fluorescent species present in a sample in fluorescence microscopy, the method comprising:

• (a) illuminating the sample with at least one light source;
• (b) recording more than two images of the illuminated sample over a period of time, each image comprising a plurality of pixels, wherein each pixel corresponds to a location in the sample and records a degree of fluorescence at the location in the sample at a particular point in time;
• and
• (c) determining a photostability characteristic of the degree of fluorescence at each pixel over the period of time over which the more than two images were recorded, wherein the photostability characteristic of the degree of fluorescence at each pixel is used to distinguish between the more than one fluorescent species present in the sample.

A system configured to distinguish between more than one fluorescent species present in a sample in fluorescence microscopy, the system comprising:

• at least one light source configured to illuminate the sample;
• an image recording device configured to record more than two images of the illuminated sample over a period of time, each of the more than two images comprising a plurality of pixels, wherein each pixel corresponds to a location in the sample and records a degree of fluorescence at the location in the sample at a particular point in time;
• a processor programmed to determine a photostability characteristic of the degree of fluorescence at each pixel over the period of time over which the more than two images were recorded;
• and
• a display configured to present to a user the relative abundance of each fluorescent species in the sample at each pixel based on the photostability characteristic of the degree of fluorescence at each pixel.

A non-transitory computer readable medium comprising:

• computer program code, which when executed by a processor, cause the processor to distinguish between more than one fluorescent species present in a sample in fluorescence microscopy by:
• (a) causing the sample to be illuminated with at least one light source;
• (b) recording more than two images of the illuminated sample over a period of time, each image comprising a plurality of pixels, wherein each pixel corresponds to a location in the sample and records a degree of fluorescence at the location in the sample at a particular point in time;
• (c) determining a photostability characteristic of the degree of fluorescence at each pixel over the period of time over which the more than two images were recorded;
• and
• (d) causing a display, by a display device, of a relative abundance map for each fluorescent species in the sample at each pixel based on the photostability characteristic of the degree of fluorescence at each pixel.