Imaging a target fluorophore in a biological material in the presence of autofluorescence
First Claim
1. A method for extracting an image of a target fluorophore in a biological material wherein a waveband for emission of the target fluorophore overlaps a waveband for emission of a second fluorophore in the biological material, the method comprising:
- illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both fluorescence of the target fluorophore and fluorescence of the second fluorophore and with a second excitation light to induce a second fluorescence emission arising from only the fluorescence of the second fluorophore;
acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission; and
processing the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore, wherein relative intensities of the first and second excitation lights are modulated prior to acquiring the first and second fluorescence images.
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Abstract
Methods and systems are disclosed for extracting an image of a target fluorophore in a biological material, which involve inducing both autofluorescence of the biological material and fluorescence of the fluorophore, acquiring an image arising from both the autofluorescence of the biological material and the fluorophore, and an image arising only from the autofluorescence, subtracting the two images to produce an image representing only the fluorophore, wherein relative intensities of the excitation light used to induce the autofluorescence and the fluorescence are modulated prior to acquiring the images.
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Citations
38 Claims
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1. A method for extracting an image of a target fluorophore in a biological material wherein a waveband for emission of the target fluorophore overlaps a waveband for emission of a second fluorophore in the biological material, the method comprising:
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illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both fluorescence of the target fluorophore and fluorescence of the second fluorophore and with a second excitation light to induce a second fluorescence emission arising from only the fluorescence of the second fluorophore; acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission; and processing the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore, wherein relative intensities of the first and second excitation lights are modulated prior to acquiring the first and second fluorescence images. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A system for extracting an image of a target fluorophore in a biological material wherein a waveband for emission of the target fluorophore overlaps a waveband for emission of a second fluorophore in the biological material, the system comprising:
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a light source configured to illuminate the biological material with a first excitation light to induce a first fluorescence emission arising from both fluorescence of the target fluorophore and fluorescence of the second fluorophore and with a second excitation light to induce a second fluorescence emission arising from only the fluorescence of the second fluorophore; an image acquisition assembly configured to acquire a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission; a modulator configured to modulate relative intensities of the first and second excitation lights prior to acquisition of the first and second fluorescence images; and a processor assembly configured to process the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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Specification