Fluorescence measurement
AssigneeThe Science and Technology Facilities Council
InventorsStephen Edwin Dominic Webb, Marisa Martin-Fernandez
Life of this patent
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Abstract
Methods and apparatus for measuring fluorescence, are described. The method includes measuring at least one profile of at least a portion of a surface of a sample, the surface extending substantially transverse an axis, and illuminating the portion of the sample surface with radiation for stimulating fluorescence. The intensity of the radiation varies with position along the axis. Values indicative of the spatial intensity distribution of fluorescence emitted from said portion of the sample surface are measured. The measured values of fluorescence are modified to take account of the spatial variation in intensity of radiation incident upon the surface by utilising the measured profile.
Claims17
A method of measuring fluorescence from a sample, comprising the steps of
- measuring at least one profile of at least a portion of a surface of a sample, the surface extending substantially transverse an axis;
- illuminating said portion of the surface of the sample with radiation for stimulating fluorescence, the intensity of the radiation varying with position along the axis;
- measuring values indicative of the spatial intensity distribution of fluorescence emitted from said portion of the sample surface;
- and
- modifying the measured values of fluorescence to take account of the spatial variations in intensity of radiation incident upon the surface by utilising said measured profile.
A method as claimed in any one of the above claims, wherein:
- said step of illuminating said portion of the sample surface comprises providing said radiation in a first polarisation state, and
- said step of measuring values comprises measuring values indicative of the spatial intensity distribution of fluorescence emitted in a first polarisation state from said portion of the sample surface, and measuring values indicative of the spatial intensity distribution of fluorescence emitted in a second, different polarisation state from said portion of the sample surface.
A method as claimed in any one of the above claims, wherein said step of measuring values comprises:
- measuring values indicative of the spatial intensity distribution of fluorescence emitted in a first wavelength range from said portion of the sample surface, and
- measuring values indicative of the spatial intensity distribution of fluorescence emitted in a second, different wavelength range from said portion of the sample surface.
A method as claimed in any one of the above claims, wherein the steps of measuring said profile and measuring said values indicative of the spatial intensity distribution of fluorescence are both repeated over a period of time, for monitoring changes in the conformation of said sample.
A method as claimed in any one of the above claims, wherein the steps of measuring said profile and measuring values indicative of the spatial intensity distribution of fluorescence are performed simultaneously.
A method as claimed in any one of the above claims, wherein the step of measuring said profile comprises:
- illuminating said portion of the sample with a radiation beam, and measuring the spatial variation in intensity of the reflected radiation beam.
A method as claimed in any one of the above claims, wherein said step of measuring said profile comprises measuring the topography of said at least a portion of the surface of the sample.
A method as claimed in any one of the above claims, wherein said sample is a biological cell, the surface of which is defined by a cell membrane.
An apparatus for measuring fluorescence from a sample, the apparatus comprising:
- a sample holder for holding a sample such that at least a portion of a surface of the sample extends substantially transverse a predetermined axis;
- a profilometer for measuring at least one profile of said portion of the surface of the sample;
- a radiation source for illuminating said portion of the surface of the sample with radiation for stimulating fluorescence, the intensity of the radiation varying with position along the axis;
- a detector arranged to measure values indicative of the spatial intensity distribution of fluorescence emitted from said portion of the sample surface;
- and
- a processor arranged to modify the measured values of fluorescence to take account of the spatial variations in intensity of radiation incident upon the surface by utilising the measured profile.
Dependencies are inferred from the claim text.