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AssigneeDigital Optical Imaging Corporation
InventorCalum E. MacAulay
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Apparatus and methods that improve the depth resolution of confocal microscopy images using out-of-focus information from within the focal plane of interest (from the x-y direction) and/or from planes above and below the focal plane of interest (from the z-direction). The interaction of (a) a reflective surface or other light-emanating material and (b) the PSF formed by a confocal microscope results in “out-of-focus” information in, above and below the focal plane; this “out-of-focus” information can be measured. Comparing the measurements in the x-y plane, preferably at a plurality of z-positions, can improve the resolution along each of the x, y, and z-axes, increase the number of the photons used in the system, thus improving the signal to noise ratio, and help correct for aberrations, such as spherical aberrations or other optical aberrations, in the optical system of a microscope.
A confocal microscope comprising a light detection and analysis system, the system comprising a light detector disposed downstream from a sample substantially in a conjugate image plane of the sample, wherein the detector comprises a central detection pixel positioned to detect and measure in-focus light emanating from a discrete illumination pixel of the sample to provide in-focus data and at least one adjacent detection pixel in an x-y plane relative to the sample and positioned to independently detect and measure out-of-focus light emanating from the discrete illumination pixel of the sample in the x-y plane to provide out-of-focus data in the x-y plane, the system further comprising a controller operably connected to the detector and containing computer implemented programming that compiles and combines the in-focus data and the out-of-focus data to enhance resolution of the discrete illumination pixel of the sample when compared to a resolution obtained without using the out-of-focus data, wherein the detector comprises a plurality of adjacent detection pixels that surround the central detection pixel and that independently detect and measure out-of-focus light emanating from the discrete illumination pixel of the sample, and wherein the detector is movably connected to the sample along a z-axis of the sample such that movement of the detector relative to the sample permits the detector to detect and measure in-focus data from a focal plane of the sample along the z-axis and out-of-focus data from above or below the focal plane along the z-axis.
A microscope comprising a light detection and analysis system, the system comprising a light detector disposed downstream from a sample substantially in a conjugate image plane of the sample, wherein the detector is movably connected to the sample along a z-axis of the sample such that movement of the detector relative to the sample permits the detector to detect and measure in-focus data from a focal plane of the sample along the z-axis and out-of-focus data from above and below the focal plane along the z-axis, the system further comprising a controller operably connected to the detector and containing computer implemented programming that compiles and combines the in-focus data from along the z-axis and the out-of-focus data from along the z-axis to enhance resolution of the discrete illumination pixel of the sample when compared to a resolution obtained without using the out-of-focus data from along the z-axis.
A system for a confocal microscope, the system comprising a controller operably connected to a digital light detector disposed downstream from a sample substantially in a conjugate image plane of a sample, the detector comprising a central detection pixel positioned to detect and measure in-focus light emanating from a discrete illumination pixel of the sample to provide in-focus data and at least one adjacent detection pixel in an x-y plane relative to the sample and positioned to independently detect and measure out-of-focus light emanating from the discrete illumination pixel of the sample in the x-y plane to provide out-of-focus data in the x-y plane, wherein the controller further contains computer implemented programming that compares the in-focus data and the out-of-focus data to enhance resolution of the discrete illumination pixel of the sample when compared to a resolution obtained without using the out-of-focus data, wherein the detector of the microscope is movably connected to the sample along a z-axis of the sample such that movement of the detector relative to the sample permits the detector to detect and measure in-focus data from a focal plane of the sample along the z-axis and out-of-focus data from above and below the focal plane along the z-axis, wherein the controller further contains computer implemented programming that compiles and combines the in-focus data from along the z-axis and the out-of-focus data from along the z-axis to enhance resolution of the discrete illumination pixel of the sample when compared to a resolution obtained without using the out-of-focus data from along the z-axis.
A controller suitable for operable connection to a microscope comprising a light detector disposed downstream from a sample substantially in a conjugate image plane of a sample and movably connected to the sample along a z-axis of the sample such that movement of the detector relative to the sample permits the detector to detect and measure in-focus data from a focal plane of the sample along the z-axis and out-of-focus data from above and below the focal plane along the z-axis, wherein the controller contains computer implemented programming that compiles and combines the in-focus data from along the z-axis and the out-of-focus data from along the z-axis to enhance resolution of the discrete illumination pixel of the sample when compared to a resolution obtained without using the out-of-focus data from along the z-axis.
A confocal microscope comprising
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