Optical coherence microscope and methods of use for rapid in vivo three-dimensional visualization of biological function

A method of visualizing a structure beneath a surface of a biological sample comprising the steps of:

• providing light having a wavelength between 700 and 1500 nm;
• dividing the light into a sample light path and a reference light path;
• modulating the length of at least one of the light paths with a piezoelectric device, wherein the modulation of the path length occurs at a frequency, f, of at least 50 kHz and amplitude of modulation is less than 0.50 of the wavelength;
• directing light from the sample path into the biological sample with a focusing lens, wherein the light enters the biological sample and tapers to a beam waist within the sample,
• scanning the beam waist across a first plane substantially normal to the direction of the incident light beam;
• combining a light beam from the sample light path scattered back from the biological sample with a light beam from the reference light path reflected by a reference mirror, wherein the combination of light beams produces an interference fringe signal on a photodetector when the sample light path length and the reference light path length are substantially the same to within the coherence length of the light source;
• moving the beam waist to a different depth within the sample and scanning another plane while the position of the reference mirror is translated to keep the equal path lengths of the sample and reference light paths coincident with the beam waist;
• summing the powers in the fringe signal at a fundamental frequency, f, and a second harmonic of the fundamental frequency, 2f, to provide a measure of the amplitude of the fringes;
• and
• recording the fringe amplitude at each volume element, voxel, during the scan of the sample resulting in a three-dimensional data set that is volume-rendered to provide a three-dimensional visualization of the sample.

A high resolution optical coherence microscope system for visualizing structures below a surface of a biological sample, the system comprising:

• a light source emitting light in a wavelength of between 700 and 1500 nm;
• a reference mirror:
• a focusing lens for focusing light on the biological sample;
• a photodetector;
• a sample optical path for receiving light from the light source and directing the light to the focusing lens, said sample path having a sample path length, wherein the light directed along the sample path enters the biological sample and tapers to a beam waist diameter within the sample;
• a reference optical path for receiving light from the light source and directing the light to the reference mirror, said reference path having a reference path length
• a piezoelectric device, wherein the length of at least one of the optical paths is modulated by the piezoelectric device, and wherein a light beam from the sample optical path scattered back from the biological sample is combined with a light beam from the reference path reflected from the reference mirror, wherein the combination of light beams produces an interference fringe signal on the photodetector when the sample path length and the reference path length are substantially the same to within the coherence length of the light source, wherein an amplitude of modulation is equal to or less than 1.0 of the wavelength, and the powers in a fringe signal at two adjacent harmonic frequencies of the fundamental frequency, f, are summed to provide a measure of the amplitude of the fringes.
• a scanner ranged to scan the beam waist across a first plane substantially normal to the direction of the incident light beam, and to then move the beam waist deeper into the sample and to scan another plane while the position of the reference mirror is translated to keep the equal path lengths of the sample and reference paths coincident with the beam waist, and that the fringe amplitude is recorded at each volume element, voxel, during the scan of the sample resulting in a three-dimensional data set that is volume-rendered to provide a three-dimensional visualization of the sample.