N. Zhadin

Second-harmonic tomography of tissues

A novel noninvasive second-harmonic-generation tomographic method of mapping the structure of animal tissues by use of 100-fs laser pulses at 625nm is described. Subsurface structures were measured with this approach, which is potentially a symmetry-sensitive tool for optical histological reconstruction.

Correction of the Internal Absorption Effect in Fluorescence Emission and Excitation Spectra from Absorbing and Highly Scattering Media: Theory and Experiment

Fluorescence spectra measured from biological samples, such as tissues or cell suspensions, are usually distorted due to the light absorption by intrinsic chromophores. These distortions are aggravated by strong scattering of light inside the samples. A new method is described for a fast correction of these spectral distortions, using only steady-state spectroscopic measurements. The method is based on the formulas derived from a simplified photon diffusion model, in the isotropic one-dimensional approximation applied to a semi-infinite, highly scattering, and moderately absorbing medium with a refractive-index-matched boundary. The formulas describe the spectral distortions of the fluorescence emission and excitation spectra, together with the diffuse reflectance spectrum, as the functions of one spectral characteristic of the medium, the darkness, which is the ratio of absorption coefficient and reduced scattering coefficient. The algorithm does not involve any iterative procedures, and offers a direct, simple, and fast method for real-time spectral correction. The true fluorescence emission or excitation spectrum is directly calculated from a pair of experimental spectra: the fluorescence emission or excitation spectrum and the diffuse reflectance spectrum, measured from the same position on a sample. The correction produces the profile of the true fluorescence spectrum, the same as the one measured from the corresponding sample with an infinitely low absorption and no scattering. The restoration of the spectral profiles of true fluorescence emission and excitation spectra was tested experimentally, using highly scattering phantoms with a fluorescent dye and a deliberately added nonfluorescent dye producing strong inner-filter distortions.

Two-photon excitation of fluorescence from chicken tissue

We have measured UV fluorescence excited through two-photon absorption from native chicken tissue, using 600-nm, 500-fs pulses from a R6G dye laser. The observed emission signal was found to depend quadratically on the excitation intensity. The two-photon excitation-induced fluorescence spectrum is attributed to tryptophan residues in proteins.

Biochemical distinctions between normal and cancerous human breast tissues obtained from fluorescence spectroscopy

A novel method for correcting the fluorescence emission and excitation spectra is applied to native fluorescence spectra from normal and cancerous human breast tissues. The method effectively eliminates the distortions produced by internal light-absorption and allows a direct, real-time, correction without any iterative procedures. A simplified photon- diffusion model was used to develop the method. An analysis of both the true fluorescence spectra, and the diffuse reflectance spectra transformed into the ratio of absorption and reduced scattering coefficients, shows distinctive biochemical differences between cancerous and normal breast tissues. The fluorescence spectra feature a lower contribution of NADH and, possibly, collagen and elastin in cancerous tumor tissues as compared with normal tissues. The fluorescence spectra from cancerous tumors also show a lower degree of variability than the spectra from normal tissues. The corrected spectra from cancerous tumors show a greater similarity in their profiles than the non-corrected fluorescence spectra distorted by the internal light- absorption.

Enhancement of the fluorescence cancer diagnostic method of tissues using diffuse reflectance and the analysis of oxygenation state

We report on an analysis of diffuse reflectance spectra measured in conjunction with the fluorescence from normal human breast tissues and malignant breast tumors. The diffuse reflectance spectra from excised, air-equilibrated, human breast tissue samples show lower fractions of oxygenated hemoglobin and higher content of ferric (Fe3+) heme in malignant breast tumor samples than in normal breast tissues. Normal tissues are found to be easily deoxygenated and reoxygenated, but malignant tumors usually do not change their state as much. An analysis of tissue oxygenation parameters is discussed with respect to an enhancement of predictive power of fluorescence diagnostic method. The oxygenation state of tissues may be used as an additional marker in cancer diagnostics.

Nonlinear optical histological spectroscopy and imaging of biological tissues

We report on nonlinear optical (NLO) spectroscopy and tomographic imaging using second harmonic generation (SHG) and two-photon excitation of fluorescence (TPF) in highly scattering native animal and human tissues.

Second harmonic and two-photon fluorescence histology of tissues

Second harmonic and two photon fluorescence generated by focused 10 fs ultrashort laser pulses were used to image subsurface structures of highly scattering biological tissues. By scanning the focal point of the incident laser beam, 2D sectional images of the tissues subsurface structure properties, such as symmetry and molecular compositions are obtained.