The critical optical properties of a Gaussian laser beam in two-photon or multiphoton fluorescence imaging, including the beam spot size, depth of focus, and intensity profile, are investigated for spatially locating nanoscale solutes in and surrounding the microvessels of rat brain.

Photon entanglement, the cornerstone of quantum correlations, provides a level of coherence that is not present in classical correlations. Harnessing it by study of its passage through organic matter may offer new possibilities for medical diagnosis technique. In this work, we study the preservation of photon entanglement in polarization, created by spontaneous parametric down-conversion, after one entangled photon propagates through multiphoton-scattering brain tissue slices with different thickness. The Tangle-Entropy (TS) plots show the strong preservation of entanglement of photons propagating in brain tissue. By spatially filtering the ballistic scattering of an entangled photon, we find that its polarization entanglement is preserved and non-locally correlated with its twin in the TS plots. The degree of entanglement correlates better with structure and water content than with sample thickness.

We studied the ability to detect small absorbing objects embedded in a highly scattering medium. Absorbing spheres of varying size, from 0.8 mm to 6.8 mm radius, submerged in a solution of highly scattering, low absorbing liquid: skim milk, were studied in a trans-illumination geometry. Groups of more than one sphere and a single circular disk, with radius identical to that of one of the spheres, were also studied. Single linear raster scans in the plane of the sphere, with the spheres centered between the source and detector, were made. Data was taken in the frequency-domain, yielding profiles of the objects in each of the three measurable quantities: dc intensity, phase, and modulation. The diffraction pattern form the sphere differed from that of the disk, demonstrating a volume effect associated with photon diffusion. The diffraction pattern of multiple spheres differed from that of single spheres.

Light spectroscopy in the frequency-domain has been used to study the optical properties of biological tissues. We have analyzed the possibility of using light emitting diodes (LEDs) as intensity modulated light sources for frequency-domain spectroscopy. The use of LEDs presents several advantages: One LED's output covers a spectral region of about 80 nm, and commercially available LEDs allow for the coverage of the spectral range from 550 to 900 nm, which is a region of interest in near infrared medical applications; the light output of an LED is stable with respect to that of lasers and lamps; the wide angular disthbution make LEDs safe for in vivo studies. Furthermore, LED frequency-domain spectroscopy is a relatively inexpensive technique. We describe some circuits we used to modulate the intensity of LEDs at radio frequency, and point out the possibility of building a multi-source spectrometer. Some applications of LED frequency-domain spectroscopy, both in vitro and in vivo, are shown.

Two-photon (2P) excitation of the second singlet (S₂) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S₂ state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S₂ state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

We have investigated the accuracy of the frequency-domain, diffusion equation Green's function in modeling optical signals in turbid media. Our optical measurements were conducted in strongly scattering media having absorption coefficients in the range 0.035-0.14 cm-1 and reduced scattering coefficients in the range 5-22 cm-1. The optical signal was both delivered to and collected from the sample by means of optical fibers. We have verified that the r-independent factor in the Green's function does not agree with the experimental data. This discrepancy shows that the photons launched in the medium by an optical fiber cannot be modeled by a point source located at a fixed position. These results have no influence on multi-distance measurement protocols (which only employ the r-dependent part of the solution), while they must be considered when calibration measurements on a known sample are performed.

Optical inhomogeneities embedded in a turbid medium are characterized not only by their absorption and reduced scattering coefficients, but also by their index of refraction relative to the background medium. Although in diffusion theory it is impossible to separate the index of refraction from the absorption and reduced scattering coefficients in an infinite homogeneous medium, application of boundary conditions for an inhomogeneity adds enough information to separately determine these optical properties. A mismatched index of refraction affects diffuse photon propagation in two ways: photons travel at a different speed inside the inhomogeneity, and photons entering and leaving the inhomogeneity are influenced by Fresnel reflections at the surface of the object. We have integrated these two effects into the analytical solution to the diffusion equation for a cylinder in an infinite medium. Theoretical results are compared with experimental data, and the effect of index of refraction mismatch is evaluated for different combinations of optical properties.

In recent years researchers have made significant progress in understanding the physics of fluorescence in highly scattering materials such as tissues in the near-infrared. We have quantitatively verified a model which describes fluorescence in ideal (homogeneous and infinite) tissue-like media. Given the quantitative accuracy of this model, one can use measurements of the fluorescence of a tissue with a homogeneous distribution of fluorophore to obtain the quantum yield, lifetime of the probe, and the absorption and scattering coefficients of the tissue at the fluorescent wavelength. We demonstrate that this can be done with a simple measurement of the photon density as a function of source-detector separation at the excitation and emission wavelengths. To verify our approach we present the lifetime, quantum yield of the fluorescent probe (rhodamine B), and the absorption and scattering coefficients of the medium at the emission peak wavelength that are obtained by a fit of the model to experimental measurements.

We have developed a high-bandwidth frequency-domain photon migration (FDPM) instrument which capable of noninvasively determining the optical properties of biological tissues in near-real-time. This portable, inexpensive, diode-based instrument is unique in the sense that we employ direct diode laser modulation avalanche photodiode detection. Diffusion models were used to extract the optical properties (absorption and transport scattering coefficients) of tissue-simulating solution from the 300 kHz to 1 GHz photon density wave data.

In this study, label-free fluorescence spectroscopy was used for the first time to determine spectral profiles of tryptophan, reduced nicotinamide adenine dinucleotide (NADH), and flavin denine dinucleotide (FAD) in fresh brain samples of a mouse model of Alzheimer's disease (AD). Our results showed that the emission spectral profile levels of tryptophan and NADH were higher in AD samples than normal samples. The intensity ratio of tryptophan to NADH and the change rate of fluorescence intensity with respect to wavelength also increased in AD brain. These results yield an optical method for detecting early stage of AD by comparing spectral profiles of biomolecules.