We present spatial light interference microscopy (SLIM) as a new optical microscopy technique, capable of measuring nanoscale structures and dynamics in live cells via interferometry. SLIM combines two classic ideas in light imaging: Zernike’s phase contrast microscopy, which renders high contrast intensity images of transparent specimens, and Gabor’s holography, where the phase information from the object is recorded. Thus, SLIM reveals the intrinsic contrast of cell structures and, in addition, renders quantitative optical path-length maps across the sample. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. We illustrate the novel insight into cell dynamics via SLIM by experiments on primary cell cultures from the rat brain. SLIM is implemented as an add-on module to an existing phase contrast microscope, which may prove instrumental in impacting the light microscopy field at a large scale.
The refractive index of human skin tissues is an important parameter in characterizing the optical response of the skin. We extended a previously developed method of coherent reflectance curve measurement to determine the in vitro values of the complex refractive indices of epidermal and dermal tissues from fresh human skin samples at eight wavelengths between 325 and 1557 nm. Based on these results, dispersion relations of the real refractive index have been obtained and compared in the same spectral region.
Fourier transform light scattering (FTLS) is a novel experimental approach that combines optical microscopy, holography, and light scattering for studying inhomogeneous and dynamic media. In FTLS the optical phase and amplitude of a coherent image field are quantified and propagated numerically to the scattering plane. Because it detects all the scattered angles (spatial frequencies) simultaneously in each point of the image, FTLS can be regarded as the spatial equivalent of Fourier transform infrared spectroscopy, where all the temporal frequencies are detected at each moment in time.
We constructed an automated reflectometry system for accurate measurement of coherent reflectance curves of turbid samples and analyzed the presence of coherent and diffuse reflection near the specular reflection angle. An existing method has been validated to determine the complex refractive indices of turbid samples on the basis of nonlinear regression of the coherent reflectance curves by Fresnel's equations. The complex refractive indices of fresh porcine skin epidermis and dermis tissues and Intralipid solutions were determined at eight wavelengths: 325, 442, 532, 633, 850, 1064, 1310, and 1557 nm.
We derive two mathematical relations between quantitative phase images of thin slices of inhomogeneous media and the scattering parameters of the bulk, i.e., scattering mean free path, l(s), and anisotropy factor, g. The l(s) turns out to be inversely proportional to the spatial variance of the phase shift, and g is related to the variance of the phase gradient. These formulas, referred collectively to as the scattering-phase theorem, allow for extracting l(s) and g in a spatially resolved manner and across an entire tissue section, that is, mapping large cross sections of tissues in terms of l(s) and g.
The lack of a primary method for determination of optical parameters remains a significant barrier in optical study of turbid media. We present a complete system of experimental setups and Monte Carlo modeling tools for fast and accurate solution of the inverse problem from the measured signals of homogeneous turbid samples. The calibration of the instrument and validation of the Monte Carlo modeling have been carried out to ensure the accuracy of the inverse solution. We applied this method to determine the optical parameters of turbid media of 10% intralipid between 550 and 940 nm and 20% intralipid between 550 and 1630 nm.
We employ Fourier-transform light scattering, a technique recently developed in our laboratory, to study the scattering properties of rat organ tissues. Using the knowledge of the complex field associated with high-resolution microscope images of tissue slices, we extracted the scattering mean-free path l(s) and anisotropy factor g, which characterize the bulk tissue for three different rat organs. This "bottom up" approach to measuring tissue scattering parameters allows for predicting the wave transport phenomena within the organ of interest at a multitude of scales-from organelle to organ level.
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