Analysis of tissue microstructure with Mueller microscopy: logarithmic decomposition and Monte Carlo modeling.

Significance: Definitive diagnostics of many diseases is based on the histological analysis of thin tissue cuts with optical white light microscopy. Extra information on tissue structural properties obtained with polarized light would help the pathologist to improve the accuracy of his diagnosis.

Aim: We report on using Mueller matrix microscopy data, logarithmic decomposition, and polarized Monte Carlo (MC) modeling for qualitative and quantitative analysis of thin tissue cuts to extract the information on tissue microstructure that is not available with a conventional white light microscopy.

High-resolution cost-effective compact portable inverted light microscope.

Commercial high-resolution optical microscopes are essential for microscopy imaging; however, they are expensive and bulky, which limits their use in point-of-care devices, resource-limited areas, and real-time imaging of a sample in a large apparatus. In this study, we report a novel compact (10 cm × 5 cm × 5 cm, without the light source) lightweight (∼0.5 kg) submicron-resolution inverted optical microscope at low cost (∼$ 300). Our technique utilises the proximity of the image sensor to a commercial microscope objective lens for compactness of the microscope. The use of an image sensor with a small pixel size helps to reduce the information loss, which provides high-resolution images. Moreover, our technique offers a freedom to tailor the design of microscope according to the required resolution, cost, and portability for specific applications, which makes it a suitable candidate for affordable point-of-care devices. Images of several micron-to-submicron scale patterns and spherical beads are acquired to observe the resolution and quality of the images obtained using our microscope. In addition, we demonstrate the applications of our microscope in various fields such as recording of high-speed water microdroplet formation inside a microfluidic device, high-resolution live cell imaging inside an incubator, and real-time imaging of crack propagation in a sample under stretching by a material testing system (MTS). Therefore, this portable and inexpensive microscope provides the essential functionalities of a bulky expensive high-performance microscope at a lower cost. LAY DESCRIPTION: Microscope is an essential tool in research allowing for observation of microsized objects and life forms. Contemporary commercial high-resolution microscopes have long optical paths involving series of lenses and filters. Although this configuration precisely corrects for optical distortions and produces clear images, it makes modern microscopes very costly and bulky, restricting their usage to low-funded research laboratories and at remote places. We have developed a simple digital microscope with high-resolution but with much smaller size and lighter in weight at low cost by removing the long optical terrain. Our microscope consists of a commercial microscope objective lens for magnification and semiconductor image sensor with small pixels placed right after the lens, both of which are affordable and easily available. The small pixel size helps to translate the magnified analogue sample image to high-resolution digital image. In our paper, we show that our microscope can view micro and submicron-sized patterns and beads. Moreover, our fist-sized microscope can be placed inside an incubator for real-time imaging of cells or rotated sideways for recording submicron-sized crack generation due stretching of novel materials, both of which could not be accomplished with the 2 feet tall laboratory microscopes.

Quantitative assessment of birefringent skin structures in scattered light confocal imaging using radially polarized light.

The polarization characteristics of birefringent tissues could be only partially obtained using linearly polarized light in polarization sensitive optical imaging. Here we analyze the change in polarization of backscattered light from birefringent structures versus the orientations of the incident polarizations using linearly, circularly and radially polarized light in a cross-polarized confocal microscope. A spatially variable retardation plate composed of eight sectors of λ/2 wave plates was used to transform linearly polarized light into a radially polarized light. Based on the experimental data obtained from ex-vivo measurements on human scalp hairs and in-vivo measurements on hair and skin, we exemplify that the underestimation of the birefringence content resulting from the orientation related effects associated with the use of linearly polarized light for imaging tissues containing wavy birefringent structures could be minimized by using radially polarized light.

A simplified, low-cost method for polarized light microscopy.

Malaria pigment is an intracellular inclusion body that appears in blood and tissue specimens on microscopic examination and can help in establishing the diagnosis of malaria. In simple light microscopy, it can be difficult to discern from cellular background and artifacts. It has long been known that if polarized light microscopy is used, malaria pigment can be much easier to distinguish. However, this technique is rarely used because of the need for a relatively costly polarization microscope. We describe a simple and economical technique to convert any standard light microscope suitable for examination of malaria films into a polarization microscope.

Mueller matrix decomposition for polarized light assessment of biological tissues.

The Mueller matrix represents the transfer function of an optical system in its interactions with polarized light and its elements relate to specific biologically or clinically relevant properties. However, when many optical polarization effects occur simultaneously, the resulting matrix elements represent several "lumped" effects, thus hindering their unique interpretation. Currently, no methods exist to extract these individual properties in turbid media. Here, we present a novel application of a Mueller matrix decomposition methodology that achieves this objective. The methodology is validated theoretically via a novel polarized-light propagation model, and experimentally in tissue simulating phantoms. The potential of the approach is explored for two specific biomedical applications: monitoring of changes in myocardial tissues following regenerative stem cell therapy, through birefringence-induced retardation of the light's linear and circular polarizations, and non-invasive blood glucose measurements through chirality-induced rotation of the light's linear polarization. Results demonstrate potential for both applications.

Simulation of polarization-sensitive optical coherence tomography images by a Monte Carlo method.

We introduce a new Monte Carlo (MC) method for simulating optical coherence tomography (OCT) images of complex multilayered turbid scattering media. We demonstrate, for the first time of our knowledge, the use of a MC technique to imitate two-dimensional polarization-sensitive OCT images with nonplanar boundaries of layers in the medium like a human skin. The simulation of polarized low-coherent optical radiation is based on the vector approach generalized from the iterative procedure of the solution of Bethe-Saltpeter equation. The performances of the developed method are demonstrated both for conventional and polarization-sensitive OCT modalities.

Orthogonal polarization technique in the assessment of human skin microcirculation.

BACKGROUND: The "gold standard" for the study of the in vivo microcirculation is intravital microscopy. The recently developed method of orthogonal polarization of light [orthogonal polarization spectral (OPS) imaging] allows for the in vivo transcutaneous evaluation of the microcirculation without the need for invasive surgical procedures. METHODS: The application of polarized light originating from a 100 W halogen tungsten lamp is able to penetrate tissues at a depth of up to 3 mm, and generates reissued light from this depth. The evaluation of this depolarized light, from a deeper origin, may be carried out separately from the light reflected by the more superficial layers of the tissue under study because this light retains photon polarization, whereas the former light undergoes real depolarization. RESULTS: The process of validation of the OPS technique, when compared with intravital microscopy, the "gold standard" for the in vivo observation of the microcirculation, has shown that it is as effective and reliable as the gold standard, reaching the same resolution level in the visualization of blood vessels, but without the need for invasive surgical procedures. CONCLUSIONS: The OPS technique is a very promising tool for dermatologists and researchers, especially in the study of vasculitis, chronic venous insufficiency, and skin tumors.

Polarized light propagation in multiply scattering media exhibiting both linear birefringence and optical activity: Monte Carlo model and experimental methodology.

A Monte Carlo model for polarized light propagation in birefringent, optically active, multiply scattering media is developed in an effort to accurately represent the propagation of polarized light in biological tissue. The model employs the Jones N-matrix formalism to combine both linear birefringence and optical activity into a single effect that can be applied to photons as they propagate between scattering events. Polyacrylamide phantoms with strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres as scattering particles are used for experimental validation. Measurements are made using a Stokes polarimeter that detects scattered light in different geometries, and compared to the results of Monte Carlo simulations run with similar parameters. The results show close agreement between the experimental measurements and Monte Carlo calculations for phantoms exhibiting turbidity and birefringence, as well as for phantoms exhibiting turbidity, birefringence, and optical activity. Other scattering-independent polarization properties can be incorporated into the developed Jones N-matrix formalism, enabling quantification of the polarization effects via an accurate polarization-sensitive Monte Carlo model.

Perturbation and differential Monte Carlo methods for measurement of optical properties in a layered epithelial tissue model.

The use of perturbation and differential Monte Carlo (pMC/dMC) methods in conjunction with nonlinear optimization algorithms were proposed recently as a means to solve inverse photon migration problems in regionwise heterogeneous turbid media. We demonstrate the application of pMC/dMC methods for the recovery of optical properties in a two-layer extended epithelial tissue model from experimental measurements of spatially resolved diffuse reflectance. The results demonstrate that pMC/dMC methods provide a rapid and accurate approach to solve two-region inverse photon migration problems in the transport regime, that is, on spatial scales smaller than a transport mean free path and in media where optical scattering need not dominate absorption. The pMC/dMC approach is found to be effective over a broad range of absorption (50 to 400%) and scattering (70 to 130%) perturbations. The recovery of optical properties from spatially resolved diffuse reflectance measurements is examined for different sets of source-detector separation. These results provide some guidance for the design of compact fiber-based probes to determine and isolate optical properties from both epithelial and stromal layers of superficial tissues.

Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study.

Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis in vivo. Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg x bw(-1)) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral imaging. Data for functional vessel density correlated excellently with data obtained by fluorescence microscopy (y=0.99x+0.48, r2=0.97, R(S)=0.98, precision: 8.22 cm(-1) and bias: -0.32 cm(-1)). Correlation parameters for diameter of microvessels and red blood cell velocity were similar (r2=0.97, R(S)=0.99 and r2=0.93, R(S)=0.94 for diameter of microvessels and red blood cell velocity, respectively). Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8+/-2.1 and 87.2+/-10.2 cm(-1) compared to values of control animals of 66.6+/-10.1 and 147.4+/-13.2 cm(-1), respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours.

New microscope gives scientists the inside scoop on living cells.

Scientists at Pacific Northwest National Laboratory in Richland, Washington, have developed a unique imaging system capable of focusing on a single living cell within an organism. This new technology will be used in what the multidisciplinary team has termed a "cellular observatory" to study the effect of environmental insults to live cells.