CYCLIC TISSUE CLEARING Nataliya Kozintseva MEPHI Moscow Russia

CYCLIC TISSUE CLEARING Nataliya Kozintseva, MEPHI, Moscow, Russia Arkady S. Abdurashitov, Saratov State University, Saratov, Russia; Tomsk State University, Tomsk, Russia Valery V. Tuchin, Saratov State University, Saratov, Russia; Tomsk State University, Tomsk, Russia; Institute of Precision Mechanics and Control RAS, Saratov, Russia ITMO University, St. Petersburg, Russia

Introduction � Scattering determines the spectral and angular characteristics of light interacting with tissues, as well as the depth of its penetration into the tissue. The optical scattering properties of biological tissue can be controlled by various physical or chemical influences, for example, by compression or stretching of biological tissue, UV irradiation or coagulation, exposure to low temperatures, impregnation with chemical agents. It is well known that the transparency of a dispersed physical system can be effectively controlled by ensuring that the refractive indices of the scattering elements and the base material are matched. This is the socalled immersion technique. The matching of refractive indices manifests itself in a decrease in the scattering coefficient (µs→ 0) and an increase in the directivity of radiation (g→ 1).

Object of research � The human dura mater is a membrane tissue that encompasses the brain. It is a typical fibrous connective tissue that is relatively thick and dense. The dura mater can be thought of as a flat, isotropic material composed of elastic collagen fibers embedded in a matrix composed mainly of mucopolysaccharides and water. Dura mater of a laboratory rat. Samples were removed immediately prior to the experiment and stored for a short time in saline.

Method of research � � The speckle phenomenon is a three-dimensional interference effect that exists at all points in space, where reflected or transmitted waves from an optically rough surface or volume intersect. The presence of particles moving at an arbitrary speed in the structure of the object leads to a local change in the scattering properties of the object in time, which in turn leads to local fluctuations in the intensity of the speckle field. By numerically analyzing the degree of contrast of the intensity distribution, one can judge the presence of moving particles in the object and their speed. Speckle contrast is usually defined as the ratio of the root-mean-square deviation of intensity fluctuations to the average value of these fluctuations in a certain local area: K = σ / ˂I˃

Installation and it’s schematic representation (1 -camera, 2 -laser, 3 sample on a glass plate, 4 -PC, 5 -optical fiber)

Tissue clearing algorithm � � The impact of OCA on tissue is a reversible process. The transition from multiple to single scattering, i. e. the onset of optical transparency and the establishment of linear polarization of the transmitted light is reversible. To demonstrate reversibility, after exposure to OCA (glycerol) for a time sufficient for significant clearing, the sample was immersed in saline for several minutes to wash out the OCA and restore the initial scattering characteristics of the sample (low transmission), and then the saline was replaced with OCA for re-enlightenment. This cyclic tissue clearing was performed several times. The kinetics of optical clearing of tissue using OPA is determined by the characteristic response time, in the case of the dura mater as a test object and glycerol as OCA, from about 1 to 5 minutes. In practice, optical clearing with the help of an OCA is preferable, since it gives more opportunities to control the response and efficiency by using different chemical agents; in addition, the action of these agents can be combined.

The result of the dura mater clearing

Processing of the obtained data � There are two main approaches to analyzing speckle images. It is a temporal and spatial contrast calculation algorithm. In the spatial case, the speckle is calculated - the contrast inside the sliding window. It should be noted that the window size is inversely proportional to the spatial resolution and directly proportional to the reliability of the results obtained. This algorithm has a high temporal resolution. When calculating speckle contrast using a time algorithm, instead of calculating the contrast within one frame, the intensity fluctuations in each pixel of the image are tracked at different times. This algorithm has good spatial resolution. In this study, a spatial speckle contrast calculation algorithm is used to process the obtained images.

� Figures on this slide show speckle patterns at different time intervals, a significant increase in the intensity of the transmitted light is obvious. A decrease in contrast indicates a decrease in scattering, that is, an increase in the degree of enlightenment. When the tissue is re-immersed in the OCA after the next washing, the degree of clearing increases in comparison with the previous dynamics. Thus, using a cyclic clearing algorithm, it is possible to achieve an increase in clearing than with a single immersion of the sample. Speckle pattern recorded at the beginning of the experiment (left) and speckle pattern obtained at the end of the experiment (the result of cyclic clearing, right)

Figure 6. Dependence of speckle contrast on time, red lines show the sample washes in saline

Conclusion � Tissue lightening is one of the main methods of non-invasive diagnosis of diseases. In this work, tissue clarification is combined with laser speckle imaging, which is a powerful, accurate and, at the same time, relatively not expensive and uncomplicated instrument for non-invasive research. In the experiment, glycerin was used as a clarifying agent and the dura mater was used as an object. The object is cleared according to a cyclical algorithm: until significantly noticeable clearing, the object is in glycerin, then it is placed in a saline solution for a couple of minutes until the scattering properties are restored, then the object is again placed in the OCA. Such a multi-stage method leads to a higher degree of clearing of the dura mater, as evidenced by the data obtained as a result of the experiment.