Two-photon microscopy methods have been actively developing in the last two decades. In particular, various approaches are being developed to analyze metabolic activity obtained by laser microscopy with high temporal resolution. One such approach is ploting the autofluorescence lifetime signal data to the phasor plot and observe deviations from the normal state. This tool has proven itself well for analyzing the metabolic activity of biological objects, including bacteria. This study analyzes the antimicrobial activity for MRSA bacteria by phasor plot approach. It was possible to show that, the lifetime of autofluorescence in the phasor plane changes depending on the type of nanoparticles used, with which bacteria are incubated. This result can be used in the future for rapid assessment of the antimicrobial activity of nanoparticles.
Terahertz (THz) spectroscopy of skin pathologies associated with water migration and content were analyzed using the method of immersion optical clearing. It shows different behavior THz spectra for healthy and diabetic skin after application of an optical clearing agent. It method can be used to identify skin pathologies. There is a migration of water and contents from various layers of the skin and dehydration of the skin due to the application of the optical clearing agent.
A model of lymphedema in laboratory animals was realized and analyzed with multiphoton microscopy (MPM). Computer vision methods were used to describe the textures of lymphedema tissue MPM images, then informative features of the fibrosis development were extracted. Besides, the ratios of the autofluorescence lifetime to the second harmonic signal were estimated.
Terahertz medical imaging is an expanding field of research in the diagnosis of several medical conditions, including wound healing. Study was carried out in rat models where a THz imaging system was used to mark the difference in absorption spectra between a wound and a healthy tissue. Absorption spectra of the wound and healthy tissues have been measured in the 0.3-1.5 THz range.
Non-invasive diagnosis of diseases using analysis of exhaled air are actively developing in medical practice. The aim of work is to compare a specifics groups of patients with pulmonary diseases. As features of the participants' state, absorption spectra of exhaled air samples were used. The analyzed spectrum is a bar chart which describes the dependence of the absorption coefficient on the wavelength. The problem to be solved is the choice of informative sub-ranges of spectra to improve the classification of the studied groups and the subsequent classification of several spectra for one person by voting methods. An integrated approach was used to solve this problem using the principal component analysis, support vector machine with RBF core and the subsequent voting technique.
Measurements and modeling of the optical properties of adipose tissue and its components in the terahertz range with a change in tissue temperature were carried out. It was shown that the optical density (OD) of adipose tissue samples decreases with increasing temperature, which can be mainly associated with dehydration of the sample. We can also expect some contribution to the decrease in the OD of suppression of THz wave scattering when matching the refractive indices of scatterers and their environment due to the intake of free fatty acids secreted by adipocytes due to thermally induced cell lipolysis. It is shown that in the experimental model, the difference between the THz absorption spectra of water and oil allows us to estimate the water content in adipose tissue. A comparison of the measurement results and molecular modeling in the terahertz region confirmed the hypothesis about the reasons for the change in the optical properties of heated adipose tissue.
The terahertz (THz) attenuated total reflectance (ATR) imaging of normal and pathological skin under the action of various dehydration agents was carried out in vivo. Studies were conducted on animal models (the mouse), patients with diabetes, and healthy volunteers. For measurements, each animal was leaned against the ATR prism of the skin surface, and several locations in the skin of each animal were analyzed. Places on the skin for analysis were chosen so that the intensity spectra of the THz signal were practically the same for selected points. THz spectra measurements were carried out every 10 minutes within 45 minutes interval under the action of a dehydration agent. 40% glucose was shown to provide the most effective improving tissue optical clearing effect in the THz range.
The ability of diagnostics of melanoma and nevus based on spectral analysis of paraffin-embedded tissues in the 0.3-1.5 terahertz (THz) range has been carried out. The principal component analysis was applied to reduce the dimension of the feature space. A comparison of these spectra shows evident differences between samples. The possibility of applying the optical clearing to paraffin-embedded tissue for improving the visualization of the internal structure of tissue for diagnostic and research purposes is shown. In the studies, a sufficiently strong effect of optical clearing of paraffin-embedded muscle was obtained (63%).
The results of studies of exosome visualization obtained using multiphoton fluorescence spectroscopy for healthy volunteers and patients with colorectal cancer are presented. Standard centrifuging technology was used to isolate exosomes from blood plasma. Exosome samples were investigated using the femtosecond laser pumping at the wavelength of 760 nm with and without FITC (Fluorescein isothiocyanate) labels. In particular, the exosomes with CD9 surface glycoprotein of the tetraspanin family was shown to be studied without labeling using FLIM (fluorescent lifetime microscopy).
A model of lymphedema was developed using laboratory animals. During lymphatic edema enhancement, a study was made of changes in the structure of biological tissues by interference optical methods in the near infrared region of the spectrum. Statistical methods were applied for describing textures of the experimental images of the affected tissues, informative features of the dynamics of fibrosis development were identified.
Methods of two-photon microscopy are widely used in the study of biological objects, in particular, skin, due to the possibility to study objects both on the surface and at depth without attracting additional fluorophores due to endogenous autofluorescence. In this paper, the methods of image analysis of the AF signal and SHG signal are applied to assess the condition of the skin during the development of lymphedema. It is shown that for groups of healthy tissue and lymphedematous using SAAID distribution histograms, changes in tissues can be detected.
The of multiphoton microscopy (MPM) based on two-photon fluorescence and second-harmonic generation is promising for diagnosing neoplastic transformation of tissues. The results of MPM studies of tissues with prostate adenocarcinoma embedded in paraffin blocks are presented. The tissue fluorescence was also analyzed using the fluorescence lifetime (Fluorescence-lifetime imaging microscopy - FLIM) option. The relationships between the fluorescence peculiarities and the second harmonic generation of the prostate gland tissues were obtained for different morphological images.
In the present study, observations of temperature-mediated phase transitions of the components of adipose tissue were done. Pieces of pork abdominal fat were used as samples of adipose tissue. These samples were heated using a temperature-controlled table and observed using a MPTflex two-photon microscope (JenLab GmbH) with fluorescence lifetime imaging microscopy (FLIM) mode. The analysis of FLIM data was done with a software package “Becker&Hickl”. The developed approach has a great potential for obtaining accurate information on the phase-transition processes occurring during metabolism alterations.
Last years the development of computer-aided diagnostic systems for medical image analysis has become a hot topic. A key step is connected with informative features extraction. Here, we discussed multiphoton microscopy and optical coherent tomography lymphedema tissue images analysis using gradient processing methods.
The problem of extracting useful information for medical diagnosis from 2D and 3D optical imaging experimental data is of great importance. We are discussing challenges and perspectives of medical diagnosis using machine learning analysis of NIR and THz tissue imaging. The peculiarities of tissue optical clearing for tissue imaging in NIR and THz spectral ranges aiming the improvement of content data analysis, methods of extracting of informative features from experimental data and creating of prognostic models for medical diagnosis using machine learning methods are discussed.
In our study we used rank-order filter, the emissions filter on the base of the criteria of Pearson, Gaussian filter and median filterfor improving the is fluorescence lifetime imaging microscopy (FLIM) data. The data obtained with the FLIM technology are the distribution with a pronounced peak, while during measurement the peak value is measured with an error. According to the analysisthe Gaussian filter is more useful to improve quality of FLIM data.Spatial filtering allows to reduce the noise component, obtained in the course of measurements, including reduction the influence of the individual bursts. Filtering in time scale allows to determine a peak value of intensity more accurately.This research was carried out using the equipment of Tomsk Regional Common Use Center of Tomsk State University.
The kernel construction for the biomedical data classification using Support Vector Machine based on Green function for Ornstein-Uhlenbeck equation is presented. Quantitative estimates of classification quality of exhaled air samples absorption spectra for patients with chronic obstructive pulmonary disease and healthy volunteers were carried out.
We examined possibilities of the Kalman filter for reducing the noise effects in the analysis of absorption spectra of gas samples, in particular, for samples of the exhaled air. It has been shown that when comparing groups of patients with broncho-pulmonary diseases on the basis of the absorption spectra analysis of exhaled air samples the data preprocessing with the Kalman filtering can improve the classification sensitivity using a support vector kernel with mpl.
The results of numerical simulation of application principal component analysis to absorption spectra of breath air of patients with pulmonary diseases are presented. Various methods of experimental data preprocessing are analyzed.
The results of comparison of quality of two classificators – SVM (support vector machine) and SIMCA (soft independent modelling of class analogies) on model data contained profiles of absorbtion specra of exhalted air are presented. It is shown, that SVM classification results can be improved by preprocessing if input data with principal component analysis method.
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