Urszula Zabarylo

Fluorescence Imaging of Heat-Stress Induced Mitochondrial Long-Term Depolarization in Breast Cancer Cells

Various thermotherapies are based on the induction of lethal heat in target tissues. Spatial and temporal instabilities of elevated temperatures induced in therapy targets require optimized treatment protocols and reliable temperature control methods during thermotherapies. Heat-stress induced effects on mitochondrial transmembrane potentials were analyzed in breast cancer cells, species MX1, using the potential sensor JC-1 (Molecular Probes, Invitrogen, Germany). Potential dependant labeling of heat-stressed cells was imaged and evaluated by fluorescence microscopy and compared with control cells. JC-1 stains mitochondria in cells with high mitochondrial potentials by forming orange-red fluorescent J-aggregates while in cells with depolarized or damaged mitochondria the sensor dye exists as green fluorescent monomers. In MX1 cells orange-red and green fluorescence intensities were correlated with each other after various heat-stress treatments and states of mitochondrial membrane potentials were deduced from the image data. With increasing stress temperatures the intensity of red fluorescent J-aggregates decreased while the green fluorescence intensity of JC-1 monomers increased. This heat-stress response happened in a nonlinear manner with increasing temperatures resulting in a nonlinear increase of red/green fluorescence ratios. These data indicated that mitochondria in MX1 cells were increasingly depolarized in response to increasing ambient temperatures.

LED-based near infrared sensor for cancer diagnostics

Optical spectroscopic technologies are increasingly used for cancer diagnostics. Feasibility of differentiation between malignant and healthy samples of human kidney using Fluorescence, Raman, MIR and NIR spectroscopy has been recently reported . In the present work, a simplification of NIR spectroscopy method has been studied. Traditional high-resolution NIR spectrometry was replaced by an optical sensor based on a set of light-emitting diodes at selected wavelengths as light sources and a photodiode. Two prototypes of the sensor have been developed and tested using 14 in-vitro samples of seven kidney tumor patients. Statistical evaluation of results using principal component analysis and partial least-squares discriminant analysis has been performed. Despite only partial discrimination between tumor and healthy tissue achieved by the presented new technique, the results evidence benefits of LED-based near-infrared sensing used for oncological diagnostics. Publishers Note: This paper, originally published on 4 March, 2016, was replaced with a corrected/revised version on 7 April, 2016. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.

Diagnosis of rheumatoid arthritis using light: correction of motion artefacts and color visualization of multispectral images

State-of-the-art image-processing methods offer new possibilities for diagnosing diseases using scattered light. The optical diagnosis of rheumatism is taken as an example to show that the diagnostic sensitivity can be improved using overlapped pseudocolored images of different wavelengths, provided that multispectral images are recorded to compensate for any motion-related artefacts that occur during examination.

Comparison of Photo Optical Imaging with Musculoskeletal Ultrasound and Clinical Examination in the Assessment of Inflammatory Activity in Proximal Interphalangeal Joints in Rheumatoid Arthritis and Osteoarthritis.

Objective. Lightscan is a novel, rapid, low-cost, easily operated and noninvasive imaging technology used to assess inflammatory activity in proximal interphalangeal (PIP) joints. The results are calculated automatically. To our knowledge, this is the first comparative study of photo optical imaging (POI), with clinical examination (CE), disease activity score at 28 joints (DAS28)-erythrocyte sedimentation rate (ESR), and musculoskeletal ultrasonography (US) in healthy subjects and patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Methods. There were 688 PIP joints of both hands examined in 87 subjects (38 RA, 21 OA, 28 healthy) by Lightscan and compared with CE for clinically swollen and tender joints, DAS28-ESR (only RA), and US. Results. With US as reference, POI had a sensitivity of 74% and a specificity of 93%. In the receiver-operating curve (ROC) analysis, the Lightscan showed a higher sensitivity and specificity [area under the curve (AUC) 0.879] for the distinction of healthy subjects versus patients (OA, RA) than US in greyscale (GSUS; AUC 0.797) and power Doppler (PDUS; AUC 0.67). POI correlated significantly with GSUS (r 0.473, p < 0.01) and PDUS (r 0.486, p < 0.01). The agreement rates between POI and GSUS were up to 79%, between POI and PDUS up to 92%, and between POI and CE up to 66%. POI did not correlate with DAS28-ESR. Conclusion. The Lightscan is a new technology offering sensitive imaging detection of inflammatory changes in subjects with RA and OA with PIP arthritis. POI was more sensitive than CE and correlated significantly to GSUS and PDUS, while presenting a higher sensitivity and specificity for the detection of healthy subjects versus patients (RA, OA) based on the ROC analysis.

Registration of scattered laser images and radiographs of small finger joints

Abstract Image processing methods for the registration of scattered light pictures and X-ray radiographs can improve the diagnostic yield of rheumatoid joints as compared to a separate interpretation of radiographs and optical images. The image fusion described uses thin plate spline interpolation techniques. This article also describes the image pre-processing techniques necessary to identify intrinsic landmarks that are a prerequisite for a point-based registration technique. Zusammenfassung Bildverarbeitungsmethoden für die Registrierung von Streulichtbildern und Röntgenaufnahmen können die Diagnostik der rheumatoiden Arthritis im Vergleich zur separaten Interpretation der Röntgenbilder und der optischen Bilder verbessern. Die Fusionierung der Bilder wird am Beispiel des sogenannten „Thin Plate Spline“ RBF-Interpolationsverfahrens dargestellt. Der Artikel beschreibt die notwendigen Vor-Verarbeitungsmethoden zur Bestimmung der intrinsischen Landmarker als Voraussetzung einer punktbasierten Registrierung.

Development and Testing of an LED-Based Near-Infrared Sensor for Human Kidney Tumor Diagnostics

Optical spectroscopy is increasingly used for cancer diagnostics. Tumor detection feasibility in human kidney samples using mid- and near-infrared (NIR) spectroscopy, fluorescence spectroscopy, and Raman spectroscopy has been reported (Artyushenko et al., Spectral fiber sensors for cancer diagnostics in vitro. In Proceedings of the European Conference on Biomedical Optics, Munich, Germany, 21–25 June 2015). In the present work, a simplification of the NIR spectroscopic analysis for cancer diagnostics was studied. The conventional high-resolution NIR spectroscopic method of kidney tumor diagnostics was replaced by a compact optical sensing device constructively represented by a set of four light-emitting diodes (LEDs) at selected wavelengths and one detecting photodiode. Two sensor prototypes were tested using 14 in vitro clinical samples of 7 different patients. Statistical data evaluation using principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) confirmed the general applicability of the LED-based sensing approach to kidney tumor detection. An additional validation of the results was performed by means of sample permutation.

Optical clearing of human skin for the enhancement of optical imaging of proximal interphalangeal joints

We are proposing a new method for enhancement of optical imaging of proximal interphalangeal (PIP) joints in humans at skin using optical clearing technique. A set of illuminating laser diodes with the wavelengths 670, 820, and 904 nm were used as a light source. The laser diodes, monochromatic digital CCD camera and specific software allowed for detection of the finger joint image in a transillumination mode. The experiments were carried out in vivo with human fingers. Dehydrated glycerol and hand cream with urea (5%) were used as optical clearing agents (OCAs). The contrast of the obtained images was analyzed to determine the effect of the OCA. It was found that glycerol application to the human skin during 60 min caused the decrease of contrast in 1.4 folds for 670 nm and the increase of contrast in 1.5 and 1.7 folds for 820 nm and 904 nm, respectively. At the same time, the hand cream application to the human skin during 60 min caused the decrease of contrast in 1.1 folds for 670 nm and the increase of contrast in 1.3 and 1.1 folds for 820 nm and 904 nm, respectively. The results have shown that glycerol and the hand cream with 5% urea allow for obtaining of more distinct image of finger joint in the NIR. Obtained data can be used for development of optical diagnostic methods of rheumatoid arthritis.

Optical monitoring of rheumatoid arthritis : Monte Carlo generated reconstruction kernels

Optical imaging in biomedicine is governed by the light absorption and scattering interaction on microscopic and macroscopic constituents in the medium. Therefore, light scattering characteristics of human tissue correlate with the stage of some diseases. In the near infrared range the scattering event with the coefficient approximately two orders of magnitude greater than absorption plays a dominant role. When measuring the optical parameters variations were discovered that correlate with the rheumatoid arthritis of a small joint. The potential of an experimental setup for transillumination the finger joint with a laser diode and the pattern of the stray light detection are demonstrated. The scattering caused by skin contains no useful information and it can be removed by a deconvolution technique to enhance the diagnostic value of this non-invasive optical method. Monte Carlo simulations ensure both the construction of the corresponding point spread function and both the theoretical verification of the stray light picture in rather complex geometry.

Single- and multispectral fiber probes as a solution for guided diagnostics and therapy (Conference Presentation)

Medical fiber optics as a part of laboratory and clinical diagnostic tools, surgical instrumentation and endoscopy should satisfy modern biomedical requirements. The flexibility, small size, bio-compatibility and feasibility to use sterilisable or disposable parts allow to apply fiber optic probes in clinical environment both ex-vivo and in-vivo. To enable spectroscopic differentiation cancer and normal tissue, we developed and applied various single and combined fiber optic probes using key spectroscopy methods such as Raman scattering, Mid IR-absorption, Diffuse NIR-reflection, and fluorescence in order to compare them and select the best combination for malignant detection of tissue in clinical environment. All four spectroscopic methods have been tested on biopsies of health and malignant tissues (colon and kidney) and bioliquids (serum, plasma and urine) of patients before and after surgery. The tiny Raman probe with 1.5 mm diameter has been developed for experimental ex-vivo tests. Further multivariate data analysis of spectroscopic data, both individual techniques and their combinations has provided a reliable cancer recognition for colon and kidney biopsies. The best synergic gain was observed of combining Mid IR-absorption and fluorescence spectroscopy (98% Sensitivity vs 63% or 88% for fluorescence or Mid IR-absorption correspondingly). Based on obtained results, both techniques were implemented within the same fiber probe to provide a simultaneous measurement of exactly the same spot at the sample surface. An innovative combi Fluo-ATR probe was designed, fabricated and tested. Diffuse NIR-reflection and fluorescence spectroscopy as the fastest measurement techniques were used for investigation of surgically removed colorectal tissue samples in a few minutes after resection. Taking into account the necessity of rapid measurement process, we developed multispectral NIR-UV probe that enables efficient excitation and collection of NIR diffuse reflectance and fluorescence spectra of the same point. Needle probe design with diameter 0.7 mm allows to penetrate in some depth of tissue and obtain most reliable spectra avoiding artefacts related to surface drying. Multivariate data analysis proved the increased sensitivity of methods combination in one dataset (94% Sensitivity vs 85% or 85% for fluorescence or NIR reflection correspondingly).

Synergy Effect of Combining Fluorescence and Mid Infrared Fiber Spectroscopy for Kidney Tumor Diagnostics

Matching pairs of tumor and non-tumor kidney tissue samples of four patients were investigated ex vivo using a combination of two methods, attenuated total reflection mid infrared spectroscopy and fluorescence spectroscopy, through respectively prepared and adjusted fiber probes. In order to increase the data information content, the measurements on tissue samples in both methods were performed in the same 31 preselected positions. Multivariate data analysis revealed a synergic effect of combining the two methods for the diagnostics of kidney tumor compared to individual techniques.