O. Minet

Heat Stress Induced Redistribution of Fluorescent Quantum Dots in Breast Tumor Cells

The probing of living cells in different colors over extended periods of time can be used to see the complicated processes that take place during carcinogenesis or heat stress, for example. Since most therapeutic laser tissue interactions are based on thermal effects a detailed characterization of thermal tissue damages in the cellular and sub-cellular levels is important. In order to study such microdosimetry laser-induced fluorescences of Quantum dots provide a suitable approach. Streptavidin conjugated Qdot™ 605 (Quantum Dot Corp., USA) were used in combination with the concanavalin A-biotin labeling system (Molecular Probes, NL) to observe membrane associated thermal lesions. Fluorescent Qdot conjugates are a promising alternative to organic dyes. The extinction coefficient of Qdot™ 605 streptavidin conjugate is 650,000 M−1 cm−1 at 600 nm. Red fluorescent Qdots™ 605 were selected because autofluorescence of cells in the red spectral range is not relevant. Fluorescence detection was performed with a confocal laser scan microscope LSM410 (Carl Zeiss, Germany). Breast cancer cells were used in the thermal stressing experiments performed at 40°C, 42°C, 45°C, 50°C or 56°C for 30 min, each. In this methodical approach Qdot mediated labeling of heat stressed cells were demonstrated to show alterations of plasma membrane organizations and integrities, respectively.

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.

Microscopical heat stress investigations under application of quantum dots

Heat stress responses are analyzed in cancer cells by applying different microscopy techniques for targeting various fluorescently labeled or native structures. Thermotreatments are performed at 40, 45, 50, and 56 degrees C, respectively, for 30 min each, while controls were kept at 37 degrees C. Actin cytoskeletons labeled with Alexa Fluor 488-conjugated phalloidin are imaged by wide-field fluorescence microscopy (WFFM). Structural plasma membrane stabilities are labeled with fluorescent quantum dots and analyzed by laser scanning microscopy (LSM). High-resolution atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to study morphological features and surface structures. Fluorescence images reveal F-actin to be a comparatively thermolabile cell component showing distinctive alteration after heat treatment at 40 degrees C. Destabilization of actin cytoskeletons proceed with increasing stress temperatures. Active reorganization of plasma membranes coincidental to heat-induced shrinkage and rounding of cell shapes, and loosening of monolayered tissue are observed after treatment at 45 or 50 degrees C. Active stress response is inhibited by stress at 56 degrees C, because actin cytoskeletons as well as plasma membranes are destroyed, resulting in necrotic cell phenotypes. Comparing data measured with the same microscopic technique and comparing the different datasets with each other reveal that heat stress response in MX1 cells results from the overlap of different heat-induced subcellular defects.

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.

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.

Comparison of retina damage thresholds simulating the femtosecond-laser in situ keratomileusis (fs-LASIK) process with two laser systems in the CW- and fs-regime

The femtosecond-laser in situ keratomileusis procedure affords the opportunity to correct ametropia by cutting transparent corneal tissue with ultra-short laser pulses. Thereby the tissue cut is generated by a laser-induced optical breakdown in the cornea with ultra-short laser pulses in the near-infrared range. Compared to standard procedures such as photorefractive keratectomy and laser in-situ keratomileusis with the excimer laser, where the risk potential for the eye is low due to the complete absorption of ultraviolet irradiation from corneal tissue, only a certain amount of the pulse energy is deposited in the cornea during the fs-LASIK process. The remaining energy propagates through the eye and interacts with the retina and the strong absorbing tissue layers behind. The objective of the presented study was to determine and compare the retina damage thresholds during the fs-LASIK process simulated with two various laser systems in the CW- and fs-regime.

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.

Dosimetric investigations of laser-induced phase transition of MX1-cell membranes by use of quantum dots

It is well known that laser scattered-light applicators when applied for laser-induced tumor therapy allow the precise thermal destruction of metastases. Using laser radiation in the NIR spectral range (usually, Nd:YAG laser systems λ = 1064 nm), a penetration depth of 5–10 cm (1/e is the decrease in radiation intensity) is achieved in biological tissues. The major tissue-optical parameters, i.e., absorption coefficient μa, scattering coefficient μs, and the anisotropy factor g, show biological tissues to be strongly scattering media which have a so-called optical window in the NIR. As a consequence, the therapeutic laser radiation is scattered and absorbed at a deeper level, leading to a virtual enlargement of the laser applicator. The thermal sclerotization and the thermal cell damage originate within the absorbing volume of the laser radiation and spread outward by thermal diffusion. There are three dosimetrically relevant zones of thermal and biological damage: (1) a zone of thermal coagulation; (2) a threshold of partial necrosis (destruction of all metabolic processes in the cell is the maintenance of essential parts of the cytoskeleton and the plasma membrane); this is characterized by a specific temperature range, the so-called phase transition, which refers to the transition from the gel phase of the biomembrane to the fluid phase; the determination of this temperature zone is an integral part of the following experimental investigations on MX1 cells; (3) an external zone of thermal effects made up of partial and multiple damage with a statistical chance of survival. This paper describes the investigations on heat stress in cancer cells to verify the maximum phase transition of the outer MX1 cell membranes and the related results. For this purpose, a novel method of quantum dot fluorescence dosimetry was developed. The evaluation of the measured laser-induced fluorescences yields a first approximation of the determination of the phase transition on MX1 cells.

Vitality of the MeWo melanoma cell line during intense nanosecond-pulsed NIR laser radiation

Knowledge of the limiting photo-thermal energy deposition in cells is of primary importance for new diagnostic applications of near infrared lasers. The influence of high-energy near infrared (NIR)-optical parametric oscillator (OPO)-irradiation on the MeWo melanoma cell line was investigated for different periods of illumination. Minor decreases of cell viability of 10% and 13% after 1 min and 5 min, respectively, were observed. After 15 min OPO irradiance the cell viability was reduced significantly. The results of CellTiter-Blue® viability assay were confirmed with a new lab-on-a-chip technology.