Quantifying the Dynamics of Bacterial Biofilm Formation on the Surface of Soft Contact Lens Materials Using Digital Holographic Tomography to Advance Biofilm Research.

The increase in bacterial resistance to antibiotics in recent years demands innovative strategies for the detection and combating of biofilms, which are notoriously resilient. Biofilms, particularly those on contact lenses, can lead to biofilm-related infections (e.g., conjunctivitis and keratitis), posing a significant risk to patients. Non-destructive and non-contact sensing techniques are essential in addressing this threat. Digital holographic tomography emerges as a promising solution. This allows for the 3D reconstruction of the refractive index distribution in biological samples, enabling label-free visualization and the quantitative analysis of biofilms. This tool provides insight into the dynamics of biofilm formation and maturation on the surface of transparent materials. Applying digital holographic tomography for biofilm examination has the potential to advance our ability to combat the antibiotic bacterial resistance crisis. A recent study focused on characterizing biofilm formation and maturation on six soft contact lens materials (three silicone hydrogels, three hydrogels), with a particular emphasis on Staphylococcus epidermis and Pseudomonas aeruginosa, both common culprits in ocular infections. The results revealed species- and time-dependent variations in the refractive indexes and volumes of biofilms, shedding light on cell dynamics, cell death, and contact lens material-related factors. The use of digital holographic tomography enables the quantitative analysis of biofilm dynamics, providing us with a better understanding and characterization of bacterial biofilms.

Multimodal study of CHI3L1 inhibition and its effect on angiogenesis, migration, immune response and refractive index of cellular structures in glioblastoma.

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721-0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721-0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour.

Superficial temperature distribution patterns before and after physical activity in school children are indicative for personalized exercise coaching and disease prevention.

BACKGROUND: Thermoregulation is highly individual and predictive for potentially cascading pathologies. Altered and deficient thermoregulation is considered an important diagnostic indicator which can be of great clinical utility for specialized screening programs and individualized prediction and prevention of severe pathologies triggered early in life. WORKING HYPOTHESIS: Individual thermoregulation can be objectively assessed by thermovision camera before and after exercises in school children stratified by age and gender that may be of great clinical utility for personalized training early in life in the framework of 3P medicine. STUDY DESIGN: In this study, 60 female and male primary school children were exposed to physical exercises in the form of 45-min general fitness training. The subjects under examination were stratified by age: group 1 (7-year-olds), group 2 (9-year-olds), and group 3 (12-year-olds). Superficial body temperature patterns were measured by means of thermovision camera before and immediately after exercises, as well as after the 15-min recovery time. Temperature patterns were analyzed in 12 areas of the body front and back, covering trunk and upper and lower limbs. RESULTS: The obtained results revealed an individual and age-depended difference in response of the body to exercises. The first measurement prior to exercise (measurement 1) revealed no statistically significant differences in the mean surface temperature of all analyzed areas between 7- and 9-year-old children. Further, 7- and 9-year-old children did not differ significantly in the mean temperature recorded in the trunk compared to the 12-year-old children. However, in 12-year-old children, statistically significant higher values of the mean temperature of the upper and lower limbs, were observed compared to the group of 7-year-olds and significantly higher values of the mean temperature of the lower limbs compared to the group of 9-year-olds. Immediately after exercises (measurement 2), a statistically significant decrease in the temperature was noted in all groups and in all areas of the body. The greatest temperature change was observed in 12-year-olds, while the least one was measured in the youngest subjects. The statistically significant relation between the average trunk temperature of 7-year-old and 12-year-old children was observed: lower values of the mean temperature of the front and back of the trunk were noted in the group of 12-year-old children compared to the group of 7-year-olds. A significantly lower average temperature of the back of the trunk compared to the youngest group was also recorded in 9-year-old children. The study performed after the 15-min recovery time (measurement 3) showed an increase in the average temperature of all analyzed areas. In all subjects, the mean temperature recorded in measurement 3 did not differ significantly from the initial ones (measurement 1, prior to exercises). Only the mean temperature of the trunk back of 12-year-old children was significantly lower after the rest period compared to the initial examination. In all groups, the temperatures after exercises followed by a 15-min recovery returned to the initial ones, except of the trunk backs of 12-year-old children, where the temperature was lower than before exercises. CONCLUSIONS AND EXPERT RECOMMENDATIONS IN THE FRAMEWORK OF 3PM: Thermovision analysis is an effective tool to assess individual thermoregulation and to stratify school children for personalized exercise coaching. Body exercise-based disease prevention early in life is effective when tailored to the person: multi-parametric guidance for prescribing exercises individually is needed. Contextually, proposed individualized training approach should be adapted to the age-dependent particularities and individual thermoregulation.

Caution, "normal" BMI: health risks associated with potentially masked individual underweight-EPMA Position Paper 2021.

An increasing interest in a healthy lifestyle raises questions about optimal body weight. Evidently, it should be clearly discriminated between the standardised "normal" body weight and individually optimal weight. To this end, the basic principle of personalised medicine "one size does not fit all" has to be applied. Contextually, "normal" but e.g. borderline body mass index might be optimal for one person but apparently suboptimal for another one strongly depending on the individual genetic predisposition, geographic origin, cultural and nutritional habits and relevant lifestyle parameters-all included into comprehensive individual patient profile. Even if only slightly deviant, both overweight and underweight are acknowledged risk factors for a shifted metabolism which, if being not optimised, may strongly contribute to the development and progression of severe pathologies. Development of innovative screening programmes is essential to promote population health by application of health risks assessment, individualised patient profiling and multi-parametric analysis, further used for cost-effective targeted prevention and treatments tailored to the person. The following healthcare areas are considered to be potentially strongly benefiting from the above proposed measures: suboptimal health conditions, sports medicine, stress overload and associated complications, planned pregnancies, periodontal health and dentistry, sleep medicine, eye health and disorders, inflammatory disorders, healing and pain management, metabolic disorders, cardiovascular disease, cancers, psychiatric and neurologic disorders, stroke of known and unknown aetiology, improved individual and population outcomes under pandemic conditions such as COVID-19. In a long-term way, a significantly improved healthcare economy is one of benefits of the proposed paradigm shift from reactive to Predictive, Preventive and Personalised Medicine (PPPM/3PM). A tight collaboration between all stakeholders including scientific community, healthcare givers, patient organisations, policy-makers and educators is essential for the smooth implementation of 3PM concepts in daily practice.

Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging.

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria-photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.

Impact of Liposomal Drug Formulations on the RBCs Shape, Transmembrane Potential, and Mechanical Properties.

Liposomal technologies are used in order to improve the effectiveness of current therapies or to reduce their negative side effects. However, the liposome-erythrocyte interaction during the intravenous administration of liposomal drug formulations may result in changes within the red blood cells (RBCs). In this study, it was shown that phosphatidylcholine-composed liposomal formulations of Photolon, used as a drug model, significantly influences the transmembrane potential, stiffness, as well as the shape of RBCs. These changes caused decreasing the number of stomatocytes and irregular shapes proportion within the cells exposed to liposomes. Thus, the reduction of anisocytosis was observed. Therefore, some nanodrugs in phosphatidylcholine liposomal formulation may have a beneficial effect on the survival time of erythrocytes.

On the application of multi-parametric optical phenotyping of bacterial colonies for multipurpose microbiological diagnostics.

The development of new diagnostics techniques and modalities is critical for early detection of microbial contamination. In this study, the novel integrated system for multi-parametric optical phenotyping and characterization of bacterial colonies, is presented. The system combines Mach-Zehnder interferometer with a spectral imaging system for capturing multispectral diffraction patterns and multispectral two-dimensional transmission maps of bacterial colonies, along with the simultaneous interferometric profilometry. The herein presented investigation was carried out on five representative bacteria species and nearly 3000 registered multispectral optical signatures. The interferograms were analyzed by four-step phase shift algorithm to reconstruct the colony profile to enable the obtaining of the comparable optical signatures. The dedicated image processing algorithms were used for extraction of quantitative features of these signatures. The random forest algorithm was applied for selection of the most predictive set of features, which were used in classification model based on Support-Vector Machine. Obtained results have shown that the use of multiple multispectral optical signatures provide a multi-parametric bacteria identification at an exceptionally high accuracy (99.4-100%), significantly better than in case of classification based on each of these signatures (multispectral diffraction patterns, two-dimensional transmission coefficient maps), separately. Obtained results revealed that analysis of multispectral signatures can also be applied for characterisation of physical, physicochemical and chemical properties of the bacterial colonies in the presence of the antimicrobial factors. Therefore, the proposed label-free, non-destructive optical technique has perspectives to be exploited in the multipurpose diagnostics and it can be used as a pre-screening tool in microbiological laboratories.

Antibody CD133 Biofunctionalization of Ammonium Acryloyldimethyltaurate and Vinylpyrrolidone Co-Polymer-Based Coating of the Vascular Implants.

Current vascular stents, such as drug eluting stents (DES), have some serious drawbacks, like in stent restenosis and thrombosis. Therefore, other solutions are sought to overcome these post-implantations complications. These include the strategy of biofunctionalization of the stent surface with antibodies that facilitate adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs). Rapid re-endothelialization of the surface minimizes the risk of possible complications. In this study, we proposed ammonium acryloyldimethyltaurate/vinylpyrrolidone co-polymer-based surface (AVC), which was mercaptosilanized in order to expose free thiol groups. The presence of free thiol groups allowed for the covalent attachment of CD133 antibodies by disulfide bridges formation between mercaptosilanized surface and cysteine of the protein molecule thiol groups. Various examinations were performed in order to validate the procedure, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and Fourier transform Raman spectroscopy (FT-Raman), atomic force microscopy (AFM) and scanning electron microscopy (SEM). By means of ATR-FTIR spectroscopy presence of the CD133 antibody within coating was confirmed. In vitro studies proved good biocompatibility for blood cells without induction of hemolytic response. Thus, proposed biofunctionalized CD133 antibody AVC surface has shown sufficient stability for adapting as cardiovascular implant coating and biocompatibility. According to conducted in vitro studies, the modified surface can be further tested for applications in various biological systems.

Development of the Correction Algorithm to Limit the Deformation of Bacterial Colonies Diffraction Patterns Caused by Misalignment and Its Impact on the Bacteria Identification in the Proposed Optical Biosensor.

Recently proposed methods of bacteria identification in optical biosensors based on the phenomenon of light diffraction on macro-colonies offer over 98% classification accuracy. However, such high accuracy relies on the comparable and repeatable spatial intensity distribution of diffraction patterns. Therefore, it is essential to eliminate all non-species/strain-dependent factors affecting the diffraction patterns. In this study, the impact of the bacterial colony and illuminating beam misalignment on the variation of classification features extracted from diffraction patterns was examined. It was demonstrated that misalignment introduced by the scanning module significantly affected diffraction patterns and extracted classification features used for bacteria identification. Therefore, it is a crucial system-dependent factor limiting the identification accuracy. The acceptable misalignment level, when the accuracy and quality of the classification features are not affected, was determined as no greater than 50 µm. Obtained results led to development of image-processing algorithms for determination of the direction of misalignment and concurrent alignment of the bacterial colonies' diffraction patterns. The proposed algorithms enable the rigorous monitoring and controlling of the measurement's conditions in order to preserve the high accuracy of bacteria identification.

Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs.

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

Implementation of artificial intelligence and non-contact infrared thermography for prediction and personalized automatic identification of different stages of cellulite.

BACKGROUND: Cellulite is a common physiological condition of dermis, epidermis, and subcutaneous tissues experienced by 85 to 98% of the post-pubertal females in developed countries. Infrared (IR) thermography combined with artificial intelligence (AI)-based automated image processing can detect both early and advanced cellulite stages and open up the possibility of reliable diagnosis. Although the cellulite lesions may have various levels of severity, the quality of life of every woman, both in the physical and emotional sphere, is always an individual concern and therefore requires patient-oriented approach. OBJECTIVES: The purpose of this work was to elaborate an objective, fast, and cost-effective method for automatic identification of different stages of cellulite based on IR imaging that may be used for prescreening and personalization of the therapy. MATERIALS AND METHODS: In this study, we use custom-developed image preprocessing algorithms to automatically select cellulite regions and combine a total of 9 feature extraction methods with 9 different classification algorithms to determine the efficacy of cellulite stage recognition based on thermographic images taken from 212 female volunteers aged between 19 and 22. RESULTS: A combination of histogram of oriented gradients (HOG) and artificial neural network (ANN) enables determination of all stages of cellulite with an average accuracy higher than 80%. For primary stages of cellulite, the average accuracy achieved was more than 90%. CONCLUSIONS: The implementation of computer-aided, automatic identification of cellulite severity using infrared imaging is feasible for reliable diagnosis. Such a combination can be used for early diagnosis, as well as monitoring of cellulite progress or therapeutic outcomes in an objective way. IR thermography coupled to AI sets the vision towards their use as an effective tool for complex assessment of cellulite pathogenesis and stratification, which are critical in the implementation of IR thermographic imaging in predictive, preventive, and personalized medicine (PPPM).

Functionalization with a VEGFR2-binding antibody fragment leads to enhanced endothelialization of a cardiovascular stent in vitro and in vivo.

Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.

Photoactive Pore Matrix for In Situ Delivery of a Photosensitizer in Vascular Smooth Muscle Cells Selective PDT.

In this study we present the porous silica-based material that can be used for in situ drug delivery, offering effective supply of active compounds regardless its water solubility. To demonstrate usability of this new material, three silica-based materials with different pore size distribution as a matrix for doping with Photolon (Ph) and Protoporphyrin IX (PPIX) photosensitizers, were prepared. These matrices can be used for coating cardiovascular stents used for treatment of the coronary artery disease and enable intravascular photodynamic therapy (PDT), which can modulate the vascular response to injury caused by stent implantation-procedure that should be thought as an alternative for drug eluting stent. The FTIR spectroscopic analysis confirmed that all studied matrices have been successfully functionalized with the target photosensitizers. Atomic force microscopy revealed that resulting photoactive matrices were very smooth, which can limit the implantation damage and reduce the risk of restenosis. No viability loss of human peripheral blood lymphocytes and no erythrocyte hemolysis upon prolonged incubations on matrices indicated good biocompatibility of designed materials. The suitability of photoactive surfaces for PDT was tested in two cell lines relevant to stent implantation: vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC). It was demonstrated that 2 h incubation on the silica matrices was sufficient for uptake of the encapsulated photosensitizers. Moreover, the amount of the absorbed photosensitizer was sufficient for induction of a phototoxic reaction as shown by a rise of the reactive oxygen species in photosensitized VSMC. On the other hand, limited reactive oxygen species (ROS) induction in HUVECs in our experimental set up suggests that the proposed method of PDT may be less harmful for the endothelial cells and may decrease a risk of the restenosis. Presented data clearly demonstrate that porous silica-based matrices are capable of in situ delivery of photosensitizer for PDT of VSMC.

Photoactive Liposomal Formulation of PVP-Conjugated Chlorin e6 for Photodynamic Reduction of Atherosclerotic Plaque.

BACKGROUND: Liposomes serve as delivery systems for biologically active compounds. Existing technologies inefficiently encapsulate large hydrophilic macromolecules, such as PVP-conjugated chlorin e6 (Photolon). This photoactive drug has been widely tested for therapeutic applications, including photodynamic reduction of atherosclerotic plaque. METHODS: A novel formulation of Photolon was produced using "gel hydration technology". Its pharmacokinetics was tested in Sus scrofa f. domestica. Its cellular uptake, cytotoxicity, and ability to induce a phototoxic reaction were demonstrated in J774A.1, RAW264.7 macrophages, and vascular smooth muscle (T/G HA-VSMC) as well as in vascular endothelial (HUVEC) cells. RESULTS: Developed liposomes had an average diameter of 124.7 ± 0.6 nm (polydispersity index (PDI) = 0.055) and contained >80% of Photolon). The half-life of formulation in S. scrofa was 20 min with area under the curve (AUC) equal to 14.7. The formulation was noncytotoxic in vitro and was rapidly (10 min) and efficiently accumulated by macrophages, but not T/G HA-VSMC or HUVEC. The accumulated quantity of photosensitizer was sufficient for induction of phototoxicity in J774A.1, but not in T/G HA-VSMC. CONCLUSIONS: Due to the excellent physical and pharmacokinetic properties and selectivity for macrophages, the novel liposomal formulation of Photolon is a promising therapeutic candidate for use in arteriosclerosis treatment when targeting macrophages but not accompanying vascular tissue is critical for effective and safe therapy.

Integrated multi-channel optical system for bacteria characterization and its potential use for monitoring of environmental bacteria.

The potential use of a novel multichannel optical system towards fast and non-destructive bacteria identification and its application for environmental bacteria characterisation on the strain level is presented. It is the first attempt to use the proposed optical method to study various bacteria species (Gram-negative, Gram-positive) commonly present in the environment. The novel configuration of the optical system enables multichannel examination of bacterial colonies and provides additional functionality such as registration of two-dimensional (2D) distribution of monochromatic transmission coefficient of examined colonies, what can be used as a novel optical signature for bacteria characterization. Performed statistical analysis indicates that it is possible to identify representatives of environmental soil bacteria on the species level with the 98.51% accuracy and in case of two strains of Rahnella aquatilis bacteria on the strain level with the 98.8% accuracy. The proposed method is an alternative to the currently used preliminary bacteria examination in environment safety control with the advantage of being fast, reliable, non-destructive and requiring minimal sample preparation.

Functionalized CD133 antibody coated stent surface simultaneously promotes EPCs adhesion and inhibits smooth muscle cell proliferation-A novel approach to prevent in-stent restenosis.

We report a multistep strategy of biochemical surface modifications that resulted in the synthesis of new, effective and biocompatible intravascular implants coating with immobilized anti-CD133 antibodies, that proved to be the most effective in endothelial progenitor cells capture and reduced smooth muscle cells growth. Biomolecules were immobilized on differently functionalized surfaces. The distribution, nanostructural characteristics and intramolecular interactions of anti-CD133 molecules as well as their ability to bind EPCs was evaluated. We also tempted to build a molecular model of the CD133 protein to study antigen-antibody interactions. CD133 protein is expressed in endothelial progenitor cells (EPCs). Absence of preferential interaction site on CD133, but rather a presence of a small binding area, may be the specificity of reconnaissance sequence, thus importantly increasing the probability of CD133 protein binding. After all, regarding our molecular model, we are convinced that specific, and large enough interactions between anti-CD133 coating stent surface and CD133 present on EPCs will reduce risk of restenosis by favoring the endothelial growth. Additionally, the safety study of the vivo performance of modified titania based surface was performed using small animal models. No allergological or toxical local or systemic adverse effects of the developed coatings were noted.

Biocompatible Carbon-Based Coating as Potential Endovascular Material for Stent Surface.

Stainless steel 316L is a material commonly used in cardiovascular medicine. Despite the various methods applied in stent production, the rates of in-stent restenosis and thrombosis remain high. In this study graphene was used to coat the surface of 316L substrate for enhanced bio- and hemocompatibility of the substrate. The presence of graphene layers applied to the substrate was investigated using cutting-edge imaging technology: energy-filtered low-voltage FE-SEM approach, scanning electron microscopy (SEM), Raman spectroscopy, and atomic force microscopy (AFM). The potential of G-316L surface to influence endothelial cells phenotype and endothelial-to-mesenchymal transition (EndoMT) has been determined. Our results show that the bio- and hemocompatible properties of graphene coatings along with known radial force of 316L make G-316L a promising candidate for intracoronary implants.

Alterations of biomechanics in cancer and normal cells induced by doxorubicin.

Mechanical properties of biological structures play an important role in regulating cellular activities and are critical for understanding metabolic processes in cancerous cells and the effects of drugs. For some cancers, such as acute myeloid leukaemia, chemotherapy is one of preferential methods. However, due to the lack of selectivity to cancer cells, cytostatic agents cause toxicity to normal tissues. Here, we study the effect of doxorubicin (DOX) on the mechanical properties of DNA molecules, leukemic blast cells and erythrocytes, using optical tweezers. In addition, we controlled the subcellular distribution of the drug by confocal microscopy. Our results indicated that doxorubicin affects mechanical properties of cellular structures. In all cases the drug reduced mechanical strength of examined objects. For the leukemic cells the drug subcellular distribution was predominantly nuclear with some particulate cytoplasmic fluorescence. In erythrocytes, doxorubicin showed fluorescence mainly in cytoplasm and plasma membrane. The lowering of blast cells stiffness may be due to the interaction of doxorubicin with nuclear structures, especially with nucleic acids, as our studies with DNA confirmed. In addition, it is known that DOX inhibits the polymerization of actin and thus cytoskeletal modification may also be important in reducing of cell mechanical strength. In the case of erythrocytes - the non-nucleated cells, a significant effect on the decrease of cell stiffness, besides the cytoskeleton, may have the interaction of the drug with the cell membrane. Experiments with model phospholipid membranes confirmed that observed increase in cell elasticity originates, among other things, from the drug incorporation in the lipid membrane itself. The lowering of mechanical strength of leukemic cells may have an significant impact on the effectiveness of chemotherapy. However, the fact that doxorubicin interacts not only with proliferating cancer cells, but also with the health ones may explains the high toxicity of the drug at the therapeutic concentrations. Our observations also suggest that chemotherapy with doxorubicin may decrease the risk of vascular complications in acute leukemia, due to increasing the cell elasticity.

Towards dosimetry for photodynamic diagnosis with the low-level dose of photosensitizer.

BACKGROUND: Contemporary medicine does not concern the issue of dosimetry in photodynamic diagnosis (PDD) but follows the photosensitizer (PS) producers recommendation. Most preclinical and clinical PDD studies indicate a considerable variation in the possibility of visualization and treatment, as e.g. in case of cervix lesions. Although some of these variations can be caused by the different histological subtypes or various tumor geometries, the issue of varying PS concentration in the tumor tissue volume is definitely an important factor. Therefore, there is a need to establish the objective and systematic PDD dosimetry protocol regarding doses of light and photosensitizers. METHODS: Four different irradiation sources investigated in PDD (literature) were used for PS excitation. The PS luminescence was examined by means of the non-imaging (spectroscopic) and imaging (wide- and narrow-field of view) techniques. The methodology for low-level intensity photoluminescence (PL) characterization and dedicated image processing algorithm for PS luminescence images analysis were proposed. Further, HeLa cells' cultures penetration by PS was studied by a confocal microscopy. RESULTS: Reducing the PS dose with the choice of proper photoexcitation conditions decreases the PDD procedure costs and the side effects, not affecting the diagnostic efficiency. We determined in vitro the minimum incubation time and photosensitizer concentration of Photolon for diagnostic purposes, for which the Photolon PL can still be observed. It was demonstrated that quantification of PS concentration, choice of proper photoexcitation source, appropriate adjustment of light dose and PS penetration of cancer cells may improve the low-level luminescence photodynamic diagnostics performance. CONCLUSIONS: Practical effectiveness of the PDD strongly depends on irradiation source parameters (bandwidth, maximum intensity, half-width) and their optimization is the main conditioning factor for low-level intensity and low-cost PDD.

Novel Perspectives on the Characterization of Species-Dependent Optical Signatures of Bacterial Colonies by Digital Holography.

The use of light diffraction for the microbiological diagnosis of bacterial colonies was a significant breakthrough with widespread implications for the food industry and clinical practice. We previously confirmed that optical sensors for bacterial colony light diffraction can be used for bacterial identification. This paper is focused on the novel perspectives of this method based on digital in-line holography (DIH), which is able to reconstruct the amplitude and phase properties of examined objects, as well as the amplitude and phase patterns of the optical field scattered/diffracted by the bacterial colony in any chosen observation plane behind the object from single digital hologram. Analysis of the amplitude and phase patterns inside a colony revealed its unique optical properties, which are associated with the internal structure and geometry of the bacterial colony. Moreover, on a computational level, it is possible to select the desired scattered/diffracted pattern within the entire observation volume that exhibits the largest amount of unique, differentiating bacterial features. These properties distinguish this method from the already proposed sensing techniques based on light diffraction/scattering of bacterial colonies. The reconstructed diffraction patterns have a similar spatial distribution as the recorded Fresnel patterns, previously applied for bacterial identification with over 98% accuracy, but they are characterized by both intensity and phase distributions. Our results using digital holography provide new optical discriminators of bacterial species revealed in one single step in form of new optical signatures of bacterial colonies: digital holograms, reconstructed amplitude and phase patterns, as well as diffraction patterns from all observation space, which exhibit species-dependent features. To the best of our knowledge, this is the first report on bacterial colony analysis via digital holography and our study represents an innovative approach to the subject.