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.

Label-Free Quantitative Phase Imaging Reveals Spatial Heterogeneity of Extracellular Vesicles in Select Colon Disorders.

Three-dimensional (3D) imaging and quantitative analysis of extracellular vesicles (EVs) remain largely unexplored, mainly because of limitations in detection techniques. In this study, EVs from patients diagnosed with colorectal cancer (CRC) and ulcerative colitis were examined. To investigate the spatial heterogeneity and 3D refractive index (RI) distribution of single EVs, a label-free digital holographic tomography technique was used at a submicrometer spatial resolution. The presented image-processing algorithms were used in quantitative analysis with digital staining and 3D visualization, the determination of the EV size distribution and extraction of fractions with different RIs. Reconstructed 3D RI distributions revealed variations in the spatial heterogeneity of EVs related to tissue specificity, such as CRC, normal colonic mucosa, and ulcerative colitis, as well as the isolation procedures used. The RI values of EVs isolated from solid tissues of frozen CRC samples were also dependent on the tumor grade and cancer cell proliferation. The simultaneous examination of cell culture models confirmed the association of the RI of EVs with the tumor grade. 3D-RI data analysis generates new perspectives with the optical, contact-free, label-free examination of the individual EVs. Depending on the specific tissue and isolation method, EVs exhibit significant spatial heterogeneity. The optical parameters of single EVs enabled their classification into two unique subgroups with different RI values.

The Enhancement of Antimicrobial Photodynamic Therapy of Escherichia Coli by a Functionalized Combination of Photosensitizers: In Vitro Examination of Single Cells by Quantitative Phase Imaging.

The prevention of biofilm formation is crucial for the limitation of bacterial infections typically associated with postoperative infections, complications in bedridden patients, and a short-term prognosis in affected cancer patients or mechanically ventilated patients. Antimicrobial photodynamic therapy (aPDT) emerges as a promising alternative for the prevention of infections due to the inability of bacteria to become resistant to aPDT inactivation processes. The aim of this study was to demonstrate the use of a functionalized combination of Chlorin e6 and Pheophorbide as a new approach to more effective aPDT by increasing the accumulation of photosensitizers (PSs) within Escherichia coli cells. The accumulation of PSs and changes in the dry mass density of single-cell bacteria before and after aPDT treatment were investigated by digital holotomography (DHT) using the refractive index as an imaging contrast for 3D label-free live bacteria cell imaging. The results confirmed that DHT can be used in complex examination of the cell-photosensitizer interaction and characterization of the efficiency of aPDT. Furthermore, the use of Pheophorbide a as an efflux pomp inhibitor in combination with Chlorin e6 increases photosensitizers accumulation within E. coli and overcomes the limited penetration of Gram-negative cells by anionic and neutral photosensitizers.

Photolon Nanoporous Photoactive Material with Antibacterial Activity and Label-Free Noncontact Method for Free Radical Detection.

The worldwide increase in bacterial resistance and healthcare-associated bacterial infections pose a serious threat to human health. The antimicrobial photodynamic method reveals the opportunity for a new therapeutic approach that is based on the limited delivery of photosensitizer from the material surface. Nanoporous inorganic-organic composites were obtained by entrapment of photosensitizer Photolon in polysiloxanes that was prepared by the sol-gel method. The material was characterized by its porosity, optical properties (fluorescence and absorbance), and laser-induced antimicrobial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The permanent encapsulation of Photolon in the silica coating and the antimicrobial efficiency was confirmed by confocal microscope and digital holotomography. The generation of free radicals from nanoporous surfaces was proved by scanning Kelvin probe microscopy. For the first time, it was confirmed that Kelvin probe microscopy can be a label-free, noncontact alternative to other conventional methods based on fluorescence or chemiluminescence probes, etc. It was confirmed that the proposed photoactive coating enables the antibacterial photodynamic effect based on free radicals released from the surface of the coating. The highest bactericidal efficiency of the proposed coating was 87.16%. This coating can selectively limit the multiplication of bacterial cells, while protecting the environment and reducing the risk of surface contamination.

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.

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.

Bacteria identification in an optical system with optimized diffraction pattern registration condition supported by enhanced statistical analysis.

It is possible to identify bacteria species basing on their diffraction patterns followed by statistical analysis. The new approach exploits two steps: optimization of the recording conditions and introduction of new interpretable features for the identification. First, optimal diffraction registration plane, was determined. Next, results were verified by the analysis workflow based on ANOVA and Fisher divergence for feature selection, QDA and SVM models for classification and identification and CV with stratified sampling, sensitivity and specificity for performance assessment of the identification process. The proposed approach resulted in high sensitivity 0.9759 and specificity 0.9903 with very small identification error 1.34%.

Degeneration of Fraunhofer diffraction on bacterial colonies due to their light focusing properties examined in the digital holographic microscope system.

The degeneration of Fraunhofer diffraction conditions in the optical system with converging spherical wave illumination for bacteria species identification based on diffraction patterns is analyzed by digital holographic methods. The obtained results have shown that the colonies of analyzed bacteria species act as biological lenses with the time-dependent light focusing properties, which are characterized and monitored by means of phase retrieval from sequentially captured digital holograms. This significantly affects the location of Fraunhofer patterns observation plane, which is continuously shifted across optical axis in time.

Bacteria species identification by the statistical analysis of bacterial colonies Fresnel patterns.

It was demonstrated that statistical analysis of bacteria colonies Fresnel patterns recorded in the optical system with converging spherical wave illumination is suitable for highly effective bacteria species classification. The proposed method includes Fresnel patterns recording followed by image processing and the statistical analysis based on feature extraction, feature selection, classification and classification performance methods. Examination performed on various bacteria species (Salmonella enteritidis, Staphylococcus aureus, Staphylococcus intermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Citrobacter freundii) revealed that the proposed method achieved very high accuracy of over 98%.

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.

Quantitative Phase Imaging Detecting the Hypoxia-Induced Patterns in Healthy and Neoplastic Human Colonic Epithelial Cells.

Hypoxia is a frequent phenomenon during carcinogenesis and may lead to functional and structural changes in proliferating cancer cells. Colorectal cancer (CRC) is one of the most common neoplasms in which hypoxia is associated with progression. The aim of this study was to assess the optical parameters and microanatomy of CRC and the normal intestinal epithelium cells using the digital holotomography (DHT) method. The examination was conducted on cancer (HT-29, LoVo) and normal colonic cells (CCD-18Co) cultured in normoxic and hypoxic environments. The assessment included optical parameters such as the refractive index (RI) and dry mass as well as the morphological features. Hypoxia decreased the RI in all cells as well as in their cytoplasm, nucleus, and nucleoli. The opposite tendency was noted for spheroid-vesicular structures, where the RI was higher for the hypoxic state. The total volume of hypoxic CCD-18Co and LoVo cells was decreased, while an increase in this parameter was observed for HT-29 cells. Hypoxia increased the radius and cell volume, including the dry mass of the vesicular content. The changes in the optics and morphology of hypoxic cells may suggest the possibility of using DHT in the detection of circulating tumor cells (CTCs).

Influence of various growth conditions on Fresnel diffraction patterns of bacteria colonies examined in the optical system with converging spherical wave illumination.

The novel optical system based on converging spherical wave illumination for analysis of bacteria colonies diffraction patterns, is proposed. The complex physical model of light transformation on bacteria colonies in this system, is presented. Fresnel diffraction patterns of bacteria colonies Escherichia coli, Salmonella enteritidis, Staphylococcus aureus grown in various conditions, were examined. It was demonstrated that the proposed system enables the characterization of morphological changes of colony structures basing on the changes of theirs Fresnel diffraction patterns.

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.

Molecular profiling of the intestinal mucosa and immune cells of the colon by multi-parametric histological techniques.

The impact of the post-mortem interval (PMI) on the optical molecular characteristics of the colonic mucosa and the gut-associated lymphoid tissue (GALT) were examined by multi-parametric measurements techniques. Inflammatory cells were identified by immunohistochemical staining. Molecular parameters were estimated using the Raman spectroscopy (RS) and Fourier Transform Infrared (FTIR) spectroscopic imaging. The 3D refractive index (3D-RI) distributions of samples were determined using the digital holographic tomography. The distribution of immune cells between post-mortem (PM) and normal controls did show significant differences for CD4 (P = 0.0016) or CD8 (P < 0.0001), whose expression level was decreased in PM cases. No association was found between individual PMI values and inflammatory cell distribution. However, there was a tendency for a negative correlation between CD4+ cells and PMI (r = - 0.542, P = 0.032). The alterations ongoing in post-mortem tissue may suggest that PMI has a suppressive effect on the effector properties of the cell-mediated immunity. Moreover, it was confirmed that spectroscopic and digital holotomographic histology are also a useful technique for characterization of the differences in inflammation of varying intensity and in GALT imaging in a solid tissue. Anatomical location of immune cells and methods of tissue fixation determine the molecular and optical parameters of the examined cases.

Image processing guided analysis for estimation of bacteria colonies number by means of optical transforms.

A novel method for evaluation of bacterial colonies number (Colony Forming Units--CFU), is described. Proposed algorithm, based on the Mellin transform, allows the CFU evaluation, invariant for the spatial orientation and scale changes. The proposed method involves image recording of bacteria grown in Petri dishes, calculation of the Fourier spectrum followed by coordinates transformation, and determination of the Mellin transform. It was proved that there is a high correlation between CFU and maxima of Mellin spectra. The method was practically implemented for evaluation of antibacterial activity of silver-based nanomaterials and the effect of an additional laser light irradiation.

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.

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.

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.