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

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 &micro;m. Obtained results led to development of image-processing algorithms for determination of the direction of misalignment and concurrent alignment of the bacterial colonies&rsquo; diffraction patterns. The proposed algorithms enable the rigorous monitoring and controlling of the measurement&rsquo;s conditions in order to preserve the high accuracy of bacteria identification

The schematic set-up of the PSDIHS configuration.

<p>Light generated by a coherent light source (LS) with a wavelength of 405 nm and output power of 25 mW is focused onto a 0.5-μm-diameter pinhole (PH), which acts as the point source emitting the divergent spherical wave. The wave illuminates an examined object, bacterial colonies grown on nutrient medium in a Petri dish on a sample holder with an X-Y translation stage, and forms magnified diffraction pattern in the recording plane (C) of the CMOS camera with the sensing area: 2048 × 2048 pixels (pixel size: 6 × 6 μm), mounted on a Z translation stage.</p

Novel Perspectives on the Characterization of Species-Dependent Optical Signatures of Bacterial Colonies by Digital Holography - Fig 3

<p>Exemplary reflection absorbance spectra of the bacterial colonies of (a) <i>S</i>. <i>intermedius</i> and (b) <i>E</i>. <i>coli</i>.</p

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)

The Enhancement of Antimicrobial Photodynamic Therapy of <i>Escherichia Coli</i> 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

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&mdash;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

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