Aggregates of misfolded α-Synuclein in the brain represent a hallmark of Parkinson’s disease (PD). In patients and animal models, phosphorylated α-Synuclein was detected in the gut, hence, raising the hypothesis that early-stage PD could be diagnosed based on colon tissues. Marker-independent technologies represent an ideal method to monitor disease progression and potentially detect early-stage aggregated α-Synuclein in vivo. Here, formalin-fixed, paraffinembedded colon tissues of a transgenic rat model were analyzed using Raman imaging. Detailed spectral and imagebased analysis was performed indicating the major spectral shifts that alter in PD rat tissues in the amide I region. Peak fitting and multivariate analysis specified an increase of β-sheet proteins in transgenic rat colon compared with wild-type colon. In summary, Raman imaging is capable to detect α-Synuclein aggregates in colon tissues of a PD rat model, indicating that it could be a useful tool to support diagnosis in PD pathology.
Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.
Bone-marrow mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medicine.
However, it is challenging to determine whether isolated BM-MSC populations are free of fibroblasts. We
employed traditional methods and Raman spectroscopy to distinguish BM-MSCs and human dermal
fibroblasts (hDFs). Although in vitro differentiation assays revealed the multipotent character of BM-MSCs,
long culture periods are a major disadvantage. Using Raman spectroscopy, we could quickly distinguish
between BM-MSCs and hDFs. Therefore we conclude that this method is sufficient for the rapid detection of
fibroblastic contaminations in BM-MSC cultures.
KEYWORDS: Tissues, In vivo imaging, Collagen, Heart, Skin, Near infrared, Optical fibers, Second-harmonic generation, Optical tomography, In vitro testing
Multiphoton optical tomography based on NIR (near-infrared) femtosecond laser pulses provides non-invasive optical sectioning of skin with high spatial intracellular resolution and high tissue penetration. The imaging system DermaInspect was used to perform this technology in clinical studies in vivo on patients with suspicious melanoma. Pigmented cell clusters based on non-linear luminescence were clearly distinguished from non-pigmented cells in the epidermis using the autofluorescence of endogenous fluorophores like NAD(P)H, flavins, keratin, elastin, collagen and melanin. Some of the investigated tissues showed differences in the structure of the epidermal layers and the presence of dendritic cells compared to normal skin. Multiphoton laser microscopy was used to visualize extracellular matrix (ECM) structures of native and tissueengineered heart valves. The quality of the resulting 3-D images allowed an exact differentiation between collagenous and elastic fibers. The analysis of heart valve tissues of patients with cardiomyopathy revealed a dramatic loss of its capability to generate SH (second harmonic), indicating a structural deformation of the collagenous fibers, which was virtually impossible to obtain by routine histological or immunohistological staining. These results indicate that NIR femtosecond laser scanning systems can be employed as novel non-invasive optical technology for 3-D resolved ECM component imaging and in vitro and in vivo tissue diagnosis.
Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.
Near infrared (NIR) femtosecond laser imaging systems represent a novel and very promising diagnostic technology for non-invasive cross-sectional analysis of living biological tissues. In this study 3D multiphoton imaging with submicron resolution has been performed for non-invasive analysis of living native and tissue-engineered (TE) heart valves and blood vessels. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous structures and elastic fibers were demonstrated using multiphoton excitation at two different wavelengths. Non-invasive optical sections have been obtained without the need of staining or embedding. The quality of the resulting three-dimensional images allowed exact differentiation between collagenous structures and elastic fibers. These experimental results are very encouraging for NIR femtosecond laser scanning microscopy as a useful tool for future non-destructive monitoring and characterization of vital and intact TE cardiovascular structures.
Near infrared (NIR) femtosecond laser scanning microscopy represents a novel and very promising medical diagnostic imaging technology for non-invasive cross-sectional analysis of living biological tissues. In this study multiphoton imaging has been performed to analyze the structural features of extracellular matrix (ECM) components, e.g. collagen and elastin, of living pulmonary and aortic heart valves. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and non-invasive optical sectioning, without the need of embedding or staining. The quality of the resulting three-dimensional images allowed exact differentiation of the ECM components. These experimental results indicated that NIR femtosecond laser scanning microscopy may prove to be a useful tool for the non-destructive monitoring and characterization of cardiovascular structures.
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