Definition and Quantification of Three-Dimensional Imaging Targets to Phenotype Pre-Eclampsia Subtypes: An Exploratory Study.

Pre-eclampsia is a severe placenta-related complication of pregnancy with limited early diagnostic and therapeutic options. Aetiological knowledge is controversial, and there is no universal consensus on what constitutes the early and late phenotypes of pre-eclampsia. Phenotyping of native placental three-dimensional (3D) morphology offers a novel approach to improve our understanding of the structural placental abnormalities in pre-eclampsia. Healthy and pre-eclamptic placental tissues were imaged with multiphoton microscopy (MPM). Imaging based on inherent signal (collagen, and cytoplasm) and fluorescent staining (nuclei, and blood vessels) enabled the visualization of placental villous tissue with subcellular resolution. Images were analysed with a combination of open source (FIJI, VMTK, Stardist, MATLAB, DBSCAN), and commercially (MATLAB) available software. Trophoblast organization, 3D-villous tree structure, syncytial knots, fibrosis, and 3D-vascular networks were identified as quantifiable imaging targets. Preliminary data indicate increased syncytial knot density with characteristic elongated shape, higher occurrence of paddle-like villous sprouts, abnormal villous volume-to-surface ratio, and decreased vascular density in pre-eclampsia compared to control placentas. The preliminary data presented indicate the potential of quantifying 3D microscopic images for identifying different morphological features and phenotyping pre-eclampsia in placental villous tissue.

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures.

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

Metabolic Interventions to Prevent Hypertrophy-Induced Alterations in Contractile Properties In Vitro.

(1) Background: The exact mechanism(s) underlying pathological changes in a heart in transition to hypertrophy and failure are not yet fully understood. However, alterations in cardiac energy metabolism seem to be an important contributor. We characterized an in vitro model of adrenergic stimulation-induced cardiac hypertrophy for studying metabolic, structural, and functional changes over time. Accordingly, we investigated whether metabolic interventions prevent cardiac structural and functional changes; (2) Methods: Primary rat cardiomyocytes were treated with phenylephrine (PE) for 16 h, 24 h, or 48 h, whereafter hypertrophic marker expression, protein synthesis rate, glucose uptake, and contractile function were assessed; (3) Results: 24 h PE treatment increased expression of hypertrophic markers, phosphorylation of hypertrophy-related signaling kinases, protein synthesis, and glucose uptake. Importantly, the increased glucose uptake preceded structural and functional changes, suggesting a causal role for metabolism in the onset of PE-induced hypertrophy. Indeed, PE treatment in the presence of a PAN-Akt inhibitor or of a GLUT4 inhibitor dipyridamole prevented PE-induced increases in cellular glucose uptake and ameliorated PE-induced contractile alterations; (4) Conclusions: Pharmacological interventions, forcing substrate metabolism away from glucose utilization, improved contractile properties in PE-treated cardiomyocytes, suggesting that targeting glucose uptake, independent from protein synthesis, forms a promising strategy to prevent hypertrophy and hypertrophy-induced cardiac dysfunction.

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds.

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.

Molecular Imaging in Oncology: Advanced Microscopy Techniques.

Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.

Biomass Pretreatment and Enzymatic Hydrolysis Dynamics Analysis Based on Particle Size Imaging.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 µm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Spectral morphological analysis of skin lesions with a polarization multispectral dermoscope.

Dermoscopy is the conventional technique used for the clinical inspection of human skin lesions. However, the identification of diagnostically relevant morphologies can become a complex task. We report on the development of a polarization multispectral dermoscope for the in vivo imaging of skin lesions. Linearly polarized illumination at three distinct spectral regions (470, 530 and 625 nm), is performed by high luminance LEDs. Processing of the acquired images, by means of spectral and polarization filtering, produces new contrast images, each one specific for melanin absorption, hemoglobin absorption, and single scattering. Analysis of such images could facilitate the identification of pathological morphologies.

Thermal transitions of fibrillar collagen unveiled by second-harmonic generation microscopy of corneal stroma.

The thermal transitions of fibrillar collagen are investigated with second-harmonic generation polarization anisotropy microscopy. Second-harmonic generation images and polarization anisotropy profiles of corneal stroma heated in the 35-80°C range are analyzed by means of a theoretical model that is suitable to probe principal intramolecular and interfibrillar parameters of immediate physiological interest. Our results depict the tissue modification with temperature as the interplay of three destructuration stages at different hierarchical levels of collagen assembly including its tertiary structure and interfibrillar alignment, thus supporting and extending previous findings. This method holds the promise of a quantitative inspection of fundamental biophysical and biochemical processes and may find future applications in real-time and postsurgical functional imaging of collagen-rich tissues subjected to thermal treatments.

Intravital microscopy for real-time monitoring of drug delivery and nanobiological processes.

Intravital microscopy (IVM) expands our understanding of cellular and molecular processes, with applications ranging from fundamental biology to (patho)physiology and immunology, as well as from drug delivery to drug processing and drug efficacy testing. In this review, we highlight modalities, methods and model organisms that make up today's IVM landscape, and we present how IVM - via its high spatiotemporal resolution - enables analysis of metabolites, small molecules, nanoparticles, immune cells, and the (tumor) tissue microenvironment. We furthermore present examples of how IVM facilitates the elucidation of nanomedicine kinetics and targeting mechanisms, as well as of biological processes such as immune cell death, host-pathogen interactions, metabolic states, and disease progression. We conclude by discussing the prospects of IVM clinical translation and examining the integration of machine learning in future IVM practice.

Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae.

Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy-imaging the sample from different angles followed by 3D image reconstruction-was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

Time- and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ.

Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.

Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging.

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues.

Clinical nonlinear laser imaging of human skin: a review.

Nonlinear optical microscopy has the potential of being used in vivo as a noninvasive imaging modality for both epidermal and dermal imaging. This paper reviews the capabilities of nonlinear microscopy as a noninvasive high-resolution tool for clinical skin inspection. In particular, we show that two-photon fluorescence microscopy can be used as a diagnostic tool for characterizing epidermal layers by means of a morphological examination. Additional functional information on the metabolic state of cells can be provided by measuring the fluorescence decay of NADH. This approach allows differentiating epidermal layers having different structural and cytological features and has the potential of diagnosing pathologies in a very early stage. Regarding therapy follow-up, we demonstrate that nonlinear microscopy could be successfully used for monitoring the effect of a treatment. In particular, combined two-photon fluorescence and second-harmonic generation microscopy were used in vivo for monitoring collagen remodeling after microablative fractional laser resurfacing and for quantitatively monitoring psoriasis on the basis of the morphology of epidermal cells and dermal papillae. We believe that the described microscopic modalities could find in the near future a stable place in a clinical dermatological setting for quantitative diagnostic purposes and as a monitoring method for various treatments.

In vivo non-invasive monitoring of collagen remodelling by two-photon microscopy after micro-ablative fractional laser resurfacing.

Non-linear optical microscopy is becoming popular as a non-invasive in vivo imaging modality in dermatology. In this study, combined TPF and SHG microscopy were used to monitor collagen remodelling in vivo after micro-ablative fractional laser resurfacing. Papillary dermis of living subjects, covering a wide age range, was imaged immediately before and forty days after treatment. A qualitative visual examination of acquired images demonstrated an age-dependent remodelling effect on collagen. Additional quantitative analysis of new collagen production was performed by means of two image analysis methods. A higher increase in SHG to TPF ratio, corresponding to a stronger treatment effectiveness, was found in older subjects, whereas the effect was found to be negligible in young, and minimal in middle age subjects. Analysis of collagen images also showed a dependence of the treatment effectiveness with age but with controversial results. While the diagnostic potential of in vivo multiphoton microscopy has already been demonstrated for skin cancer and other skin diseases, here we first successfully explore its potential use for a non-invasive follow-up of a laser-based treatment.

Combined fluorescence-Raman spectroscopic setup for the diagnosis of melanocytic lesions.

Two optical fibre-based probes for spectroscopic measurements on human tissues were designed and developed. The two probes combine fluorescence and Raman spectroscopy in a multimodal approach. The fluorescence excitation was provided by two laser diodes emitting in the UV (378 nm) and in the visible (445 nm) range, while a third source in the NIR (785 nm) was used for Raman. The device was tested on freshly excised human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin. Discrimination of lesions based on their fluorescence and Raman spectra showed good correlation with the subsequent histological examination.

In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy.

Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 µm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 µm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments.

Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries.

AIMS: In engineered cells, endothelin ETA and ETB receptors can heterodimerize. We tested whether this can also be observed in native tissue. MAIN METHODS: Rat mesenteric resistance arteries (rMRA) were maintained in organ culture for 24h to upregulate ETB-mediated contractions in addition to their normal ETA-mediated responses. They were then exposed to 100 nM linear ET-1 (ETB-agonist) labeled with Oregon Green 488 (OG488/L.-ET-1) and/or to 16nM intact ET-1 (ETA/ETB-agonist) labeled with the rhodamine dye TAMRA (TAMRA/ET-1). Two photon laser scanning microscopy (TPLSM) was used for the visualization of their binding in the tissue. Fluorescence Lifetime Imaging Microscopy (FLIM) was employed for measurements of the OG488/L.-ET-1 lifetime in the absence and presence of TAMRA/ET-1. KEY FINDINGS: After incubation with the labeled ligands, medial smooth muscle cells (SMCs) were efficiently stained and became visible under TPLSM. TAMRA/ET-1 bound to all SMCs whereas OG488/L.-ET-1 stained only groups of SMCs. Interaction of the two receptor subtypes in SMC was investigated in double staining experiments. Fluorescence lifetime of OG488/L.-ET-1 was reduced in the presence of TAMRA/ET-1, which indicates the occurrence of Fluorescence Resonant Energy Transfer (FRET) and suggests close proximity of the two receptor subtypes within the arterial wall. SIGNIFICANCE: The methodology that is introduced by these new observations may be useful to assess ET-receptor heterodimerization in biopsies from relevant experimental animal models and human patients.

Multiphoton morpho-functional imaging of healthy colon mucosa, adenomatous polyp and adenocarcinoma.

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues.

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy.

Second-harmonic generation (SHG) microscopy is a fantastic tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. In fact, SHG combines the advantages of a non-linear microscopy approach with a coherent modality able to probe molecular organization. In this manuscript we review the physical concepts describing SHG from collagen, highlighting how this optical process allows to probe structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward - backward SHG detection, we then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods, highlighting advantages and limitations of the methods applied to tissue imaging and to potential clinical applications.