Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

Third harmonic generation (THG) microscopy is a label‐free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all‐nuclei‐highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification.

     

Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue

Real‐time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth‐sectioning at sub‐cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components—lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label‐free THG images. Furthermore, 2‐ and 3‐photon excited auto‐fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real‐time assessment of excised tissue during surgery.      

Detection of the T cell activation state using nonlinear optical microscopy

The ability to monitor the activation state of T‐cells during immunotherapy is of great importance. Although specific activation markers do exist, their abundance and complicated regulation cannot definitely define the activation state of the cells. Previous studies have shown that Third Harmonic Generation (THG) imaging could distinguish between activated versus resting microglia and healthy versus cancerous cells, mainly based on their lipid‐body profiles. In the present study, mitogen or antigen‐stimulated T‐cells were subjected to THG imaging microscopy. Qualitative and quantitative analysis showed statistically significant increase of THG mean area and intensity in activated versus resting T‐cells. The connection of THG imaging to chemical information was achieved using Raman spectroscopy, which showed significant differences between the activation processes and controls, correlating of THG signal area with cholesterol and lipid compounds, but not with triglycerides. The obtained results suggested a potential employment of nonlinear microscopy in evaluating of T‐cell activation, which is expected to be largely appreciated in the clinical practice.      

Low‐frequency conductivity tensor of rat brain tissues inferred from diffusion MRI

Conductivity tensor maps of the rat brain were obtained using diffusion magnetic resonance imaging (MRI). Signal attenuations in the cortex and the corpus callosum were measured using the stimulated echo acquisition mode (STEAM) sequence with b factors up to 6000 s/mm². Our previously published method was improved to infer 3 × 3 conductivity tensor at the low‐frequency limit. The conductivity tensor of the tissue was inferred from the fast component of the diffusion tensor and a fraction of the fast component. The mean conductivity (MC) of the cortex and the corpus callosum was 0.52 and 0.62 S/m, respectively. Diffusion‐weighted images were obtained with b factors up to 4500 s/mm². Conductivity tensor images were calculated from the fast diffusion tensor images. Tissues with highly anisotropic cellular structures, such as the corpus callosum, the internal capsule, and the trigeminal nerve, exhibited high anisotropy in conductivity. The resulting values corresponded to conductivities at the low‐frequency limit because our method assumed electric currents flowing only through extracellular fluid. Bioelectromagnetics 30:489–499, 2009. © 2009 Wiley‐Liss, Inc.     

Back Cover: Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth (J. Biophotonics 1/2019)

TP‐SAX microscopy images are shown in cyan, cyan hot, red, gray, and orange hot colors; TPFM images are in green color while the LSCM image is in magenta color. Our results show a spatial resolution enhancement for TP‐SAX (cyan image) even at 2.4 mm depth of a mouse brain in comparison with TPFM (green image) where scattering seriously degrades the PSF. Further details can be found in the article by Sandeep Chakraborty, Szu‐Yu Lee, Jye‐Chang Lee, Chen‐Tung Yen, and Chi‐Kuang Sun ( e201800136 ).

     

Classification of established atopic dermatitis in children with the in vivo imaging methods

Atopic dermatitis (AD) is a cutaneous disease resulting from a defective barrier and dysregulated immune response. The severity scoring of atopic dermatitis (SCORAD) is used to classify AD. Noninvasive imaging approaches supplementary to SCORAD were investigated. Cr:forsterite laser‐based microscopy was employed to analyze endogenous third‐harmonic generation (THG) and second‐harmonic generation (SHG) signals from skin. Imaging parameters were compared between different AD severities. Three‐dimensional reconstruction of imaged skin layers was performed. Finally, statistic models from quantitative imaging parameters were developed for predicting disease severity. Our data demonstrate that THG signal intensity of lesional skin in AD were significantly increased and was positively correlated with AD severity. Characteristic gray level co‐occurrence matrix (GLCM) values were observed in more severe AD. In the 3D reconstruction video, individual dermal papilla and obvious fibrosis in the upper papillary dermis were easily identified. Our estimation models could predict the disease severity of AD patients with an accuracy of nearly 85%. The THG signal intensity and characteristic GLCM patterns are associated with AD severity and can serve as quantitative predictive parameters. Our imaging approach can be used to identify the histopathological changes of AD objectively, and to complement the SCORAD index, thus improving the accuracy of classifying AD severity.      

Distinction between breast cancer cell subtypes using third harmonic generation microscopy

Third Harmonic Generation (THG) microscopy as a non‐invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2‐positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer‐specific profiles, correlating lipid raft‐corresponding spectra to THG signal, associating thus THG to chemical information.

THG imaging of a cancer cell.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Dorsal Visual Pathway Changes in Patients with Comitant Extropia

Background

Strabismus is a disorder in which the eyes are misaligned. Persistent strabismus can lead to stereopsis impairment. The effect of strabismus on human brain is not unclear. The present study is to investigate whether the brain white structures of comitant exotropia patients are impaired using combined T1-weighted imaging and diffusion tensor imaging (DTI).

Principal Findings

Thirteen patients with comitant strabismus and twelve controls underwent magnetic resonance imaging (MRI) with acquisition of T1-weighted and diffusion tensor images. T1-weighted images were used to analyze the change in volume of white matter using optimized voxel-based morphology (VBM) and diffusion tensor images were used to detect the change in white matter fibers using voxel-based analysis of DTI in comitant extropia patients. VBM analysis showed that in adult strabismus, white matter volumes were smaller in the right middle occipital gyrus, right occipital lobe/cuneus, right supramarginal gyrus, right cingulate gyrus, right frontal lobe/sub-gyral, right inferior temporal gyrus, left parahippocampa gyrus, left cingulate gyrus, left occipital lobe/cuneus, left middle frontal gyrus, left inferior parietal lobule, and left postcentral gyrus, while no brain region with greater white matter volume was found. Voxel-based analysis of DTI showed lower fractional anisotropy (FA) values in the right middle occipital gyrus and right supramarginal gyrus in strabismus patients, while brain region with increased FA value was found in the right inferior frontal gyrus.

Conclusion

By combining VBM and voxel-based analysis of DTI results, the study suggests that the dorsal visual pathway was abnormal or impaired in patients with comitant exotropia.     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.      

Nonlinear‐optical stain‐free stereoimaging of astrocytes and gliovascular interfaces

Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain.     

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

White matter changes and word finding failures with increasing age

Background

Increasing life expectancy necessitates the better understanding of the neurophysiological underpinnings of age-related cognitive changes. The majority of research examining structural-cognitive relationships in aging focuses on the role of age-related changes to grey matter integrity. In the current study, we examined the relationship between age-related changes in white matter and language production. More specifically, we concentrated on word-finding failures, which increase with age.

Methodology/Principal Findings

We used Diffusion tensor MRI (a technique used to image, in vivo, the diffusion of water molecules in brain tissue) to relate white matter integrity to measures of successful and unsuccessful picture naming. Diffusion tensor images were used to calculate Fractional Anisotropy (FA) images. FA is considered to be a measure of white matter organization/integrity. FA images were related to measures of successful picture naming and to word finding failures using voxel-based linear regression analyses. Successful naming rates correlated positively with white matter integrity across a broad range of regions implicated in language production. However, word finding failure rates correlated negatively with a more restricted region in the posterior aspect of superior longitudinal fasciculus.

Conclusions/Significance

The use of DTI-MRI provides evidence for the relationship between age-related white matter changes in specific language regions and word finding failures in old age.     

Pre‐synaptic localization of the γ‐secretase‐inhibiting protein p24α2 in the mammalian brain

Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α₂ reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α₂ in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α₂ in the mouse brain. Immunostaining showed that p24α₂ is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α₂ in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α₂ is condensed at active zone‐docked SV. During development, p24α₂ expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α₂ in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α₂ in the regulation of Aβ generation and secretion in the brain.

     

Sensitivity analysis of reaction-diffusion constraints in muscle energetics

Theoretical and experimental studies of aerobic metabolism on a wide range of skeletal muscle fibers have shown that while all fibers normally function within the reaction control regime, some fibers operate near the transition region where reaction control switches to diffusion control. Thus, the transition region between reaction and diffusion control may define the limits of muscle function, and analysis of factors that affect this transition is therefore needed. In order to assess the role of all important model parameters, a sensitivity analysis (SA) was performed to define the parameter space where muscle fibers transition from reaction to diffusion control. SA, performed on a previously developed reaction–diffusion model, shows that the maximum rate for the ATPase reaction (V_max,ATPase), boundary oxygen concentration in the capillary supply (O), the mitochondrial volume fraction (ε_mito), and the diffusion coefficient of oxygen ( ) are the most sensitive parameters affecting this transition to diffusion control. It is demonstrated that fibers are not limited by diffusion for slow reactions (V_max,ATPase < 25 mM/min), high oxygen supply for the capillaries (O ≥ 35 µM), and large amounts of mitochondria (ε_mito ≥ 0.1). These conditions are applicable to muscle cells spanning a very broad range of animals. Within the diffusion‐controlled region, the overall metabolic rate and ATP concentrations have much higher sensitivity to the diffusion coefficient of oxygen than to the diffusion coefficients of the other metabolites (ATP, ADP, P_i). Biotechnol. Bioeng. 2012; 109:559–571. © 2011 Wiley Periodicals, Inc.     

Diffusion Tensor Imaging in Patients with Glioblastoma Multiforme Using the Supertoroidal Model

Purpose

Diffusion Tensor Imaging (DTI) is a powerful imaging technique that has led to improvements in the diagnosis and prognosis of cerebral lesions and neurosurgical guidance for tumor resection. Traditional tensor modeling, however, has difficulties in differentiating tumor-infiltrated regions and peritumoral edema. Here, we describe the supertoroidal model, which incorporates an increase in surface genus and a continuum of toroidal shapes to improve upon the characterization of Glioblastoma multiforme (GBM).

Materials and Methods

DTI brain datasets of 18 individuals with GBM and 18 normal subjects were acquired using a 3T scanner. A supertoroidal model of the diffusion tensor and two new diffusion tensor invariants, one to evaluate diffusivity, the toroidal volume (TV), and one to evaluate anisotropy, the toroidal curvature (TC), were applied and evaluated in the characterization of GBM brain tumors. TV and TC were compared with the mean diffusivity (MD) and fractional anisotropy (FA) indices inside the tumor, surrounding edema, as well as contralateral to the lesions, in the white matter (WM) and gray matter (GM).

Results

The supertoroidal model enhanced the borders between tumors and surrounding structures, refined the boundaries between WM and GM, and revealed the heterogeneity inherent to tumor-infiltrated tissue. Both MD and TV demonstrated high intensities in the tumor, with lower values in the surrounding edema, which in turn were higher than those of unaffected brain parenchyma. Both TC and FA were effective in revealing the structural degradation of WM tracts.

Conclusions

Our findings indicate that the supertoroidal model enables effective tensor visualization as well as quantitative scalar maps that improve the understanding of the underlying tissue structure properties. Hence, this approach has the potential to enhance diagnosis, preoperative planning, and intraoperative image guidance during surgical management of brain lesions.     

Relationships between serotonergic and cannabinoid system in depressive‐like behavior: a PET study with [11C]‐DASB

Chronic stress represents a major environmental risk factor for mood disorders in vulnerable individuals. The neurobiological mechanisms underlying these disorders involve serotonergic and endocannabinoid systems. In this study, we have investigated the relationships between these two neurochemical systems in emotional control using genetic and imaging tools. CB₁ cannabinoid receptor knockout mice (KO) and wild‐type littermates (WT) were exposed to chronic restraint stress. Depressive‐like symptoms (anhedonia and helplessness) were produced by chronic stress exposure in WT mice. CB₁ KO mice already showed these depressive‐like manifestations in non‐stress conditions and the same phenotype was observed after chronic restraint stress. Chronic stress similarly impaired long‐term memory in both genotypes. In addition, brain levels of serotonin transporter (5‐HTT) were assessed using positron emission tomography. Decreased brain 5‐HTT levels were revealed in CB₁ KO mice under basal conditions, as well as in WT mice after chronic stress. Our results show that chronic restraint stress induced depressive‐like behavioral alterations and brain changes in 5‐HTT levels similarly to those revealed in CB₁ KO mice in non‐stressed conditions. These results underline the relevance of chronic environmental stress on serotonergic and endocannabinoid transmission for the development of depressive symptoms.

     

Adenosine transiently modulates stimulated dopamine release in the caudate–putamen via A1 receptors

Adenosine modulates dopamine in the brain via A₁ and A_2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast‐scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate–putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 μM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2 s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7%, similar to the 54 ± 6% decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 min. The A₁ receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A_2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A₁ receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain.

     

Gas exchange strategy in the Nile crocodile: a morphometric study

Summary The respiratory surface area (S_AR) per kilogram body mass (M_B), the harmonic mean thickness of the air-blood barrier (_htR) in the gas exchange tissue, and the anatomical diffusion factor (ADF=S_AR/_htR per M_B) were calculated for four juvenile Nile crocodiles. The ADF of three small specimens (mean M_B=3.59 kg) was 625 cm²·m^–1·kg^–1. The values varied considerably among individuals and were similar to that of a 5.68-kg specimen (593 cm²·m^–1·kg^–1). Only 9% of the ADF is located in the anterior third of the lung, which because of its conical shape makes up only 14 percent of the total lung volume. Particularly in the middle third of the lung, the proximal region near the intrapulmonary bronchus displays a greater ratio of respiratory/non-respiratory surface areas than do more distally located sampling sites. The _htR is also significantly smaller proximally than distally. The cumulative ADF per unit M_B is greater than that previously reported for this species on the basis of overall estimates of S_AR and _htR, but is still less than that of lizards and testudinids. The disposition of ADF between distal air storage region and the intrapulmonary bronchus is consistent with a bidirectional cross-current gas exchange model.Abbreviations ADF anatomical diffusion factor - %AR percent of S_A included in the effective respiratory zone - M _B body mass - NVP non-ventilatory period - %P percent of total lung volume containing parenchyma - S _A total surface area of intrapulmonary septa - S _ANR that portion ofS _A lying out the effective respiratory zone - S _V surface-to-volume ratio in the parenchyma - _htR harmonic mean thickness of the air-blood tissue barrier within the respiratory zone - V _P parenchymal volume - VP ventilatory period     

Hyperspectral imaging in medicine: image pre‐processing problems and solutions in Matlab

The paper presents problems and solutions related to hyperspectral image pre‐processing. New methods of preliminary image analysis are proposed. The paper shows problems occurring in Matlab when trying to analyse this type of images. Moreover, new methods are discussed which provide the source code in Matlab that can be used in practice without any licensing restrictions.

The proposed application and sample result of hyperspectral image analysis.