Strain Ultrasound Elastography of Aneurysm Sac Content after Randomized Endoleak Embolization with Sclerosing vs. Non-sclerosing Chitosan-based Hydrogels in a Canine Model.

PURPOSE: To compare the mechanical properties of aneurysm content after endoleak embolization with a chitosan hydrogel (CH) with that with a chitosan hydrogel with sodium tetradecyl sulfate (CH-STS) using strain ultrasound elastography (SUE). MATERIALS AND METHODS: Bilateral common iliac artery type Ia endoleaks were created in 9 dogs. Per animal, 1 endoleak was randomized to blinded embolization with CH, and the other, with CH-STS. Brightness-mode ultrasound, Doppler ultrasound, SUE radiofrequency ultrasound, and computed tomography were performed for up to 6 months until sacrifice. Radiologic and histopathologic studies were coregistered to identify 3 regions of interest: the embolic agent, intraluminal thrombus (ILT), and aneurysm sac. SUE segmentations were performed by 2 blinded independent observers. The maximum axial strain (MAS) was the primary outcome. Statistical analysis was performed using the Fisher exact test, multivariable linear mixed-effects models, and intraclass correlation coefficients (ICCs). RESULTS: Residual endoleaks were identified in 7 of 9 (78%) and 4 of 9 (44%) aneurysms embolized with CH and CH-STS, respectively (P = .3348). CH-STS had a 66% lower MAS (P < .001) than CH. The ILT had a 37% lower MAS (P = .01) than CH and a 77% greater MAS (P = .079) than CH-STS. There was no significant difference in ILT between treatments. The aneurysm sacs embolized with CH-STS had a 29% lower MAS (P < .001) than those embolized with CH. Residual endoleak was associated with a 53% greater MAS (P < .001). The ICC for MAS was 0.807 (95% confidence interval: 0.754-0.849) between segmentations. CONCLUSIONS: CH-STS confers stiffer intraluminal properties to embolized aneurysms. Persistent endoleaks are associated with increased sac strain, an observation that may help guide management.

Feasibility of shear wave sonoelastography to detect endoleak and evaluate thrombus organization after endovascular repair of abdominal aortic aneurysm.

PURPOSE: To investigate the feasibility of shear wave sonoelastography (SWS) for endoleak detection and thrombus characterization of abdominal aortic aneurysm (AAA) after endovascular repair (EVAR). MATERIALS AND METHODS: Participants who underwent EVAR were prospectively recruited between November 2014 and March 2016 and followed until March 2019. Elasticity maps of AAA were computed using SWS and compared to computed tomography angiography (CTA) and color Doppler ultrasound (CDUS). Two readers, blinded to the CTA and CDUS results, reviewed elasticity maps and B-mode images to detect endoleaks. Three or more CTAs per participant were analyzed: pre-EVAR, baseline post-EVAR, and follow-ups. The primary endpoint was endoleak detection. Secondary endpoints included correlation between total thrombus elasticity, proportion of fresh thrombus, and aneurysm growth between baseline and reference CTAs. A 3-year follow-up was made to detect missed endoleaks, EVAR complication, and mortality. Data analyses included Cohen's kappa; sensitivity, specificity, and positive predictive value (PPV); Pearson coefficient; and Student's t tests. RESULTS: Seven endoleaks in 28 participants were detected by the two SWS readers (k = 0.858). Sensitivity of endoleak detection with SWS was 100%; specificity and PPV averaged 67% and 50%, respectively. CDUS sensitivity was estimated at 43%. Aneurysm growth was significantly greater in the endoleak group compared to sealed AAAs. No correlation between growth and thrombus elasticity or proportion of fresh thrombus in AAAs was found. No new endoleaks were observed in participants with SWS negative studies. CONCLUSION: SWS has the potential to detect endoleaks in AAA after EVAR with comparable sensitivity to CTA and superior sensitivity to CDUS. KEY POINTS: • Dynamic elastography with shear wave sonoelastography (SWS) detected 100% of endoleaks in abdominal aortic aneurysm (AAA) follow-up that were identified by a combination of CT angiography (CTA) and color Doppler ultrasound (CDUS). • Based on elasticity maps, SWS differentiated endoleaks from thrombi within the aneurysm sac (p < 0.001). • After 3-year follow-up, no new endoleaks were observed in SWS negative examinations.

Abdominal aortic aneurysm follow-up by shear wave elasticity imaging after endovascular repair in a canine model.

OBJECTIVES: To investigate if shear wave imaging (SWI) can detect endoleaks and characterize thrombus organization in abdominal aortic aneurysms (AAAs) after endovascular aneurysm repair. METHODS: Stent grafts (SGs) were implanted in 18 dogs after surgical creation of type I endoleaks (four AAAs), type II endoleaks (13 AAAs) and no endoleaks (one AAA). Color flow Doppler ultrasonography (DUS) and SWI were performed before SG implantation (baseline), on days 7, 30 and 90 after SG implantation, and on the day of the sacrifice (day 180). Angiography, CT scans and macroscopic tissue sections obtained on day 180 were evaluated for the presence, size and type of endoleaks, and thrombi were characterized as fresh or organized. Endoleak areas in aneurysm sacs were identified on SWI by two readers and compared with their appearance on DUS, CT scans and macroscopic examination. Elasticity moduli were calculated in different regions (endoleaks, and fresh and organized thrombi). RESULTS: All 17 endoleaks (100 %) were identified by reader 1, whereas 16 of 17 (94 %) were detected by reader 2. Elasticity moduli in endoleaks, and in areas of organized thrombi and fresh thrombi were 0.2 ± 0.4, 90.0 ± 48.2 and 13.6 ± 4.5 kPa, respectively (P < 0.001 between groups). SWI detected endoleaks while DUS (three endoleaks) and CT (one endoleak) did not. CONCLUSIONS: SWI has the potential to detect endoleaks and evaluate thrombus organization based on the measurement of elasticity. KEY POINTS: • SWI has the potential to detect endoleaks in post-EVAR follow-up. • SWI has the potential to characterize thrombus organization in post-EVAR follow-up. • SWI may be combined with DUS in post-EVAR surveillance of endoleak.

Endovascular Repair of Abdominal Aortic Aneurysm: Follow-up with Noninvasive Vascular Elastography in a Canine Model.

PURPOSE: To assess the ability of noninvasive vascular elastography (NIVE) to help characterize endoleaks and thrombus organization in a canine model of abdominal aortic aneurysm after endovascular aneurysm repair with stent-grafts, in comparison with computed tomography (CT) and pathologic examination findings. MATERIALS AND METHODS: All protocols were approved by the Animal Care Committee in accordance with the guidelines of the Canadian Council of Animal Care. Stent-grafts were implanted in a group of 18 dogs with aneurysms created in the abdominal aorta. Type I endoleak was created in four aneurysms; type II endoleak, in 13 aneurysms; and no endoleak, in one aneurysm. Doppler ultrasonography and NIVE examinations were performed at baseline and at 1-week, 1-month, 3-month, and 6-month follow-up. Angiography, CT, and macroscopic tissue examination were performed at sacrifice. Strain values were computed by using the Lagrangian speckle model estimator. Areas of endoleak, solid organized thrombus, and fresh thrombus were identified and segmented by comparing the results of CT and macroscopic tissue examination. Strain values were compared by using the Wilcoxon rank-sum and Kruskal-Wallis tests. RESULTS: All stent-grafts were successfully deployed, and endoleaks were clearly depicted in the last follow-up elastography examinations. Maximal axial strains over consecutive heart cycles in endoleak, organized thrombus, and fresh thrombus areas were 0.78% ± 0.22, 0.23% ± 0.02, 0.10% ± 0.04, respectively. Strain values were significantly different between endoleak and organized or fresh thrombus areas (P < .000) and between organized and fresh thrombus areas (P < .0002). No correlation was found between strain values and type of endoleak, sac pressure, endoleak size, and aneurysm size. CONCLUSION: NIVE may be able to help characterize endoleak and thrombus organization, regardless of the size, pressure, and type of endoleak.

In Vitro and Pilot In Vivo Evaluation of a Bioactive Coating for Stent Grafts Based on Chondroitin Sulfate and Epidermal Growth Factor.

PURPOSE: To evaluate the potential of a bioactive coating based on chondroitin sulfate (CS) and tethered epidermal growth factor (EGF) for improvement of healing around stent grafts (SGs). MATERIALS AND METHODS: The impact of the bioactive coating on cell survival was tested in vitro on human vascular cells using polyethylene terephthalate films (PET) as a substrate. After being transferred onto a more "realistic" material (expanded polytetrafluoroethylene [ePTFE]), the durability and mechanical behavior of the coating and cell survival were studied. Preliminary in vivo testing was performed in a canine iliac aneurysm model reproducing type I endoleaks (three animals with one control and one bioactive SG for each). RESULTS: CS and EGF coatings significantly increased survival of human smooth muscle cells and fibroblasts compared with bare PET or ePTFE (P < .05). The coating also displayed good durability over 30 days according to enzyme-linked immunosorbent assay and cell survival tests. The coating did not affect mechanical properties of ePTFE and was successfully transferred onto commercial SGs for in vivo testing. No difference was observed on computed tomography and macroscopic examinations in endoleak persistence at 3 months, but the bioactive coating deposited on the abluminal surface of the SG (exposed to the vessel wall) increased the percentage of healed tissue in the aneurysm. No adverse effect, such as neointima formation or thrombosis, was observed. CONCLUSIONS: The bioactive coating promoted in vitro cell survival, displayed good durability, and was successfully transferred onto a commercial SG. Preliminary in vivo results suggest improved healing around bioactive SGs.

Normal range of motion at the hip show different pressure behavior in the lateral and acetabular compartments: a cadaveric investigation.

PURPOSE: The techniques used previously to assess intracapsular pressures did not allow the assessment of pressure variations in both compartments throughout the entire range of motion without puncturing the capsular tissue. Our hypothesis was that the intra-capsular pressure would be different in the lateral and acetabular compartment depending on the movement assessed. METHODS: Eight hip joints from four cadaveric specimens (78.5 ± 7.9 years) were assessed using intra-osseous tunnels reaching the lateral and acetabular compartments. Using injector adaptors, 2.7 ml of liquid were inserted in both compartments to simulate synovial liquid. Optic pressure transducers were used to measure pressure variations. We manually performed hip adduction, abduction, extension, flexion and internal rotation at 90° of flexion. RESULTS: Hip extension and internal rotation show the highest intra-capsular pressures in the lateral compartment with increases of 20.56 ± 19.29 and 19.27 ± 18.96 mmHg, respectively. Hip abduction and hip internal rotation showed depressurisations of - 16.86 ± 18.01 and - 31.88 ± 30.71 mmHg in the acetabular compartment, respectively. The pressures measured in the lateral compartment and in the acetabular compartment were significantly (P < 0.05) different for the hip abduction, 90° of flexion and internal rotation. Pressure variations showed that maximum intracapsular fluid pressures in the lateral compartment occur at maximum range of motion for all movements. CONCLUSION: As an increase in pressure may produce hip pain, clinician should assess pain at maximum range of motion in the lateral compartment. The pressure measured in the acetabular compartment vary depending on the hip position. The movements assessed are used in clinical practice to evaluate hip integrity and might bring pain. The pressure variations throughout the entire range of motion are a relevant information during hip clinical assessment and might help clinicians to better understand the manifestations of pain.

Moment arms of the deltoid, infraspinatus and teres minor muscles for movements with high range of motion: A cadaveric study.

BACKGROUND: Moment arms are an indicator of the role of the muscles in joint actuation. An excursion method is often used to calculate them, even though it provides 1D results. As shoulder movement occurs in three dimensions (combination of flexion, abduction and axial rotation), moment arms should be given in 3D. Our objective was to assess the 3D moment arms of the rotator cuff (infraspinatus and teres minor) and deltoid muscles for movements with high arm elevation. METHODS: The 3D moment arms (components in plane of elevation, elevation and axial rotation) were assessed using a geometric method, enabling to calculate the moment arms in 3D, on five fresh post-mortem human shoulders. Movement with high range of motion were performed (including overhead movement). The humerus was elevated until it reaches its maximal posture in different elevation plane (flexion, scaption, abduction and elevation in a plane 30° posterior to frontal plane). FINDINGS: We found that the anterior deltoid was a depressor and contributes to move the elevation plane anteriorly. The median deltoid was a great elevator and the posterior deltoid mostly acted in moving the elevation plane posteriorly. The infraspinatus and teres minor were the greatest external rotator of the shoulder. The position of the glenohumeral joint induces changes in the muscular moment arms. The maximal shoulder elevation was 144° (performed in the scapular plane). INTERPRETATION: The knowledge of 3D moment arms for different arm elevations might help surgeons in planning tendon reconstructive surgery and help validate musculoskeletal models.

Intracapsular pressures in the flexion-abduction-external rotation and flexion-adduction-internal rotation tests and their comparison with classic hip range of motion: A cadaveric assessment.

Background Flexion-Abduction-External-Rotation and Flexion-Adduction-Internal-Rotation tests are used to reproduce pain at the hip during clinical assessment. As pain can be elicited by high intracapsular pressure, no information has been provided regarding intracapsular pressure during these pain provocative tests. Methods Eight hip joints from four cadaveric specimens (78.5 ± 7.9 years) were assessed using intra-osseous tunnels reaching the lateral and acetabular compartments. To simulate synovial liquid, 2.7 ml of liquid were inserted in both compartments using adaptor injectors. Optic pressure transducers were used to measure pressure variations. Pressures were compared between compartments in each test and between tests for each compartment. Both tests were compared with uniplanar movements. Findings The Flexion-Adduction-Internal-Rotation test showed a significant difference between pressure measured in the lateral (27.17 ± 42.63 mmHg) and acetabular compartment (-26.80 ± 29.26 mmHg) (P < 0.006). The pressure measured in the lateral compartment during the Flexion-Adduction-Internal-Rotation test (27.17 ± 42.63 mmHg) was significantly higher than in the Flexion-Abduction-External-Rotation test (-8.09 ± 15.09 mmHg) (P < 0.010). The pressure measured in the lateral compartment in the Flexion-Abduction-External-Rotation test was significantly lower than during internal rotation (P = 0.011) and extension (P = 0.006). Interpretation High intracapsular pressure is correlated with greater pain at the hip. Clinicians should assess pain with caution during the Flexion-Adduction-Internal-Rotation test as this test showed high intracapsular pressures in the lateral compartment. The Flexion-Abduction-External-Rotation is not influenced by high intra-capsular pressures.

Sonoelastography of the Shoulder: A Narrative Review.

Sonoelastography is a relatively new non-invasive imaging tool to assess the in vivo qualitative and quantitative biomechanical properties of various tissues. Two types of sonoelastography (SE) are commonly explored: strain and shear wave. Sonoelastography can be used in multiple medical subspecialties to assess pathological tissular changes by obtaining mechanical properties, shear wave speed, and strain ratio data. Although there are various radiological imaging methods, such as MRI or CT scan, to assess musculoskeletal structures (muscles, tendons, joint capsules), SE is more accessible since this approach is of low cost and does not involve radiation. As of 2018, SE has garnered promising data in multiple studies. Preliminary clinico-radiological correlations have been established to bridge tissue biomechanical findings with their respective clinical pathologies. Specifically, concerning the shoulder complex, recent findings have described mechanical tissue changes in shoulder capsulitis. The long head of the biceps and supraspinatus SE were among the recently studied structures with conditions regarding impingement, tendinosis, and tears. Since ultrasonography has established itself as an important tool in shoulder evaluation, it completes the history and physical examination skills of the clinicians. This study will provide an update on the most recent findings on SE of shoulder structures.

Shear wave elastography potential to characterize spastic muscles in stroke survivors: Literature review.

BACKGROUND: Post-stroke spasticity contributes to impairments, disabilities and decline in quality of life. Quantitative measurements of spasticity are needed in order to assess the impact of specific treatments and to choose the more accurate technique for each patient. The aim of this review is to examine the use of shear wave ultrasound elastography as a quantitative tool for monitoring biomechanical muscle properties such as stiffness and to determine whether it is a reliable method to assess spastic muscle in stroke survivors. METHODS: Studies were sought from Academic Search Complete, CINAHL, PubMed/Medline, Scopus and SportDiscus with the following keywords: shear wave elastography, spasticity, stiffness, elasticity, hardness, stroke, cerebrovascular accident, cerebral vascular event and transient ischaemic attack. Titles and abstracts were screened, and relevant full-text articles were retrieved for further review. FINDINGS: Of the 76 screened studies, nine captured elastography data of the spastic biceps brachii (n = 6) or the plantar flexors (n = 3) with stroke victims. All consulted studies had a different way of utilizing this technology which was expected considering no guidelines had been developed. Shear wave speed values obtained are compared and discussed with clinical measures. Reliability of the devices is also discussed. INTERPRETATION: Shear wave ultrasound elastography can provide useful quantitative information on the mechanical properties of the spastic muscles in post-stroke patients. Nevertheless, new studies using common terminology and parameters are needed to develop reliable methods that could help in assessing treatment efficiency.

Glenohumeral joint capsular tissue tension loading correlates moderately with shear wave elastography: a cadaveric investigation.

PURPOSE: The purpose of this study was to investigate changes in the mechanical properties of capsular tissue using shear wave elastography (SWE) and a durometer under various tensile loads, and to explore the reliability and correlation of SWE and durometer measurements to evaluate whether SWE technology could be used to assess tissue changes during capsule tensile loading. METHODS: The inferior glenohumeral joint capsule was harvested from 10 fresh human cadaveric specimens. Tensile loading was applied to the capsular tissue using 1-, 3-, 5-, and 8-kg weights. Blinded investigators measured tissue stiffness and hardness during loading using SWE and a durometer, respectively. Intraobserver reliability was established for SWE and durometer measurements using intraclass correlation coefficients (ICCs). The Pearson product-moment correlation was used to assess the associations between SWE and durometer measurements. RESULTS: The ICC3,5 for durometer measurements was 0.90 (95% confidence interval [CI], 0.79 to 0.96; P<0.001) and 0.95 (95% CI, 0.88 to 0.98; P<0.001) for SWE measurements. The Pearson correlation coefficient values for 1-, 3-, and 5-kg weights were 0.56 (P=0.095), 0.36 (P=0.313), and -0.56 (P=0.089), respectively. When the 1- and 3-kg weights were combined, the ICC3,5 was 0.72 (P<0.001), and it was 0.62 (P<0.001) when the 1-, 3-, and 5-kg weights were combined. The 8-kg measurements were severely limited due to SWE measurement saturation of the tissue samples. CONCLUSION: This study suggests that SWE is reliable for measuring capsular tissue stiffness changes in vitro at lower loads (1 and 3 kg) and provides a baseline for the non-invasive evaluation of effects of joint loading and mobilization on capsular tissues in vivo.

A novel ex vivo, in situ method to study the human brain through MRI and histology.

BACKGROUND: MRI-histology correlation studies of the ex vivo brain mostly employ fresh, extracted (ex situ) specimens, aldehyde fixed by immersion, which has several disadvantages for MRI scanning (e.g. deformation of the organ). A minority of studies are done ex vivo-in situ (unfixed brain), requiring an MRI scanner readily available within a few hours of the time of death. NEW METHOD: We propose a new technique, exploited by anatomists, for scanning the ex vivo brain: fixation by whole body perfusion, which implies fixation of the brain in situ. This allows scanning the brain surrounded by fluids, meninges, and skull, preserving the structural relationships of the brain in vivo. To evaluate the proposed method, five heads perfused-fixed with a saturated sodium chloride solution were employed. Three sequences were acquired on a 1.5 T MRI scanner: T1weighted, T2weighted-FLAIR, and Gradient-echo. Histology analysis included immunofluorescence for myelin basic protein and neuronal nuclei. RESULTS: All MRIs were successfully processed through a validated pipeline used with in vivo MRIs. All cases exhibited positive antigenicity for myelin and neuronal nuclei. COMPARISON WITH EXISTING METHODS: All scans registered to a standard neuroanatomical template in pseudo-Talairach space more accurately than an ex vivo-ex situ scan. The time interval to scan the ex vivo brain in situ was increased to at least 10 months. CONCLUSIONS: MRI and histology study of the ex vivo-in situ brain fixed by perfusion is an alternative approach that has important procedural and practical advantages over the two standard methods to study the ex vivo brain.

Shear wave elasticity imaging for residual endoleak and thrombus characterisation after endoleak embolisation following endovascular aneurysm repair: a canine animal study.

BACKGROUND: To evaluate residual endoleak and thrombus organisation with shear wave imaging (SWI) after endoleak embolisation through an animal study. METHODS: This prospective experimental study involved eight dogs with creation of 16 iliac aneurysms and type I endoleak after endovascular aneurysm repair (EVAR). Embolisation agents were injected into the sac to seal endoleak. SWI and colour flow Doppler ultrasound (DUS) were performed at implantation, one week, and one and three months after implantation; for three dogs, SWI and DUS were also performed six months after implantation. Digital subtraction angiography and contrast-enhanced computed tomography were performed at sacrifice. Macroscopic and histopathological analyses were processed to identify regions of interest (ROIs) for endoleak, fresh thrombus, organised thrombus and embolisation agent, where SWI elasticity moduli were compared. RESULTS: At sacrifice, nine aneurysms had residual endoleak, while seven were sealed. Ten had a fresh and 15 had an organised thrombus. SWI was able to detect all endoleaks, including two cases undetected with DUS. Elasticity moduli of 0.2 kPa ± 0.1 kPa (mean ± SD), 9.5 kPa ± 3.3 kPa, 48.1 kPa ± 21.3 kPa and 44.9 kPa ± 23.7 kPa were found in the ROIs positioned in endoleaks, fresh thrombi, organised thrombi and embolisation agent, respectively. Elasticity values of endoleak and fresh thrombus were lower than those of organised thrombi and embolisation agent (p < 0.001). Stiffness of fresh thrombus at one week (8.7 kPa ± 3.6 kPa) increased at three months (30.2 kPa ± 13.8 kPa), indicating thrombus maturation (p < 0.001). CONCLUSIONS: In a dog model of iliac EVAR, SWI was able to identify endoleak, thrombus maturation and embolising agents after endoleak embolisation.

Chitosan-Sodium Tetradecyl Sulfate Hydrogel: Characterization and Preclinical Evaluation of a Novel Sclerosing Embolizing Agent for the Treatment of Endoleaks.

PURPOSE: To compare the efficacy of an embolization agent with sclerosing properties (made of chitosan and sodium tetradecyl sulfate, CH-STS) with a similar embolization agent but without sclerosing properties (made of chitosan, CH) in treating endoleaks in a canine endovascular aneurysm repair model. METHODS: Two chitosan-based radiopaque hydrogels were prepared, one with STS and one without STS. Their rheological, injectability, and embolizing properties were assessed in vitro; afterwards, their efficacy in occluding endoleaks was compared in a canine bilateral aneurysm model reproducing type I endoleaks (n = 9 each). The primary endpoint was endoleak persistence at 3 or 6 months, assessed on a CT scan and macroscopic examination. Secondary endpoints were the occurrence of stent-graft (SG) thrombosis, the evolution of the aneurysm mean diameter, as well as aneurysm healing and inflammation scores in pathology examinations. RESULTS: In vitro experiments showed that both products gelled rapidly and presented initial storage moduli greater than 800 Pa, which increased with time. Both gels were compatible with microcatheter injection and occlude flow up to physiological pressure in vitro. In a type I endoleak model, the injection of CH-STS sclerosing gel tended to reduce the risk of occurrence of endoleaks, compared to CH non-sclerosing agent (2/9 vs. 6/9, p = 0.069). No case of SG thrombosis was observed. Moderate inflammation was found around both gels, with a comparable intensity score in both CH and CH-STS groups (2.6 ± 0.9 and 2.7 ± 0.9, respectively; p = 0.789). CONCLUSIONS: Flow occlusion combined with chemical endothelial denudation appears promising for the treatment of endoleaks. LEVEL OF EVIDENCE: N/A.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 µm, while in the transverse direction it is ∼1-15 µm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano-cylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced.

Visualization of mouse neuronal ganglia infected by Herpes Simplex Virus 1 (HSV-1) using multimodal non-linear optical microscopy.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.

Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

The structural origin of second harmonic generation in fascia.

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.