Laser speckle contrast imaging versus microvascular Doppler sonography in aneurysm surgery: A prospective study.

Objective: This study aimed to compare microvascular Doppler sonography (MDS) and laser speckle contrast imaging (LSCI) for assessing vessel patency and aneurysm occlusion during microsurgical clipping of intracranial aneurysms. Methods: MDS and LSCI were used after clip placement during six neurovascular procedures including six patients, and agreement between the two techniques was assessed. LSCI was performed in parallel or right after MDS evaluation. The Doppler response was assessed through listening while flow in the LSCI videos was evaluated by three blinded neurovascular surgeons after the surgery. Statistical analysis determined the agreement between the techniques in assessing flow in 18 regions of interest (ROIs). Results: Agreement between MDS and LSCI in assessing vessel patency was observed in 87 % of the ROIs. LSCI accurately identified flow in 93.3 % of assessable ROIs, with no false positive or negative measurements. Three ROIs were not assessable with LSCI due to motion artifacts or poor image quality. No complications were observed. Conclusions: LSCI demonstrated high agreement with MDS in assessing vessel patency during microsurgical clipping of intracranial aneurysms. It provided continuous, real-time, full-field imaging with high spatial resolution and temporal resolution. While MDS allowed evaluation of deep vascular regions, LSCI complemented it by offering unlimited assessment of surrounding vessels.

Cortical perfusion measurements with laser speckle contrast imaging during adenosine induced cardiac arrest for aneurysm clipping: a case report.

Adenosine induced cardiac arrest (AiCA) is one of the methods used to facilitate microsurgical aneurysm clipping by providing more visibility and less pressure in the aneurysmal sac and neighboring vessels. We report the use of laser speckle contrast imaging (LSCI) during AiCA to monitor the changes in pulsation and perfusion on the cortical surface during adenosine induced cardiac arrest for aneurysm clipping surgery. Application of this technology for perfusion monitoring may improve workflow and surgical guidance and provide valuable feedback continuously throughout the procedure. ClinicalTrials.gov identifier: NCT0502840.

Sedation with dexmedetomidine decreases skin perfusion in cats.

OBJECTIVE: To evaluate skin perfusion in cats receiving dexmedetomidine compared to a placebo. ANIMALS: 9 healthy adult research cats. METHODS: A randomized, blinded, placebo-controlled study design was used. Two sites, the dorsal metatarsus (site: limb) and lateral flank (site: flank), were evaluated with laser speckle contrast imaging (LSCI) at baseline and following administration of dexmedetomidine (1, 3, or 5 mcg/kg, IV) or a placebo (0.9% saline, IV). Mean speckle contrast (MSC), a surrogate for perfusion, was obtained from LSCI and compared between treatments. Heart rate, sedation score, and body temperature were recorded. Skin perfusion to the flank and limb, reported as MSC, was assessed via LSCI at baseline and at 5, 10, and 15 minutes posttreatment. RESULTS: There was a significant decrease in heart rate (P < .001) in cats receiving 1, 3, and 5 mcg/kg dexmedetomidine compared to placebo. There was a significant increase in median sedation score at all time points postsedation compared to baseline (P < .018). Changes in MSC for the metatarsus were not significantly different between treatments at any time point (P = .12). For the flank, MSC was significantly higher for cats treated with dexmedetomidine compared to baseline (P ≤ .01). Skin perfusion to the flank decreased as early as 5 minutes posttreatment with dexmedetomidine and persisted for at least 15 minutes, regardless of dexmedetomidine dose. CLINICAL RELEVANCE: Dexmedetomidine decreased skin perfusion in cats, even at low doses. Veterinarians may elect for an alternative sedative medication when decreased skin perfusion is a concern.

Intraoperative Laser Speckle Contrast Imaging to Assess Vessel Flow in Neurosurgery: A Pilot Study.

BACKGROUND AND OBJECTIVES: Laser speckle contrast imaging (LSCI) has emerged as a promising tool for assessment of vessel flow during neurosurgery. We aimed to investigate the feasibility of visualizing vessel flow in the macrocirculation with a new fully microscope-integrated LSCI system and assess the validity and objectivity of findings compared with fluorescence angiography (FA). METHODS: This is a single-center prospective observational study enrolling adult patients requiring microsurgical treatment for brain vascular pathologies or brain tumors. Three independent raters, blinded toward findings of FA, reviewed regions of interest (ROIs) placed in exposed vessels and target structures. The primary end point was the validity of LSCI for assessment of vessel flow as measured by the agreement with FA. The secondary end point was objectivity, measured as the inter-rater agreement of LSCI findings. RESULTS: During 18 surgical procedures, 23 observations using FA and LSCI were captured simultaneously. Using LSCI, vessel flow was assessable in 62 (86.1%) and not assessable in 10 (13.9%) ROIs. The agreement between LSCI and FA was 86.1%, with an agreement coefficient of 0.85 (95% CI: 0.75-0.94). Disagreement between LSCI and FA was observed in the 10 ROIs that were not assessable. The agreement between ROIs that were assessable using LSCI and FA was 100%. The inter-rater agreement of LSCI findings was 87.9%, with an agreement coefficient of 0.86 (95% CI: 0.79-0.94). CONCLUSION: Fully microscope-integrated LSCI is feasible and has a high potential for clinical utility. Because of its characteristics, LSCI can be viewed as a full-field visual micro-Doppler that can be used as a complementary method to FA for assessing vessel flow during neurosurgery. Despite technical limitations related to the early development phase of the fully microscope-integrated system, we demonstrated reasonable validity and objectivity of findings compared with FA. Further research and refinement of the system may enhance its value in neurosurgical applications.

Subventricular zone cytogenesis provides trophic support for neural repair in a mouse model of stroke.

Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here, we characterize the identity of cells arising from the SVZ after stroke and uncover a mechanism through which they facilitate neural repair and functional recovery. With genetic lineage tracing, we show that SVZ-derived cells that migrate towards cortical photothrombotic stroke in mice are predominantly undifferentiated precursors. We find that ablation of neural precursor cells or conditional knockout of VEGF impairs neuronal and vascular reparative responses and worsens recovery. Replacement of VEGF is sufficient to induce neural repair and recovery. We also provide evidence that CXCL12 from peri-infarct vasculature signals to CXCR4-expressing cells arising from the SVZ to direct their ectopic migration. These results support a model in which vasculature surrounding the site of injury attracts cells from the SVZ, and these cells subsequently provide trophic support that drives neural repair and recovery.

Continuous hemodynamics monitoring during arteriovenous malformation microsurgical resection with laser speckle contrast imaging: case report.

AVM surgery is challenging due to progressive and often unforeseeable flow changes during its resection which involve both the AVM and the surrounding brain tissue. Hence, accurate monitoring of blood flow is crucial to minimize complications and improve outcomes. The following case report illustrates the usefulness of complimentary non-invasive tools that can provide real time blood flow assessment. We present a case demonstrating the application of laser speckle contrast imaging (LSCI) in evaluating vessel flow dynamics during AVM surgery. A 30-year-old female presented with sudden headaches, nausea, vomiting, and vertigo. Emergency imaging revealed a ruptured cerebellar AVM necessitating surgical intervention. LSCI was integrated into the surgical workflow, providing continuous visualization of relative cerebral blood flow (rCBF) of vessels surrounding the AVM. Before AVM resection, LSCI measurements revealed the arterialized vasculature supplying the AVM nidus; measurements after AVM resection showed significant hemodynamic changes including normal flow in the initially arterialized AVM draining veins and adjacent arterial branches. LSCI also detected blood flow alterations during temporary occlusion, enabling assessment of downstream vascular regions. In conclusion, we provide an example supporting the utility of LSCI for real-time hemodynamic monitoring during AVM resection surgery. LSCI offers non-invasive, continuous, and immediate blood flow information, complementing conventional imaging methods like indocyanine green angiography. Additionally, our findings suggest that LSCI has the potential to provide a non-invasive means of identifying the specific superficial vessel branches or cortical areas that receive blood supply from a particular vessel.

Continuous blood flow visualization with laser speckle contrast imaging during neurovascular surgery.

Significance: Laser speckle contrast imaging (LSCI) has emerged as a promising tool for intraoperative cerebral blood flow (CBF) monitoring because it produces real-time full-field blood flow maps noninvasively and label free. Aim: We aim to demonstrate the ability of LSCI to continuously visualize blood flow during neurovascular procedures. Approach: LSCI hardware was attached to the surgical microscope and did not interfere with the normal operation of the microscope. To more easily visualize CBF in real time, LSCI images were registered with the built-in microscope white light camera such that LSCI images were overlaid on the white light images and displayed to the neurosurgeon continuously in real time. Results: LSCI was performed throughout each surgery when the microscope was positioned over the patient, providing the surgeon with real-time visualization of blood flow changes before, during, and after aneurysm clipping or arteriovenous malformation (AVM) resection in humans. LSCI was also compared with indocyanine green angiography (ICGA) to assess CBF during aneurysm clipping and AVM surgery; integration of the LSCI hardware with the microscope enabled simultaneous acquisition of LSCI and ICGA. Conclusions: The results suggest that LSCI can provide continuous and real-time CBF visualization without affecting the surgeon workflow or requiring a contrast agent. The results also demonstrate that LSCI and ICGA provide different, yet complementary information about vessel perfusion.

Mechanobiological conditioning of mesenchymal stem cells for enhanced vascular regeneration.

Using endogenous mesenchymal stem cells for treating myocardial infarction and other cardiovascular conditions typically results in poor efficacy, in part owing to the heterogeneity of the harvested cells and of the patient responses. Here, by means of high-throughput screening of the combinatorial space of mechanical-strain level and of the presence of particular kinase inhibitors, we show that human mesenchymal stem cells can be mechanically and pharmacologically conditioned to enhance vascular regeneration in vivo. Mesenchymal stem cells conditioned to increase the activation of signalling pathways mediated by Smad2/3 (mothers against decapentaplegic homolog 2/3) and YAP (Yes-associated protein) expressed markers that are associated with pericytes and endothelial cells, displayed increased angiogenic activity in vitro, and enhanced the formation of vasculature in mice after subcutaneous implantation and after implantation in ischaemic hindlimbs. These effects were mediated by the crosstalk of endothelial-growth-factor receptors, transforming-growth-factor-beta receptor type 1 and vascular-endothelial-growth-factor receptor 2. Mechanical and pharmacological conditioning can significantly enhance the regenerative properties of mesenchymal stem cells.

Superpixel Raman spectroscopy for rapid skin cancer margin assessment.

Spontaneous Raman micro-spectroscopy has been demonstrated great potential in delineating tumor margins; however, it is limited by slow acquisition speed. We describe a superpixel acquisition approach that can expedite acquisition between ~×100 and ×10 000, as compared to point-by-point scanning by trading off spatial resolution. We present the first demonstration of superpixel acquisition on rapid discrimination of basal cell carcinoma tumor from eight patients undergoing Mohs micrographic surgery. Results have been demonstrated high discriminant power for tumor vs normal skin based on the biochemical differences between nucleus, collagen, keratin and ceramide. We further perform raster-scanned superpixel Raman imaging on positive and negative margin samples. Our results indicate superpixel acquisition can facilitate the use of Raman microspectroscopy as a rapid and specific tool for tumor margin assessment.

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

Assessing metastatic potential of breast cancer cells based on EGFR dynamics.

Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells from less invasive ones. Using single-particle tracking techniques, we  developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines. By comparing the TReD results with the gene expression profiles, we found a clear negative correlation between the EGFR diffusivities and the breast cancer luminal differentiation scores (r = -0.75). Upon the induction of epithelial-mesenchymal transition (EMT), EGFR diffusivity significantly increased for the non-tumorigenic MCF10A (99%) and the non-invasive MCF7 (56%) cells, but not for the highly metastatic MDA-MB-231 cell. We believe that the reorganization of actin filaments during EMT modified the PM structures, causing the receptor dynamics to change. TReD can thus serve as a new biophysical marker to probe the metastatic potential of cancer cells and even to monitor the transition of metastasis.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

* Glioblastoma Exosomes for Therapeutic Angiogenesis in Peripheral Ischemia.

Peripheral ischemia as a result of occlusive vascular disease is a widespread problem in patients older than the age of 65. Angiogenic therapies that can induce microvascular growth have great potential for providing a long-lasting solution for patients with ischemia and would provide an appealing alternative to surgical and percutaneous interventions. However, many angiogenic therapies have seen poor efficacy in clinical trials, suggesting that patients with long-term peripheral ischemia have considerable therapeutic resistance to angiogenic stimuli. Glioblastoma is one of the most angiogenic tumor types, inducing robust vessel growth in the area surrounding the tumor. One major angiogenic mechanism used by the tumor cells to induce blood vessel growth is the production of exosomes and other extracellular vesicles that can carry pro-angiogenic and immunomodulatory signals. Here, we explored whether the pro-angiogenic aspects of glioblastoma-derived exosomes could be harnessed to promote angiogenesis and healing in the context of peripheral ischemic disease. We demonstrate that the exosomes derived from glioblastoma markedly enhance endothelial cell proliferation and increase endothelial tubule formation in vitro. An analysis of the microRNA expression using next generation sequencing identified that exosomes contained a high concentration of miR-221. In addition, we found that glioblastoma exosomes contained significant amounts of the proteoglycans glypican-1 and syndecan-4, which can serve as co-receptors for angiogenic factors, including fibroblast growth factor-2 (FGF-2). In a hindlimb ischemia model in mice, we found that the exosomes promoted enhanced revascularization in comparison to control alginate gels and FGF-2 treatment alone. Taken together, our results support the fact that glioblastoma-derived exosomes have powerful effects in increasing revascularization in the context of peripheral ischemia.

Intraoperative multi-exposure speckle imaging of cerebral blood flow.

Multiple studies have demonstrated that laser speckle contrast imaging (LSCI) has high potential to be a valuable cerebral blood flow monitoring technique during neurosurgery. However, the quantitative accuracy and sensitivity of LSCI is limited, and highly dependent on the exposure time. An extension to LSCI called multi-exposure speckle imaging (MESI) overcomes these limitations, and was evaluated intraoperatively in patients undergoing brain tumor resection. This clinical study ( n = 8) recorded multiple exposure times from the same cortical tissue area spanning 0.5-20 ms, and evaluated images individually as single-exposure LSCI and jointly using the MESI model. This study demonstrated that the MESI estimates provided the broadest flow sensitivity for sampling the flow magnitude in the human brain, closely followed by the shorter exposure times. Conservation of flow analysis on vascular bifurcations was used to validate physiological accuracy, with highly conserved flow estimates (<10%) from both MESI and 1 ms LSCI ( n = 14 branches). The MESI model had high goodness-of-fit with proper image calibration and acquisition, and was used to monitor blood flow changes after tissue cautery. Results from this study demonstrate that intraoperative MESI can be performed with high quantitative accuracy and sensitivity for cerebral blood flow monitoring.

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking.

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Glypican-1 nanoliposomes for potentiating growth factor activity in therapeutic angiogenesis.

Therapeutic angiogenesis is a highly appealing concept for treating tissues that become ischemic due to vascular disease. A major barrier to the clinical translation of angiogenic therapies is that the patients that are in the greatest need of these treatments often have long term disease states and co-morbidities, such as diabetes and obesity, that make them resistant to angiogenic stimuli. In this study, we identified that human patients with type 2 diabetes have reduced levels of glypican-1 in the blood vessels of their skin. The lack of this key co-receptor in the tissue may make the application of exogenous angiogenic growth factors or cell therapies ineffective. We created a novel therapeutic enhancer for growth factor activity consisting of glypican-1 delivered in a nanoliposomal carrier (a "glypisome"). Here, we demonstrate that glypisomes enhance FGF-2 mediated endothelial cell proliferation, migration and tube formation. In addition, glypisomes enhance FGF-2 trafficking by increasing both uptake and endosomal processing. We encapsulated FGF-2 or FGF-2 with glypisomes in alginate beads and used these to deliver localized growth factor therapy in a murine hind limb ischemia model. Co-delivery of glypisomes with FGF-2 markedly increased the recovery of perfusion and vessel formation in ischemic hind limbs of wild type and diabetic mice in comparison to mice treated with FGF-2 alone. Together, our findings support that glypisomes are effective means for enhancing growth factor activity and may improve the response to local angiogenic growth factor therapies for ischemia.

Syndecan-4 Enhances Therapeutic Angiogenesis after Hind Limb Ischemia in Mice with Type 2 Diabetes.

Delivering syndecan-4 with FGF-2 improves the effectiveness of FGF-2 therapy for ischemia in the diabetic disease state. The syndecan-4 proteoliposomes significantly enhance in vitro tubule formation as well as blood perfusion and vessel density in the ischemic hind limbs of diseased ob/ob mice. Syndecan-4 therapy also induces a marked immunomodulation in the tissues, increasing the polarization of macrophages toward the M2 phenotype.

Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination.

Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 µm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 µs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 µm in tumour spheroids.

Intraoperative laser speckle contrast imaging with retrospective motion correction for quantitative assessment of cerebral blood flow.

Although multiple intraoperative cerebral blood flow (CBF) monitoring techniques are currently available, a quantitative method that allows for continuous monitoring and that can be easily integrated into the surgical workflow is still needed. Laser speckle contrast imaging (LSCI) is an optical imaging technique with a high spatiotemporal resolution that has been recently demonstrated as feasible and effective for intraoperative monitoring of CBF during neurosurgical procedures. This study demonstrates the impact of retrospective motion correction on the quantitative analysis of intraoperatively acquired LSCI images. LSCI images were acquired through a surgical microscope during brain tumor resection procedures from 10 patients under baseline conditions and after a cortical stimulation in three of those patients. The patient's electrocardiogram (ECG) was recorded during acquisition for postprocess correction of pulsatile artifacts. Automatic image registration was retrospectively performed to correct for tissue motion artifacts, and the performance of rigid and nonrigid transformations was compared. In baseline cases, the original images had [Formula: see text] noise across 16 regions of interest (ROIs). ECG filtering moderately reduced the noise to [Formula: see text], while image registration resulted in a further noise reduction of [Formula: see text]. Combined ECG filtering and image registration significantly reduced the noise to [Formula: see text] ([Formula: see text]). Using the combined motion correction, accuracy and sensitivity to small changes in CBF were improved in cortical stimulation cases. There was also excellent agreement between rigid and nonrigid registration methods (15/16 ROIs with [Formula: see text] difference). Results from this study demonstrate the importance of motion correction for improved visualization of CBF changes in clinical LSCI images.

Comparison of indocyanine green angiography and laser speckle contrast imaging for the assessment of vasculature perfusion.

BACKGROUND: Assessment of the vasculature is critical for overall success in cranial vascular neurological surgery procedures. Although several methods of monitoring cortical perfusion intraoperatively are available, not all are appropriate or convenient in a surgical environment. Recently, 2 optical methods of care have emerged that are able to obtain high spatial resolution images with easily implemented instrumentation: indocyanine green (ICG) angiography and laser speckle contrast imaging (LSCI). OBJECTIVE: To evaluate the usefulness of ICG and LSCI in measuring vessel perfusion. METHODS: An experimental setup was developed that simultaneously collects measurements of ICG fluorescence and LSCI in a rodent model. A 785-nm laser diode was used for both excitation of the ICG dye and the LSCI illumination. A photothrombotic clot model was used to occlude specific vessels within the field of view to enable comparison of the 2 methods for monitoring vessel perfusion. RESULTS: The induced blood flow change demonstrated that ICG is an excellent method for visualizing the volume and type of vessel at a single point in time; however, it is not always an accurate representation of blood flow. In contrast, LSCI provides a continuous and accurate measurement of blood flow changes without the need of an external contrast agent. CONCLUSION: These 2 methods should be used together to obtain a complete understanding of tissue perfusion.