Name: Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model (Video) | JoVE Journal
Uploaded: 2024-03-01T14:15:00.000+00:00
Duration: 7 min 25 s
Description: Learn two-photon intravital microscopy for real-time imaging of nanoparticle delivery to glioblastoma in a mouse model. This technique reveals drug distribution patterns across the blood-brain barrier to optimize therapeutic strategies.

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5 minutes

We would like to thank the Stanford Wu Tsai Neuroscience Microscopy Service, the Stanford Center for Innovations in In Vivo Imaging (SCi3) - small animal imaging center, NIH S10 Shared Instrumentation Grant (S10RR026917-01, PI Michael Moseley, Ph.D.), and Stanford Preclinical Imaging Facility at Porter Drive for providing the equipment and infrastructure for this project. This work was supported by a grant from the National Institute for Child Health and Human Development, grant number R01HD103638. We would like to thank the Schnitzer Group, Stanford University; the Zuo lab, University of Santa Cruz; and the Neurovascular Imaging Laboratory, Boston Photonic Center, University of Boston, for educational discussions on two-photon imaging and cranial window models.

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

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</ol>

The authors declare that they have no conflict of interest.

We present a method for real-time in vivo NP tracking using 2P-IVM through a cranial window to evaluate the tumor delivery of fluorescent-labeled iron oxide NPs. The surgical technique for this procedure requires a steady hand and advanced experimental surgical skills. It is advisable to practice using carcasses or phantoms before moving forward to live animal experience. As an alternative, Hoeferlin et al. implemented a robotic drill to reduce thermal damage, minimize surgical technique variability, and standardize surgery22.
The size of the window represents another critical parameter. A larger window would enable imaging of a bigger portion of the tumor with 2P-IVM. In the literature, sizes between 3-7 mm have been described23. However, larger windows cannot accommodate for the brain curvature, which can lead to increased regional pressure24. To overcome this restraint, a so called &#34;glass skull&#34; can be used instead25. In comparison to traditional glass windows, this method uses curved glass, accommodating the brain curvature and thus allowing for the creation of bigger windows while reducing focal pressure on the cortex. This type of curved glass is not commercially available, and it only has limited applications in 2P-IVM, since the curved surface causes reflection artifacts, limiting the area of the window that could be imaged. Another alternative is a silicon-based window24. On one hand, this method offers more flexibility regarding the size and form of the window to be created. In addition, comparable results in terms of window failure and inflammation rates to classic glass windows have been reported. On the other hand, 2-photon imaging quality in polymer-based windows has been shown to decrease faster than in glass windows, making it not feasible for long-term applications26. A thinned skull cranial window represents another alternative. While being less invasive than the classical glass window, it does not allow for GBM cell implantation using the same technique as described here. Additionally, it is difficult to achieve a consistent skull thickness, and as a result, this can cause significant artifacts in 2P-IVM27.
The cell line and amount of GBM cells being implanted are other important considerations when planning the study timeline. The C6 rat glioma cell line is one of the most widely used cell lines in GBM research. In rats and mice, cell numbers between 5 x 104 and 5 x 106 have been reported for intracranial implantations27,28,29,30,31. As a general rule, when implanting a higher number of cells, faster tumor growth is to be expected. In this protocol, 1 x 105 cells were implanted and imaging was performed on days 11 and 12 after implantation. Xin et al. implanted 1 x 105 C6 cells in BALB/c nude mice and reported detecting advanced GBM in MRI on day 7 post-implantation and an increased mortality after 15 days30. In comparison, Jia et al. used a higher number of cells (1 x 106) and the same mouse strain and detected a small tumor mass at day 7 in MRI with a slightly disrupted blood-brain barrier (BBB), as demonstrated by a pale Evans blue stain in the GBM tissue32. On day 14, the Evans blue stain was stronger than on day 7, indicating that the BBB was highly disrupted. In turn, the tumor growth also affects how long the animals can be kept in the experiment. This is an important consideration of animal welfare for longitudinal imaging studies. Chronic cranial windows have been reported to be suitable for imaging for up to 6 months after implantation26.
The nanoparticles used in this protocol consist of iron oxide and fluorophore components. Possible applications include the investigation of the tumor delivery of novel therapeutic drugs, cellular therapeutics, and interventions on the tumor vasculature and tumor microenvironment. Different drug candidates and combination therapies can be evaluated on a cellular and molecular level. The iron oxide component of the NPs allows for multimodal imaging with MRI or magnetic particle imaging in addition to 2P-IVM. While MRI represents a clinically relevant imaging modality, its anatomical resolution is inferior to that of intravital microscopy33.
This method also has certain limitations. The brain coordinates according to a mouse brain atlas are standardized for C57BL/6J mice and must be adjusted depending on the mouse strain, sex, and age. Moreover, with two-photon imaging, only a limited depth of approximately 450 &#181;m can be accessed9. Therefore, only partial characterization of the tumor is possible with 2P-IVM, and regional differences in the tumor characteristics could be missed. Additionally, only two time points following NP administration were included. Future studies, including more time points after intravenous administration, will allow for a more detailed analysis of the spatiotemporal behavior of the NPs in the tumor microenvironment.
To conclude, we evaluated the tumor delivery of fluorescent-labeled iron oxide nanoparticles using 2P-IVM in a mouse model of GBM. This method can be easily modified to fit various areas of research and represents an important tool for in vivo brain imaging in the field of neuroscience.

Two-photon intravital microscopy (2P-IVM) is a fluorescence imaging technique that allows the visualization of living tissue1.
First developed in the 1990s, 2P-IVM has been used for in vivo analysis of the retina2, kidney3, small intestine4, cochlea5, heart6, trachea7, and the brain in various preclinical models8,9. In the field of neuroscience, 2P-IVM has gained importance as a technique for real-time imaging of the healthy brain in awake animals10, as well as studying diseases of the nervous system such as Alzheimer&#39;s11, Parkinson&#39;s12 and glioblastoma (GBM)13,14,15,16.
2P-IVM offers an elegant solution for studying the tumor microenvironment during the development of GBM. While some previous studies focused on in vitro17 and ex vivo models18, others implemented orthotopic19 and xenotropic20&#160;in vivo models for examining GBM. Madden et al. performed native imaging of CNS-1 rat glioma cell line in a mouse model13. Using an orthotopic GL261-DsRed murine model, Ricard et al. performed an intravenous administration of a fluorophore to enhance the blood vessels in the tumor region in 2P-IVM14.
Here, we apply 2P-IVM for tracking the tumor delivery of fluorescent-labeled iron oxide nanoparticles (NP) in an orthotopic mouse model of GBM. Using a cranial window, this method allows us to study the real-time spatiotemporal distribution of NPs in the brain in detail.

<table><tbody><tr><td>0.9% sodium chloride infusion solution</td><td>Baxter Corp</td><td>533-JB1301P</td><td></td></tr><tr><td>&lt;br/&gt; Dulbecco&#039;s Modified Eagle Medium</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>1 mL syringes</td><td>BD</td><td>Luer-Lok syringe, REF309628</td><td></td></tr><tr><td>10%&amp;nbsp;FBS</td><td>Thermo fisher</td><td>Cytiva SH30910.03HI</td><td></td></tr><tr><td>10% DMSO</td><td>Sigma-Aldrich</td><td>D8418-50ML</td><td></td></tr><tr><td>2-photon microscope</td><td>Prairie Technologies, Bruker</td><td>Prairie Ultima IV</td><td></td></tr><tr><td>Alcohol applicators, 70%</td><td>Medline Industries, LP</td><td>MDS093810</td><td></td></tr><tr><td>Alcohol, spray bottle</td><td>Decon Labs Inc</td><td>Decon SaniHol, 04-355-122</td><td></td></tr><tr><td>Aluminum foil</td><td>Reynold Brands</td><td>Reynold Wrap non-stick aluminum foil</td><td></td></tr><tr><td>Anesthesia machine</td><td>Patterson Scientific</td><td>SAS3</td><td></td></tr><tr><td>Anesthesia monitoring&amp;nbsp;</td><td>Kent Scientific&amp;nbsp;</td><td>MouseSTAT Jr. Rodent Pulsoxymeter</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x), liquid</td><td>Invitrogen</td><td>15240-096</td><td></td></tr><tr><td>Betadine applicators</td><td>Professional Disposables International, Inc</td><td>S41125</td><td></td></tr><tr><td>Biopsy punch, 5 mm&amp;nbsp;</td><td>Miltex</td><td>Size 5</td><td></td></tr><tr><td>Buprenorphine sustained release</td><td>Zoo Pharm</td><td>Bup SR Lab, 1.0 mg/mL</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>C6 rat glioma cell line</td><td>ATCC (American Tissue Culture Collection)</td><td>CCL-107</td><td></td></tr><tr><td>Cannulas</td><td>BD</td><td>16 G, 1.1/2&amp;rdquo;, 30 G, 1&amp;rdquo;</td><td></td></tr><tr><td>Carprofen</td><td>Pfizer&amp;nbsp;</td><td>Rimadyl, 50 mg/ml</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>Cefazoline</td><td>Sagent Pharmaceuticals</td><td>25021-101-10, 1 g/vial</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>Cell strainer, 40 &amp;micro;m</td><td>Fisher Scientific</td><td>87711</td><td></td></tr><tr><td>Cotton tip applicators, 6&amp;rdquo;&amp;nbsp;</td><td>Dyad Medical Sourcing, LLC</td><td>HCS1005</td><td></td></tr><tr><td>Dental cement</td><td>Stoelting Co</td><td>51459</td><td>Dental cement kit, clear, 2 components</td></tr><tr><td>Dexamethasone</td><td>Bimeda</td><td>138RX, 2 mg/mL</td><td>A generic drug can be used instead.&amp;nbsp;</td></tr><tr><td>DietGel</td><td>ClearH2O</td><td>Recovery, 72-06-502</td><td></td></tr><tr><td>Drape&amp;nbsp;</td><td>Cardinal Health</td><td>Bio Shield Wrap</td><td></td></tr><tr><td>Drill</td><td>Saeyang Microtech</td><td>Escort Pro, B08350</td><td></td></tr><tr><td>Drill tips&amp;nbsp;</td><td>Hager &amp;amp; Meissinger GmbH</td><td>REF310104001001005</td><td>Size 005, US 1/4</td></tr><tr><td>FIJI imaging analysis software</td><td>National Institute of Health</td><td>https://imagej.net/software/fiji/</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>13-812-41</td><td></td></tr><tr><td>Gauze</td><td>Fisher HealthCare</td><td>Sterile Cotton Gauze Pad, 4 x 4&amp;rdquo;, 22-415-469</td><td></td></tr><tr><td>Gelfoam&amp;nbsp;</td><td>Ethicon Inc.&amp;nbsp;</td><td>Surgifoam absorbable gelatin sponge, Ref. 1972</td><td></td></tr><tr><td>Germinator&amp;nbsp;</td><td>Cellpoint Scientific&amp;nbsp;</td><td>Germinator 500, No. 11688</td><td></td></tr><tr><td>Glass coverslips, 5 mm diameter</td><td>Fisher Scientific</td><td>Menzel Cover glass&amp;nbsp;</td><td></td></tr><tr><td>Gloves, non-sterile</td><td>Fisher Scientific</td><td>Nitrile powder-free medical examination gloves</td><td></td></tr><tr><td>Gloves, sterile</td><td>Medline Industries, LP</td><td>MDS104070</td><td></td></tr><tr><td>Hair removal cream</td><td>Church &amp;amp; Dwight</td><td>Nair Hair remover lotion&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton Company Inc</td><td>Gastight #1701, 10 &amp;micro;L</td><td></td></tr><tr><td>HBSS without Ca, Mg</td><td>Fisher Scientific</td><td>PI88284</td><td></td></tr><tr><td>Head bar</td><td>Hongway</td><td>5 mm inner diameter O-rings</td><td></td></tr><tr><td>Heating pad</td><td>Stoelting Co.&amp;nbsp;</td><td>Rodent warmer X2</td><td></td></tr><tr><td>Insulin syringes</td><td>Exel International Medical Products&amp;nbsp;</td><td>29G x 1/2&amp;Prime;</td><td></td></tr><tr><td>Iron oxide nanoparticles</td><td>Covis Pharma GmbH</td><td>Feraheme ferumoxytol injection, 510 mg/17 mL, 59338077501</td><td></td></tr><tr><td>Isoflurane</td><td>Dechra</td><td>26675-46-7</td><td></td></tr><tr><td>Mice</td><td>Jackson Laboratories</td><td>NSG, Strain 005557</td><td></td></tr><tr><td>Microscope (surgery)</td><td>Seiler Medical</td><td>Seiler IQ Q-100-220</td><td></td></tr><tr><td>Nanoparticles</td><td>Custom</td><td></td><td>Iron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate</td></tr><tr><td>Ophtalmic ointment</td><td>Major pharmaceuticals</td><td>Lubrifresh P.M. nighttime ointment, 203964</td><td></td></tr><tr><td>Oxygen</td><td>Linde Gas &amp;amp; Equipment Inc.&amp;nbsp;</td><td>High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540</td><td></td></tr><tr><td>Plastic cups</td><td>Georgia-Pacirif Consumer Products</td><td>Dixie Portion Cup, 2 oz., Plastic, Clear, PK2400</td><td></td></tr><tr><td>Polyethylene tubing</td><td>Braintree Scientific</td><td>50-195-5494</td><td></td></tr><tr><td>Scale</td><td>Ohaus Corp</td><td>CR2200</td><td></td></tr><tr><td>Scalpel</td><td>Integra Life Sciences Production Corp</td><td>Integra Miltex Stainless steel disposable scalpel</td><td></td></tr><tr><td>Scissors</td><td>Fisher Scientific</td><td>13-804-18</td><td></td></tr><tr><td>Sealant</td><td>Henkel Corp</td><td>Loctite 4014</td><td></td></tr><tr><td>Single use lab gown</td><td>High Tech Conversions</td><td>17-444-081</td><td></td></tr><tr><td>Stereotactic frame</td><td>Stoelting Co.&amp;nbsp;</td><td>Stoelting New Standard TM</td><td></td></tr><tr><td>Sterile Vacuum Bottle Top Filtration Systems</td><td>Fisher Scientific</td><td>S2GPU05RE, MilliporeSigma NO.:S2GPU05RE</td><td></td></tr><tr><td>Styrofoam box</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Surgical gloves</td><td>Cardinal Health</td><td>19-163-108</td><td></td></tr><tr><td>Surgical glue, 3M Vetbond tissues adhesive</td><td>3M Animal Care Prodcuts</td><td>1469SB</td><td></td></tr><tr><td>Tail vein cathether</td><td>Custom</td><td></td><td>Consists of two 30 G cannulas connected with sillicone tubing&amp;nbsp;</td></tr><tr><td>TrypLE Express (1x), no phenol red</td><td>Invitrogen</td><td>12604-039</td><td></td></tr><tr><td>Ultraviolett torch</td><td>Spring sunshine technology</td><td>Consciot</td><td></td></tr></tbody></table>

two-photon intravital microscopy of glioblastoma in a murine model

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Here, we performed cranial window surgery and engrafted C6 cells in an NSG mouse model of GBM (n = 5). A proper seal between all components involved in the creation of the window (Figure 1A) will ensure the windows' durability for long-term imaging and, additionally, reduce morbidity. Using the stage adapted for in vivo 2P-IVM (Figure 2), we could image animals under anesthesia for up to 2 h without any major motion artifacts. Approximately 10 min after intravenous application of the nanoparticles in GBM-bearing mice, 2P-IVM shows the green, fluorescent signal of vessels in the region of the tumor, consistent with intra-vascular localization of the FITC-labeled nanoparticles. Only a small amount of green-fluorescent signal is noted outside of the blood vessels, indicating the beginning extravasation of NPs (Figure 3A). An increase in the FITC signal originating from the extravascular space is seen, which is in line with advanced extravasation (Figure 3B).
<img alt="Craniotomy setup diagram; 2-photon imaging for brain study with cell implantation process shown." class="xfigimg" src="/files/ftp_upload/66304/66304fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Cranial window placement, craniotomy, implantation, and imaging. (A) Cross-section of the anatomical placement of a cranial window. Since the head bar is metal-free, it is possible to perform magnetic resonance imaging or magnetic particle imaging in addition to two-photon intravital microscopy. (B) Overview (upper row) and a detailed view (lower row) of the craniotomy (left), cell implantation (middle), and 2-photon intravital microscopy (right). Upon opening the skull, superficial bleeding occurs (left) and is quickly reabsorbed (middle). <a href="https://www.jove.com/files/ftp_upload/66304/66304fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mechanical arm static equilibrium; CAD diagram; bolt tension analysis; engineering study." class="xfigimg" src="/files/ftp_upload/66304/66304fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Computer-assisted design (CAD) of the stage. CAD of the stage used for in vivo 2-photon intravital microscopy, including a bite bar, head bar stabilization, and screws for height and angle adjustments. The 3D CAD file can be found in Supplemental Coding File 1. <a href="https://www.jove.com/files/ftp_upload/66304/66304fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Two-photon intravital microscopy of FITC nanoparticles at 10 and 24 hours, brain tumor imaging." class="xfigimg" src="/files/ftp_upload/66304/66304fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. In vivo 2-photon intravital microscopy images. Images acquired (A) 10 min and (B) 24 h after intravenous nanoparticle administration. While the GBM cells emit fluorescent signals in the red spectrum (left), the nanoparticles in the brain tissue are visualized in green (right). # indicates extravasated NPs, * indicates a blood vessel. Only a limited number of particles have undergone extravasation and are visible in the vicinity of the tumor cells 10 min after NP administration. An abundance of particles is visible in the region of GBM cells, suggesting extravasation 24 h following NP administration. The nanoparticles consist of fluorescein isothiocyanate (FITC) and iron oxide nanoparticles (Ferumoxytol). The scale bars represent 100 µm. Abbreviations: 2P-IVM: 2-photon intravital microscopy, RFP: red fluorescent protein, GBM: glioblastoma, FITC: fluorescein isothiocyanate, NP: nanoparticles. <a href="https://www.jove.com/files/ftp_upload/66304/66304fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Coding File 1: 3D CAD file of the stage. <a href="https://www.jove.com/files/ftp_upload/66304/custom%20stage%203D%20CAD.zip" target="_blank">Please click here to download this File.</a>

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

two-photon-intravital-microscopy-of-glioblastoma-in-a-murine-model

Research

JoVE Journal

Cancer Research

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

In this video, we describe fluorescence molecular tomography or FMT for in vivo imaging of fluorescent protein-tagged glioblastoma xenografts in mouse models. The technique is useful for elucidating the effects of anti-tumor therapeutics at the molecular level.

Fluorescence Molecular Tomography: An Imaging Technique for In Vivo Imaging of Fluorescent Protein-tagged Glioblastoma Xenografts in Mouse Model

Source: Clark, A.J., et al. Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma. J. Vis. Exp. (2016)This video demonstrates bioluminescence imaging to track glioblastoma progression in an immunocompetent mouse model, using luciferase-expressing tumor cells and luciferin to quantify tumor growth non-invasively.

Bioluminescence Imaging of an Immunocompetent Mouse Model for Glioblastoma

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Learn two-photon intravital microscopy for real-time imaging of nanoparticle delivery to glioblastoma in a mouse model. This technique reveals drug distribution patterns across the blood-brain barrier to optimize therapeutic strategies.

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

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