This work was supported by the American Heart Association (Post-doctoral fellowship to AYS) and the National Institutes of Health (MH085499, EB003832, and OD006831 to DK). We thank Beth Friedman and Pablo Blinder for comments on the manuscript.

1. Preparing for Surgery i
<ol>
 <li>Clean the surgical tools by sonicating in a mixture of Maxizyme and Surgical Milk in an ultrasonic cleaner. Autoclave the surgical tools before each experiment.</li>
 <li>Ensure that all necessary reagents and disposables are available. A list of reagents and disposables is provided in Table 2. Reagents and disposables that come in contact with exposed tissue should be sterile, when possible.</li>
 <li>Induce anesthesia. Typical anesthetics suitable for survival studies a...

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Two-photon imaging through a PoRTS window requires transmission through the thinned bone and the dura, which attenuates the laser light and adds optical aberrations at greater depths 8. However, despite this drawback, imaging depths up to 250 &mu;m below the pial surface can be achieved with 900 nm excitation. Greater imaging depths may in principle be possible with longer excitation wavelengths 13. A major advantage of this method is the absence of cortical inflammation that might exist transiently...

<table border="1">
<tbody>
 <tr>
 <td> Agent</td>
 <td>Route of delivery</td>
 <td>Dose for mouse</td>
 <td>Duration</td>
 <td>Notes</td>
 <td>Source</td>
 <td> Ref Ref</td>
 </tr>
 <tr>
 <td>Pentobarbital (Nembutal) </td>
 <td>IP</td>
 <td>90 &mu;g/g</td>
 <td>15-60 min</td>
 <td>Narrow safety margin. Work up to proper dose of anesthesia slowly. Supplement 10 % of induction dose as required.</td>
 <td>036093; Butler Schein</td>
 <td>7</td>
 </tr>
 <tr>
 <td>Ketamine (Ketaset)
 mixed with
 Xylazine (Anased)</td>
 <td>IP</td>
 <td>60 &mu;g/g (K)
 
 10 &mu;g/g (X) (mix in same syringe)</td>
 <td>20-30 min</td>
 <td>Xylazine is co-injected as a muscle relaxant and analgesic. Supplement only Ketamine at 50% of induction dose as required.</td>
 <td>(K) 010177, (X) 033198; Butler Schein</td>
 <td>7</td>
 </tr>
 <tr>
 <td>Isoflurane (Isothesia) </td>
 <td>Inhalation</td>
 <td>4% mean alveolar concentration (MAC) for induction;
 1-2% MAC for maintenance</td>
 <td>4-6 h. </td>
 <td>Provided in mixture of 70% oxygen and 30% nitrous oxide. Prolonged anesthesia leads to slow recovery.</td>
 <td>029403; Butler Schein</td>
 <td>26</td>
 </tr>
 </tbody>
 </table>
 Table 1. Suggested anesthetic agents for survival studies.
 <table border="1">
 <tbody>
 <tr>
 <td>ITEM</td>
 <td>COMPANY</td>
 <td>CATALOG # / MODEL</td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Butler Schein</td>
 <td>6906950</td>
 </tr>
 <tr>
 <td>Buprenorphine (Buprenex) </td>
 <td>Butler Schein</td>
 <td>031919</td>
 </tr>
 <tr>
 <td>Fluorescein isothiocyanate dextran, 2 MDa molecular weight</td>
 <td>Sigma</td>
 <td>FD2000S</td>
 </tr>
 <tr>
 <td>Isopropyl alcohol</td>
 <td>Fisher</td>
 <td>AC42383-0010</td>
 </tr>
 <tr>
 <td>Lactated Ringer's Solution</td>
 <td>Butler Schein</td>
 <td>009846;</td>
 </tr>
 <tr>
 <td>Lidocaine solution, 2 % (v/v) </td>
 <td>Butler Schein</td>
 <td>002468</td>
 </tr>
 <tr>
 <td>Saline </td>
 <td>Butler Schein</td>
 <td>009861</td>
 </tr>
 <tr>
 <td>Surgical Milk</td>
 <td>Butler Schein</td>
 <td>014325</td>
 </tr>
 <tr>
 <td>Texas Red dextran, 70 kDa molecular weight</td>
 <td>Invitrogen</td>
 <td>D1864</td>
 </tr>
 <tr>
 <td>Maxizyme</td>
 <td>Butler Schein</td>
 <td>035646</td>
 </tr>
 <tr>
 <td>DISPOSABLES</td>
 </tr>
 <tr>
 <td>Carbide burrs, 1/2 mm tip size</td>
 <td> Fine Science Tools</td>
 <td>19007-05</td>
 </tr>
 <tr>
 <td>Cottoned tip applicators </td>
 <td>Fisher Scientific</td>
 <td>23-400-100</td>
 </tr>
 <tr>
 <td>Cover Glass, no.&nbsp;0 thickness </td>
 <td>Thomas Scientific</td>
 <td>6661B40</td>
 </tr>
 <tr>
 <td>Cyanoacrylate glue </td>
 <td>ND Industries</td>
 <td>31428 H04308</td>
 </tr>
 <tr>
 <td>Gas duster</td>
 <td>Newegg</td>
 <td>N82E16848043429</td>
 </tr>
 <tr>
 <td>Grip cement powder </td>
 <td>Dentsply</td>
 <td>675571</td>
 </tr>
 <tr>
 <td>Grip cement solvent</td>
 <td>Dentsply</td>
 <td>675572</td>
 </tr>
 <tr>
 <td>Insulin syringe, 0.3 mL volume with 29.5 gauge needle </td>
 <td>Butler Schein</td>
 <td>018384</td>
 </tr>
 <tr>
 <td>Nut and bolt to secure the head </td>
 <td>Digikey</td>
 <td>Nut, H723-ND; bolt, R2-56X1/4-ND</td>
 </tr>
 <tr>
 <td>Opthalmic ointment </td>
 <td>Butler Schein</td>
 <td>039886</td>
 </tr>
 <tr>
 <td>Scalpel blades </td>
 <td>Fisher Scientific</td>
 <td>12-460-448</td>
 </tr>
 <tr>
 <td>Screws, self-tapping #000 </td>
 <td>J.I. Morris Company</td>
 <td>FF000CE125</td>
 </tr>
 <tr>
 <td>Silicone aquarium sealant</td>
 <td>Perfecto Manufacturing</td>
 <td>31001</td>
 </tr>
 <tr>
 <td>Tin oxide powder</td>
 <td>Mama's Minerals</td>
 <td>EQT-TINOX</td>
 </tr>
 <tr>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Glass scribe</td>
 <td>Fisher Scientific</td>
 <td>08-675</td>
 </tr>
 <tr>
 <td>Dissecting microscope </td>
 <td>Carl Zeiss</td>
 <td>OPMI-1 FC</td>
 </tr>
 <tr>
 <td>Electric powered drill</td>
 <td> Foredom or Osada</td>
 <td>K.1020 (Foredom) or EXL-M40 (Osada)</td>
 </tr>
 <tr>
 <td>Electrical razor</td>
 <td>Wahl</td>
 <td>Series 8900</td>
 </tr>
 <tr>
 <td>Forceps, Dumont no. 55 </td>
 <td>Fine Science Tools</td>
 <td>11255-20</td>
 </tr>
 <tr>
 <td>Feedback regulated heat pad</td>
 <td>FHC</td>
 <td>40-90-8 (rectal thermistor, 40-90-5D-02; heat pad, 40-90-2-07)</td>
 </tr>
 <tr>
 <td>Isoflurane vaporizer </td>
 <td>Ohmeda</td>
 <td>IsoTec4</td>
 </tr>
 <tr>
 <td>Pulse oximeter</td>
 <td>Starr Life Sciences</td>
 <td>MouseOx</td>
 </tr>
 <tr>
 <td>Screwdriver, miniature </td>
 <td>Garret Wade</td>
 <td>26B09.01</td>
 </tr>
 <tr>
 <td>Stereotaxic frame</td>
 <td>Kopf Instruments</td>
 <td>Model 900 (with mouse anesthesia mask and non-rupture ear bars)</td>
 </tr>
 <tr>
 <td>Surgical scissors, blunt end </td>
 <td>Fine Science Tools</td>
 <td>14078-10</td>
 </tr>
 <tr>
 <td>Ultrasonic cleaner </td>
 <td>Fisher Scientific</td>
 <td>15-335-30</td>
 </tr>
 </tbody>
 </table>
 Table 2. List of specific reagents, disposables and equipment.

a polished and reinforced thinned-skull window for long-term imaging of the mouse brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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26.6K Views.  University of California, San Diego. We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.