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 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 μm 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 μm 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.
1. Preparing for Surgery i
2. Mounting a Head Frame
3. The Generation of a Polished and Reinforced Thinned-skull (PoRTS) Window
4. Recovery
5. Imaging Preparation
6. Representative Results
A successful window will allow imaging depths up to 250 μm below the pial surface for several months. This method has been used to study in vivo capillary blood flow 4, 8, microglial activation 8, 9, and dendritic structure within the cortical parenchyma 8. In one example, we use two-photon imaging to show the cortical vasculature of an anesthetized Thy1-yellow fluorescent protein (YFP) mouse, after the blood serum is labeled by intravenous injection of Texas Red dextran (Fig. 2A). Dural vessels are often visible slightly above the cortical surface in the dura mater (Fig. 2C, arrow). Large pial arterioles and venules lie on the cortical surface (Fig 2D). Penetrating vessels branch from this surface network and dive into the cortex where they ramify into a dense capillary bed that feeds the cortical tissue (Fig. 2E to 2H). Dendritic arbors of deep YFP expressing cortical neurons, a signal endogenous to this mouse line, can be imaged concurrently in a second channel 10 (Fig. 2B to 2H). The second harmonic signal of the bone was collected in a third channel, and can be used to gauge the thickness of the thinned skull after collection of image stacks (Fig. 2A to 2C).
Cortical vascular dynamics are profoundly affected by anesthetics 11. In a second example, we show a video of spontaneous vasoactivity collected by two-photon microscopy from a habituated awake mouse. Prominent vasomotor oscillations in the lumen diameter are seen with a pial arteriole, but not with a neighboring venule. This basal range of vasoactivity is diminished with urethane anesthesia 4. To quantify spontaneous and evoked changes in blood flow, we use adapted line scanning techniques to capture both the vascular diameter and red blood cell velocity of individual vessels. Detailed resources on quantitative blood flow imaging using two-photon microscopy are available 3, 12.
Figure 1. Procedure for a PoRTS window. (A to K) Images of sequential steps in the procedure for generating a PoRTS window. See text for detailed instructions. β = bregma and λ = lambda. (L) Bolt and nut system for securing the head during imaging of anesthetized preparations. (M) Custom machined cross bar head mount for awake preparations. In this example, a connector was also implanted for repeated electrocorticogram recordings. (N) Schematic diagram showing dorsal view of the head mount and position of various components. The nut used in panel L is meant as alternative to the cross bar using in panel M. Two #000 self-tapping screws are added with the cross bar mount for added stability with awake imaging preparations. (O) Schematic diagram showing cross section of a PoRTS window.
Figure 2. Two-photon imaging of vasculature and neuronal structure in mouse cortex. All images were collected through a PoRTS window in a Thy1-YFP mouse at 2 days after window implantation 10. Maximal projection over 150 μm of tissue in the coronal orientation showing the thinned skull in relation to the vasculature (A) and dendrites (B). The bone (blue) was detected by collecting the second harmonic fluorescence at 450 nm emission with 900 nm excitation 8. The vasculature (red) was labeled by intravenously injected 70 kDa Texas Red dextran 6. The dendritic fields of neurons (green) are endogenous to the Thy1-YFP transgenic mouse line. (C-H) Maximal projections over 50 μm of tissue in the horizontal orientation at different depths below the pia. Data is from the same image stack shown in Panels A and B. Dural vessels may be visible just above the cortical surface (arrow in C).
Abbreviations
ACSF = artificial cerebral spinal fluid
PoRTS = polished and reinforced thinned skull window
YFP = yellow fluorescent protein
iEnsure that the procedures described are approved by your local Institutional Animal Care and Use Committee.
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 μm 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 in methods involving full craniotomy 14, 15. A well prepared PoRTS window should show no overt signs of angiogenesis or inflammation following implantation 8. This may be assessed in vivo during the course of the experiment by imaging vasculature structure, or by detecting morphological changes of microglia in the CX(3)CR1-GFP mouse line 8, 16, 17. Further, post hoc immunohistology should be performed to confirm the absence of astrogliosis in the superficial cortex, for example, using an antibody for glial fibrillary acidic protein 8.
The most demanding step in generating a PoRTS window is thinning the bone to 10 to 15 μm in thickness. Periodic examination of the window under a wide field fluorescence dissection microscope is also useful to gauge skull thickness during the thinning procedure. Fluorescent labels near the cortical surface should be clearly visible through the wet skull. For a more accurate measure of skull thickness, the second harmonic signal from the bone can be measured in a three-dimensional stack under two-photon microscopy (Fig. 2A to 2C). If this is done prior to application of the glue and cover glass, the bone and can be further thinned if necessary. Insufficient thinning results in poor imaging depth. Polishing will continue to thin the skull when drilling is no longer possible, and will help reduce surface irregularities and adherent bone chips. In preliminary studies, we have found that polishing improves imaging depth weeks after the initial implantation. However, a rigorous analysis on the benefit of polishing has not been performed.
The application of cyanoacrylate glue and cover glass atop the bone is a critical step to reduce light scattering and allow for deeper imaging. Due to the drilling process, the surface of the bone will be irregular, even after polishing, and therefore light scattering. The application of cyanoacrylate glue fills in these irregularities and the overlying cover glass leaves a non-scattering smooth surface. Importantly, the refractive indices for the glue and cover glass, which are 1.45 and 1.52 according to manufacturer specifications, are closely indexed matched to that of bone at 1.55 18. This is an improvement over having air or water above the thinned skull, which have refractive indices of 1.00 and 1.33. Critically, the cyanoacrylate glue further helps to impede bone re-growth. This enables longitudinal imaging for up to three months, without the need for additional maintenance after the initial surgery.
A second cause of poor imaging depth is damage to pial vessels leading to hemorrhaging or edema. Vibrations from the drill should be minimized. In a small proportion of cases, bleeding within the dura will be unavoidable. This is because the vasculature of the dura mater is continuous with the vascular plexus of the skull, which is disturbed during the thinning procedure. Preparations with any sign of sub-dural bleeding should be discarded as dural thickening and angiogenesis will ensue. The success rate of the PoRTS window can be as high as 80% with practice.
For some experiments, it may be necessary to inject dyes/viruses or insert electrodes into the tissue beneath the PoRTS window. It is possible to generate a small hole adjacent to the window, through which pipettes or electrodes can be introduced using a stereotaxic arm or Sutter manipulator 19. This hole can be resealed with bone wax if the animal is to be imaged again in future sessions. However, the introduction of electrodes can of course lead to inflammation in the cortex and acute pathology such as spreading depression.
The PoRTS window should be well suited for any imaging modality requiring optical access to the brain. This includes surface imaging techniques such as intrinsic optical imaging 20 and laser speckle imaging 21, or optical sectioning techniques such as confocal 22 or two-photon microscopy 23. Further, a PoRTS preparation should improve resolution in wide field transcranial imaging techniques such as optical coherence tomography 24 and photoacoustic microscopy 25.
Finally, the PoRTS window is suitable for imaging of awake animals as the cover glass adds rigidity to the window 4. The head mount is further stabilized by the introduction of two self-tapping #000 screws to the contralateral hemisphere prior to application of the dental cement (Fig. 1L). Habituation to head-fixation is important to reduced animal movement during imaging. A new animal can be gradually accustomed to head-restraint over a period of 3 to 7 days prior to imaging, starting with 15 min sessions and working up to several hours.
The authors have nothing to disclose.
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.
Agent | Route of delivery | Dose for mouse | Duration | Notes | Source | Ref Ref |
Pentobarbital (Nembutal) | IP | 90 μg/g | 15-60 min | Narrow safety margin. Work up to proper dose of anesthesia slowly. Supplement 10 % of induction dose as required. | 036093; Butler Schein | 7 |
Ketamine (Ketaset) mixed with Xylazine (Anased) | IP | 60 μg/g (K)
10 μg/g (X) (mix in same syringe) |
20-30 min | Xylazine is co-injected as a muscle relaxant and analgesic. Supplement only Ketamine at 50% of induction dose as required. | (K) 010177, (X) 033198; Butler Schein | 7 |
Isoflurane (Isothesia) | Inhalation | 4% mean alveolar concentration (MAC) for induction; 1-2% MAC for maintenance | 4-6 h. | Provided in mixture of 70% oxygen and 30% nitrous oxide. Prolonged anesthesia leads to slow recovery. | 029403; Butler Schein | 26 |
Table 1. Suggested anesthetic agents for survival studies.
ITEM | COMPANY | CATALOG # / MODEL |
Betadine | Butler Schein | 6906950 |
Buprenorphine (Buprenex) | Butler Schein | 031919 |
Fluorescein isothiocyanate dextran, 2 MDa molecular weight | Sigma | FD2000S |
Isopropyl alcohol | Fisher | AC42383-0010 |
Lactated Ringer’s Solution | Butler Schein | 009846; |
Lidocaine solution, 2 % (v/v) | Butler Schein | 002468 |
Saline | Butler Schein | 009861 |
Surgical Milk | Butler Schein | 014325 |
Texas Red dextran, 70 kDa molecular weight | Invitrogen | D1864 |
Maxizyme | Butler Schein | 035646 |
DISPOSABLES | ||
Carbide burrs, 1/2 mm tip size | Fine Science Tools | 19007-05 |
Cottoned tip applicators | Fisher Scientific | 23-400-100 |
Cover Glass, no. 0 thickness | Thomas Scientific | 6661B40 |
Cyanoacrylate glue | ND Industries | 31428 H04308 |
Gas duster | Newegg | N82E16848043429 |
Grip cement powder | Dentsply | 675571 |
Grip cement solvent | Dentsply | 675572 |
Insulin syringe, 0.3 mL volume with 29.5 gauge needle | Butler Schein | 018384 |
Nut and bolt to secure the head | Digikey | Nut, H723-ND; bolt, R2-56X1/4-ND |
Opthalmic ointment | Butler Schein | 039886 |
Scalpel blades | Fisher Scientific | 12-460-448 |
Screws, self-tapping #000 | J.I. Morris Company | FF000CE125 |
Silicone aquarium sealant | Perfecto Manufacturing | 31001 |
Tin oxide powder | Mama’s Minerals | EQT-TINOX |
EQUIPMENT | ||
Glass scribe | Fisher Scientific | 08-675 |
Dissecting microscope | Carl Zeiss | OPMI-1 FC |
Electric powered drill | Foredom or Osada | K.1020 (Foredom) or EXL-M40 (Osada) |
Electrical razor | Wahl | Series 8900 |
Forceps, Dumont no. 55 | Fine Science Tools | 11255-20 |
Feedback regulated heat pad | FHC | 40-90-8 (rectal thermistor, 40-90-5D-02; heat pad, 40-90-2-07) |
Isoflurane vaporizer | Ohmeda | IsoTec4 |
Pulse oximeter | Starr Life Sciences | MouseOx |
Screwdriver, miniature | Garret Wade | 26B09.01 |
Stereotaxic frame | Kopf Instruments | Model 900 (with mouse anesthesia mask and non-rupture ear bars) |
Surgical scissors, blunt end | Fine Science Tools | 14078-10 |
Ultrasonic cleaner | Fisher Scientific | 15-335-30 |
Table 2. List of specific reagents, disposables and equipment.