In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window
August 4th, 2022
The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.
This protocol shows a creation of large cranial window to measure wide-field and single-cell reservation activities in the same mouse. Using our method, large and stable cranial window can be made with food wrap at low cost. This method is effective to study the neural and glial activities during behavior at macroscopic and microscopic levels.
It is recommended to start with a small cranial window. Then if it is succeeded, increase the size of the window. Begin by removing the extra cement over the skull with a dental drill.
Mark the area to be cut with a pen. Cut into the bone using a scalpel with a blunt tip to ensure that the tip does not penetrate the skull. Scrape the bone repeatedly with a scalpel to deepen the groove until the bone in the area to be trimmed moves when lightly touched.
Remove the incised bone with fine tweezers. Be cautious to not push the bone flap into the brain which may damage the brain. To remove the dura mater, cut the dura using a pulled glass pipette with a tapered tip of about 10 micrometers.
Use a U-shaped needle to expand this cut over the entire window. Under a stereo microscope set at 60 to 100x zoom, remove the severed dura mater with ultra fine tweezers. In case of bleeding, Rinse with aCSF or use a gelatin sponge to stop the bleeding.
Then, sterilize a large piece of PVDC wrap by autoclaving and with 70%ethanol. Under a stereo microscope, use tweezers and a scalpel to cut out a wrap of the required size. Place the wrap on the brain surface while leaving the aCSF on the surface.
Suck out the aCSF from the edge of the wrap, allowing the wrap to stick firmly to the brain surface. Trim the wrap with a scalpel and tweezers such that there is approximately a one millimeter margin between the edge of the cranial window and the wrap. Once the wrap is in place, glue the edge Of the wrap to the skull with a biological adhesive.
Allow the adhesive to dry for about 30 minutes. Using a dispenser with a mixing tip, apply a transparent silicone elastomer on top of the wrap. Place the cover glass of thickness 0.12 to 0.17 millimeters on top.
Seal the perimeter of the cover glass with waterproof film, superglue, or dental cement. First, mix fibroin and AAV solutions in a one to four ratio in a small sample tube. Drop an aliquot of the solution onto the plastic wrap for the cranial window and dry it for at least three hours.
Then attach the wrap to the brain surface as shown in the previous section. Check the condition of the mice and windows regularly, two to four weeks. By this time, the genetically encoded calcium indicators will be expressed sufficiently after creating the AAV-treated window.
Using this protocol, cranial windows could be maintained in eight of 10 mice for up to 10 weeks. A wide window is created with plastic wrap, transparent silicone, and cover glass in a transgenic mouse expressing membrane-anchored genetically encoded calcium indicator in astrocytes. Wide-field calcium imaging was performed through this window and fluorescence changes were observed when air puff stimulation was applied to the contralateral whiskers.
The time course of the fluorescence change in the somatosensory cortex showed cortical activity. Two-photon imaging allowed the observation of single cell fluorescence images specific to glial cells. Fluorescence changes induced by sensory stimulation were observed in individual cells from different cortical regions.
Wrap coded with AAV expressing genetically encoded calcium indicator;and fibroin was used for making the cranial window. Wide-field calcium imaging showed cortical activity propagating across the cortex induced by sensory stimulation two to four weeks after surgery. Since the window was large, different cortical areas of the same mouse were imaged and fluorescence changes induced by sensory stimulation were seen in individual cells.
It is important to avoid damage to the brain when making cranial window. One can perform behavior tasks on mice with a large cranial window. This will allow examining the relationship between neural activity and mouse behavior.
This method can be applied to a variety of neuroscience studies. For example, it can be used to observe cortical activity during decision-making tasks, motor running, and in mass models of brain injury and disease.
Overview
This study presents a protocol for creating a large cranial window in mice using food wrap, transparent silicone, and cover glass. This window facilitates both wide-field and two-photon calcium imaging to examine neural and glial activity during behavior, offering insights into cortical dynamics and sensorimotor integration.
Key Study Components
Area of Science
- Neuroscience
- Calcium imaging
- In vivo imaging techniques
Background
- Measuring neural activity is crucial for understanding brain function.
- Cranial windows allow for chronic imaging in live subjects.
- The proposed method is cost-effective and scalable.
- Prior techniques may not accommodate large imaging areas.
Purpose of Study
- To develop a reliable method for creating large cranial windows.
- To facilitate the observation of neuronal and glial activities in real-time.
- To enable the study of behavior-response relationships in mice.
Methods Used
- Large cranial windows were created using food wrap, silicone, and cover glass.
- The model involved transgenic mice expressing genetically encoded calcium indicators.
- Detailing of the window preparation, including surgical steps and materials.
- The technique included careful handling to prevent brain damage.
- Calcium imaging was performed two to four weeks post-surgery.
Main Results
- The large window allowed observation of cortical activity induced by sensory stimulation.
- Fluorescence changes were noted, indicating neural responses across different cortical areas.
- Eight of ten mice maintained functional windows for up to ten weeks.
- Initial trials suggested starting with smaller windows for improved chances of success.
Conclusions
- This protocol enables in-depth studies of brain activity in behaving mice.
- The method has potential applications in various neuroscience research areas.
- Insights gained can advance understanding of neural plasticity and behavioral neuroscience.
Frequently Asked Questions
Executive Industry Relevance
Large cranial window preparation in mice enables simultaneous wide-field and two-photon calcium imaging, supporting multi-scale interrogation of neural and glial dynamics during behavior. This capability advances predictive confidence in preclinical neuroscience models by linking macroscopic circuit activity with single-cell resolution. The method's cost-effectiveness and stability facilitate scalable, reproducible studies relevant to early discovery and translational research pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct observation of neural and glial activity in response to behavioral stimuli.
- Supports mechanistic de-risking by correlating circuit-level and cellular responses.
- Facilitates functional target validation in disease-relevant neural systems.
Screening & Assay Development
- Provides a validated in vivo platform for quantitative calcium imaging readouts.
- Supports assay reproducibility and standardization across multiple cortical regions.
- Enables screening of genetic or pharmacological interventions with high spatial resolution.
Translational & Preclinical Research
- Aligns preclinical models with disease-relevant neural circuit dynamics.
- Enables longitudinal studies of neural plasticity and injury models.
- Supports risk-adjusted advancement decisions by linking behavior to neural activity.
Pipeline & Workflow Integration
This method bridges early discovery, lead identification, and preclinical validation by enabling multi-scale imaging in behaving mice.
- Discovery Biology: Supports hypothesis testing on neural circuit function and glial interactions.
- Screening: Delivers reproducible, quantitative imaging outputs for compound or genetic screening.
- Analytics: Provides high-content data for comparing neural responses across conditions and timepoints.
- Translational Research: Facilitates continuity from basic discovery to disease modeling and biomarker exploration.
- Enterprise Reuse: Offers a scalable, cost-effective platform adaptable to diverse neuroscience R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neural target studies.
- Operational Value: Standardizes in vivo imaging workflows and supports reproducibility across studies.
- Strategic Value: Improves go/no-go decisions and capital efficiency by enabling robust, multi-scale data collection.
- Portfolio Impact: Supports risk-adjusted prioritization of neuroscience assets and translational models.
Implementation Considerations
- Requires expertise in microsurgery and in vivo imaging techniques.
- Needs access to wide-field and two-photon microscopy infrastructure.
- Demands cross-team standardization for window preparation and imaging protocols.
- Adaptable to various transgenic or viral labeling strategies for neural and glial targets.
- Careful surgical technique is essential to avoid brain damage and ensure long-term window integrity.
