Imaging Modalities and Infrastructure
Neurotar is a leading commercial service provider of in vivo two-photon (2P) and wide-field (WF) brain imaging in mice. We offer imaging under anesthesia or in awake mice to minimize the biases introduced by anesthetics. Our setups include commercial instruments (Olympus, Bruker) and Neurotar’s own Invigilo system for wide-field imaging in head-fixed mice. These are complemented by in-house data analysis algorithms that streamline image acquisition, processing, and result interpretation.
Our flexible infrastructure allows us to investigate a broad array of experimental questions. Whether you seek to visualize fine cellular structures in subcortical areas or map functional connectivity across the entire cortex, we can help devise the most suitable imaging approach.
Core Benefits of 2P and WF imaging
Both 2P and WF imaging preserve the living brain’s complex environment and enable longitudinal studies of disease progression or therapeutic interventions over weeks and months. By using a single animal as its own control, these techniques allow for reduced variability and smaller experimental groups—optimizing resources and adhering to the 3R principles (Refine, Reduce, Replace).
Image on the left: Calcium imaging in the brain of an awake, behaving mouse using two-photon microscopy
Two-Photon Imaging (2P)
Two-photon microscopy uses femtosecond pulsed infrared laser light. Such light penetrates living tissue more effectively than traditional one-photon approaches. This enables high-resolution imaging of the mouse cortex (down to subcellular structures), while minimizing photodamage and photobleaching. As a result, 2P imaging provides the detailed insights needed to track subtle changes over extended periods.
- Cortical depth & resolution: Laser light penetrates up to 600–1000 µm in the mouse brain, allowing us to see individual neurons, dendritic spines, and even subcellular organelles. Hippocampal imaging is possible using micropericscopes.
- Longitudinal studies: Because of reduced phototoxicity, we can image the same region repeatedly for several months, collecting unmatched detail on disease progression or drug action.
- Sample readouts:
By capturing structural and functional data in vivo, 2P imaging allows us to glean critical information about a therapeutic agent’s behavior in a relevant biological context.
Wide-Field Imaging (WF)
Wide-field imaging captures whole-cortex activity at high acquisition rates (10–50 Hz). This makes it especially valuable for functional studies and rapid signaling events. Instead of focusing on individual neurons or microstructures, WF imaging spans broad cortical areas. It offers a bird’s-eye view of large-scale neuronal dynamics and functional connectivity.
- Speed & coverage: With large fields of view, WF imaging can detect synchronized cortical events and reveal global patterns, such as functional connectivity between remote brain regions.
- Ideal for:
- Monitoring calcium or BOLD signals across multiple cortical areas
- Studying network dynamics during behavioral or pharmacological interventions (including psychedelics)
- Assessing functional connectivity in real-time
- Practical considerations: WF imaging is mostly limited to superficial cortical layers.
Two-Photon vs. Wide-Field Imaging: Comparison Table
| Parameter | Two-Photon (2P) Imaging | Wide-Field (WF) Imaging |
|---|---|---|
| Light Source | Infrared femtosecond laser (point-by-point scanning) | LED (wide-field illumination) |
| Light Collection | PMTs | Video camera |
| Field of View | Up to ~0.6 x 0.6 mm | Entire cortex |
| Penetration Depth | 600–1000 µm | Superficial cortex only |
| Deep Brain Access | Possible with prisms/GRIN lenses (e.g., hippocampus) | Not feasible |
| Applications | Mostly structural (BBB, dendritic spines, Aβ, microglia) | Functional (calcium, BOLD, network connectivity) |
| Temporal Resolution | Lower (1–4 Hz per full frame) | Higher (10–50 Hz per full frame) |
| Spatial Resolution | Cellular & subcellular (<1 µm) | Regional (~10–50 µm) |
| Surgery (Success Rate) | More invasive (cranial opening), ~70% | Less invasive (transparent skull), ~90% |
| Recovery & Training | ~4 weeks | ~2 weeks |
| Analysis | Manual or semi-automated | Primarily semi-automated |
| Average Study Duration | 4–6 months | 2–3 months |
Practical Considerations
- 2P excels at revealing fine cellular and subcellular details, such as dendritic spine morphology, microglial dynamics, or changes in mitochondrial shape and function.
- WF is indispensable for assessing regional or whole-brain functional connectivity, capturing large-scale neuronal activity patterns that might be missed in smaller fields of view.
For many research questions, these two modalities can be complementary: 2P uncovers specific cellular mechanisms, while WF reveals overarching network states. By combining both approaches—or choosing one based on your primary scientific question—you can gain a multi-level understanding of brain function and pathology.
Summary
Whether you need high-resolution insights into specific cell types or broad-scale coverage of cortical activity, Neurotar’s suite of imaging technologies can be tailored to your experimental requirements. We strive to deliver clear, actionable data that help you characterize disease mechanisms, evaluate therapeutic efficacy, and unravel the complexities of neuronal networks in vivo.
Let’s talk about the best approach for your next project!