Table 2.

Advantages and disadvantages of deep optical imaging modalities

Imaging modality	Advantages	Disadvantages
Deep two-photon microscopy	High-resolution in vivo imaging of vascular structure, function, and cell types.	Deep 2PM is effective for only red/far red dyes and proteins. Typically requires introduction of exogenous fluorophores. Requires cranial windows, which can disrupt the intracranial environment Slower image acquisition is better suited for single vessel studies. Imaging is limited in field of view. Possibility of photodamage due to high laser powers.
Three-photon microscopy	High-resolution in vivo imaging of vascular structure, function and cell types. Visualization of the cortical gray/white matter boundary through intrinsic third harmonic signal generation. Multi-color imaging for concurrent investigation of neuronal activity and vascular function.
Optical coherence tomography angiography	High-resolution in vivo imaging of microvascular structure and blood flow without exogenous contrast agents. High throughput capillary velocity and flux measurement in the microvascular network	Unable to image neurovascular cell types. Typically examined as x–y projections and tracing vessel structures in 3D is problematic. Limited penetration depth for capillary velocimetry (up to 500 μm). Strong scattering caused by larger vessels at the brain surface leads to imaging artifacts at depth. Requires cranial windows.
Optical coherence microscopy	High throughput mapping of neuronal and myelinated structures.	Limited ability to visualize neurovascular cell types.
Photoacoustic imaging	Imaging of vascular structure and blood oxygen across networks with single arteriole and venular resolution. Some adaptations allow visualization of vasculature in whole brain cross sections in vivo. Imaging can be performed through an intact skull for some applications	Unable to image neurovascular cell types. Possibility of photodamage due to high laser power. Shadowing of the larger vessels at brain surface leads to imaging artifacts
Functional ultrasound imaging	Imaging of vascular structure and function through the entire rodent brain. Imaging performed through an intact skull. Skull-thinning required for high resolution. Device is portable	Unable to image neurovascular cell types. Does not allow for investigation at the level of capillary networks.