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Table 1 Comparison of spatial and temporal resolution, penetration depth and limitations of different optical imaging techniques

From: Neurovascular coupling: in vivo optical techniques for functional brain imaging

  Depth Temporal and spatial resolutions Optical source Contrast agent Limitations
2-Photons microscopy[12, 2225] Up to 1 mm Spatial resolution is up to 1 μm. The temporal resolution is variable and determined by beam-scanning methods. In specially developed high-speed 2-photon imaging systems temporal resolution can reach a few μs. Two-photon excitation wavelengths are typically around twice the usual fluorescent excitation wave- lengths. Most fluorescent probes have excitation in the 350–650 nm range, whereas the excitation laser is in the ~700–1300 nm range. Delivered from outside or genetically encoded fluorescent probes. Since a fluorescence probe can be treated with voltage- or calcium- sensitive dye, fluorescence antibodies or any kind of fluorescence biomarkers The temporal resolution of the technique is defined by the property of the imaging setup. High power laser causes photo-bleaching and even destroys cells. The method is invasive and applicable only for relatively small regions.
Laser speckle contrast imaging[2634] 0.5 - 1.0 mm Up to 10 μm, no axial/depth resolution. The temporal resolution is limited by laser scanning methods and imaging of small areas can reach few tens μs. The temporal resolution is determined by the setup and can reach up to 1 μs. Laser wavelength usually ranging from 400 to 1200 nm but very variable and is determined by the experimental goal No requirement for a chemical agent Invasive. The temporal resolution is mainly defined by the parameters of the CCD camera
Photoacoustic microscopy[3542] Up to a few centimeters, but high resolution can be reached only up to a few mm in depth. Up to 1 μm, but depends on imaging depth, acoustic transducer and optical focusing. Temporal resolution is from milliseconds to sub-milliseconds. Laser wavelength, depending on the target. For example: 570 nm is sensitive to HbT. Usually doesn’t need any contrast agents but can be combined with different biomarkers. The temporal resolution is restricted by the technical characteristics of the laser scanning system. Without the use of contrast agents it is applicable for the monitoring of the cerebral blood flow and oxygenation, but not for neural and metabolic activity.
Near-infrared spectroscopy[9, 10, 4346] Up to few centimeters transcranially; 1–2 mm in an open brain From 2–3 cm in case of human transcranial research to ~0.1 mm in open brain animal experiments. The temporal resolution can be as high as 1 ms. Monochromatic near-infrared light source, usually 700–1700 nm. Based on the difference in the light absorption of HbO and HbR and doesn’t need any contrast agents. Applicable trans-cranially as well as in open brain imaging, human research, clinical practice and animal experiments.
Voltage-sensitive dye imaging[10, 23, 24, 47, 48] Up to ~ 1 mm The spatial resolution is determined by the optical system and usually limited to 50 microns. However, in combination with 2-photon imaging it can reach 1–2 microns. The temporal resolution is limited by VSD properties and imaging rate and can reach milliseconds or even submillisecond resolution. Monochromatic light in case of conventional imaging and long wavelength laser in 2-photon imaging Voltage-sensitive dye – the chemical compounds which change their optic properties in response to the changes in the neural membrane potential. Invasive, application is limited due to toxicity and photo-bleaching. The temporal resolution is defined by the CCD camera and can reach 1 ms or less.