Fig. 1. Principle of two-photon excitation fluorescence scanning microscopy (2PLSM) and its applications in living brain imaging. (a) Jablonski diagram of single photon absorption and two-photon absorption and spatial distribution of their stimulated luminescence; (b) principle of 2PLSM imaging in mouse brain based on gain switched semiconductor laser diode
[6]; (c) installation diagram of cranial window in mouse; (d) 2PLSM imaging of cortical activity induced by visual stimulation in mouse
[24]; (e) 2PLSM imaging of cortical and hippocampal neurons in an adult mouse under gain excitation with a wavelength of 1064 nm
[6]; (f) 2PLSM imaging of cerebral cortical blood vessels in a mouse under excitation light with a wavelength of 1280 nm
[35], scaled bar: 50 μm
Fig. 2. Principle of stimulated emission depletion (STED) microscopy and its applications in living brain imaging. (a) Jablonski diagram of STED and schematics of excitation, STED, and emission spots; (b) application of STED in mouse brain imaging
[50]; (c) principle of three dimensional two-photon STED (3D-2P-STED) microscopy with aberration correction
[58]; (d) schematic of 2P-STED imaging of mouse brain
in vivo and imaging comparison of 2P-STED and 2PLSM
[57]; (e) repetitive super-resolution imaging of mouse brain cortex using STED microscopy
[56] Fig. 3. Principle of structure illumination microscopy (SIM) and its applications in living brain imaging. (a) Principle of SIM
[70]; (b) application of adaptive optics SIM (AO-SIM) in living brain imaging
[59]; (c) schematic of optical slice SIM (OS-SIM) with adaptive optics (AO) and its imaging results
[72]; (d) principle of patterned activation nonlinear SIM (PANL-SIM) and its imaging results
[74] Fig. 4. Experiments of brain activity in awake mouse. (a) Setup of an imaging system for brain activity in awake mouse based on 2PLSM
[88]; (b) setup of an imaging system for brain activity in anesthesia mouse based on optical coherence tomography (OCT)
[89]; (c) design and imaging of micro endoscope for deep imaging of the brain in a live mouse
[84]; (d) system and imaging for head-fixed awake mouse
[83] Method | Resolution /nm | Depth /μm | Field of view | Probe | Sample | Ref. |
---|
STED | ~115(xy) | | | GFP | NSM neurons of living Caenorhabditis elegans | [49] | ~70(xy) | 10-15 | | Thy1-EYFP | Molecular layer in the somatosensory cortex of a living mouse | [50] | 43-70(xy) | ~40 | | Lifeact-EYFP | Dendritic filamentous (F-) actin cytoskeleton in the visual cortex of a living mouse | [51] | ~80(xy) | ~6 | | Lifeact-mNeptune2 | Actin filaments in the cortex of a living mouse | [52] | ~84(xy) | | | PSD95-EGFP | PSD95 in the visual cortex of a living mouse | [53] | ~70(xy) | ~25 | | PSD95-HaloTag | PSD95 in the visual cortex of a living mouse | [54] | ~66-89(xy) | 5-20 | | Synaptophysin-EGFP, Myr-rsEGFP2-LDLR, PSD95-FingR-Citrine | Synaptic vesicles, dendritic membrane, and PSD95 in the visual cortex of a living mouse | [55] | ~96(xy) | 15-35 | | Thy1-GFP-M | Spine synapses in the motor cortex of a living mouse | [56] | 2P-STED | 147±8 (xy),1218±24(z) | 5-20 | 10 μm×10 μm(~1 frame/s) | Thy1-GFP-M,Thy1-YFP-H | CA1 area of the hippocampus of a living mouse | [57] | 3D-2P-STED | Subdiffraction-limit(xyz) | ~76 | 20 μm×20 μm(0.1 frame/s) | ATTO590 | Dendritic spines of a living mouse | [58] | SIM | 190±11(xy) | 25-50 | -(9.3 frame/s) | Thy1-GFP | Dendrites and synapses of a living mouse | [59] | 2P-SIM | ~119(xy) | ~120 | 21 μm×21 μm(3.5 frame/s) | Thy1-EGFP | Dendritic spines of a living mouse | [60] |
|
Table 1. Comparison of various super-resolution imaging techniques applied to living brain imaging