Two-photon calcium imaging is fluorescence microscopy in which a neuron's brightness reports its activity. Activity changes a cell's free calcium, and a genetically encoded sensor brightens when it binds calcium; the laser excites the sensor where two photons are absorbed at once, confining the signal to the focal plane. What it reads is the fluorescence change, an indirect measure from which spikes are inferred. In mouse cortical pyramidal cells, jGCaMP8 resolved spike doublets 20 ms apart using averaged responses with simultaneous electrical recording. Older GCaMP6 variants resolved burst spikes at about 50–150 ms spacing.
germline genetic engineering: Mesoscope protocol, mouse: transgenic GP5.17 mice express GCaMP6f in a subset of pyramidal neurons; GP4.3 mice express GCaMP6s in the vast majority of pyramidal neurons. Behaving activity imaging uses GP4.3 mice.; surgery: Mesoscope protocol, mouse: stainless-steel head plate and D-shaped cranial window (450 µm thick, three layers of custom-cut coverglass) spanning lambda to 3 mm anterior of Bregma and midline to 5 mm lateral over the left hemisphere.; anesthesia: Mesoscope protocol, mouse: low-magnification, high-resolution structural images (970 nm) are acquired in anesthetized mice; activity imaging (4.4 mm x 4.2 mm FOV, 1.9 Hz, layer 2/3) is in behaving mice running on a treadmill in tactile virtual reality.; genetic engineering: GCaMP6 V1 protocol, mouse: AAV2/1-syn-GCaMP-WPRE injected into visual cortex of C57BL/6J mice (1.5-2 months old) for population imaging; for sparse dendritic labeling, diluted AAV2/1-syn-Cre mixed with high-titer Cre-dependent GCaMP6s virus; injections 30 nL over 5 min at 250 µm depth at two V1 sites.; germline genetic engineering: GCaMP6 V1 protocol, mouse: PV-IRES-Cre mice receive Cre-dependent GCaMP6s AAV in visual cortex for parvalbumin interneuron labeling.; anesthesia: GCaMP6 V1 protocol, mouse: surgery after 2-4 weeks of expression under isoflurane (3% induction, 1.5-2% during surgery); imaging on a 37°C warm blanket under 0.5% isoflurane with chlorprothixene sedation (20-40 µL at 0.33 mg/ml, i.m.).; surgery: GCaMP6 V1 protocol, mouse: circular 2-3 mm craniotomy over V1 (centered 2.7 mm lateral of lambda) and custom titanium head post fixed with black dental cement. Acute: agarose (1-1.3%) plus cemented 5 mm #1 coverslip; cell-attached recording: about 1 mm 1.3% agarose without coverslip. Chronic: two-layer coverglass window (#1 piece on bone, #2 insert in craniotomy), imaging from about 1-2 weeks after implantation.; germline genetic engineering: Light-beads protocol, mouse: 6 male and female vGluT1-cre x fl-GCaMP6s (pCAG) mice express GCaMP6s in glutamatergic neurons.; genetic engineering: Light-beads protocol, mouse: 3 male and female mice express jGCaMP7f through viral transfection.; surgery: Light-beads protocol, mouse: transgenic animals carry large (8 mm diameter) cranial windows.; other: Light-beads protocol, mouse: all animals are head-fixed on a home-built treadmill with a rotation encoder on the rear axle.
Theoretical spatial resolution
350 nm (point spread)
Theoretical field of view
19.6 mm³ (reachable envelope)
Description
In laser scanning fluorescence microscopy, molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution. In the experimental characterization of this method, fluorescence emission increased quadratically with excitation intensity, so fluorescence and photobleaching were confined to the vicinity of the focal plane, as expected for cooperative two-photon excitation. Calcium imaging rests on a separate principle: neural activity causes rapid changes in intracellular free calcium, and calcium imaging experiments read out activity through these calcium changes. Calcium imaging has been used on this basis to track the activity of neuronal populations and to probe excitation of small neurons and neuronal microcompartments. Genetically encoded protein sensors can be targeted to specific cell types for imaging of identified neurons and neuronal compartments over chronic timescales. GCaMP and its progeny consist of circularly permuted green fluorescent protein (cpGFP), the calcium-binding protein calmodulin (CaM), and the CaM-interacting M13 peptide. The CaM/M13 complex lies in proximity to the chromophore inside the cpGFP beta barrel. Calcium-dependent conformational changes in CaM/M13, including modulation of solvent access and of the chromophore pKa, cause increased brightness with calcium binding.