High-speed brain-wide 3D calcium imaging of neuronal activity with sculpted light

Wide-field temporal focusing (WF-TEFO) is a two-photon imaging technique which is based on light-sculpting. It effectively decouples the parameters governing lateral size of a light beam and its axial resolution. Thus, this techniques allows exciting a large area in the lateral dimension while retaining exceptional resolution in the axial direction. In an actual setup, this is akin to creating a thin “disc” of excitation light. WF-TEFO is well suited for fast volumetric imaging, as it scanning is reduced to one dimension only.

In our latest experiments we tailored the properties of our WF-TEFO to record, with high temporal and spatial resolution, the activity of neurons in the head ganglia of C. elegans in vivo using Ca²⁺ imaging. Read below the abstract of our paper:

Recent efforts in neuroscience research seek to obtain detailed anatomical neuronal wiring maps as well as information on how neurons in these networks engage in dynamic activities. Although the entire connectivity map of the nervous system of C. elegans has been known for more than 25 years, this knowledge has not been sufficient to predict all functional connections underlying behavior. To approach this goal, we developed a two-photon technique for brain-wide calcium imaging in C. elegans using wide-field temporal focusing (WF-TEFO). Pivotal to our results was the use of a nuclear-localized, genetically encoded calcium indicator (NLS-GCaMP5K) that permits unambiguous discrimination of individual neurons within the densely-packed head ganglia of C. elegans. We demonstrate near-simultaneous recording of activity of up to 70 % of all head neurons. In combination with a lab-on-a- chip device for stimulus delivery, this method provides an enabling platform for establishing functional maps of neuronal networks.

Volumetric fluorescence imaging using wide-field two-photon light sculpting. — Figure 1: Experimental setup of volumetric fluorescence imaging using wide-field two-photon light sculpting. Schematic depicting of the light-sculpting microscope and microfluidic sample holder. The pulses at the bottom sketch the geometric dispersion in temporal focusing as a function of axial position. The inset on the top right is an artistic rendering of a C. elegans head, indicating axially scanned light discs and the imaged region. Neurons URX and BAG are also depicted. Scale bar is 15 µm. DC, dichroic mirror; OPA, optical parametric amplifier; sCMOS, scientific complementary metal–oxide–semiconductor.

To aid in cell segmentation, we use transgenic worms expressing the Ca²⁺-sensor GCaMP5K in a pan-neuronal and nucleus- bound fashion. We also used custom-designed microfluidic devices to restrain the worms while applying chemosensory stimuli. Using this approach, we demonstrate brain-wide and near-simultaneous Ca²⁺– imaging of 70 % of the neurons contained in the head ganglia.

Brain-wide WF-TEFO Ca2+-imaging in C. elegans. — Figure 2: Brain-wide WF-TEFO Ca²⁺-imaging in *C. elegans*. (A) Schematics of the microfluidic PDMS device. (I-II) Red and blue sketches indicate worm immobilization and gas delivery channel respectively. (II) Cross-section (not to scale). (III) Phase contrast image of immobilized worm inside the device. The head (tail) is at the bottom (top). Ventral is shown by the vulva’s location (blue arrow). (B) WF-TEFO imaging of head region of a Punc-31::NLS-GCaMP worm. (I) Maximum intensity projection of 14 z-planes. Dashed lines outline the locations of head ganglia as shown in (II). Scale bar represents 10 µm and refers to panels I,III-V. (II) Schematic of the left anterior head ganglia modified from ref. 9. Black lines outline neuronal nuclei. Grey lines outline the anterior ganglia as indicated. Green area indicates pharynx. (III) Single z-plane (z = 2 µm). Dashed lines indicate y-z and x-z cross- sections shown in (IV) and (V) respectively. Arrows in (III-V) indicate example neurons each seen in two projections. The crosses indicate worm orientation (A – anterior, P – posterior, D – dorsal, V – ventral, L – left, R – right).

The spatial and temporal resolution of our imaging technique is adequate for investigating global properties of the C. elegans nervous system, such as correlated activity among groups of neurons and their responses to sensory stimuli. This paves the way for future investigations on how sensory information is processed at the level of the whole brain, and for establishing a functional map of C. elegans’ nervous system and potentially also in other model organisms.

Figure 3: Data analysis of WF-TEFO Ca2+-imaging in worms during chemosensory stimulation. (A) Activity of 69 neurons under consecutively changing O2- concentrations. Rows are heat-plots of NLS-GCaMP5K fluorescence time series. Color indicates percent fluorescence changes (∆F/F₀). Colorbar indicates scaling. X-axis represents elapsed recording time. O₂-concentrations shifted as indicated by dashed lines. (B) Matrix showing correlation coefficient (R) calculated from data in (A). Color indicates the degree of correlation. Colorbar indicates scaling. The data in A-B are grouped by agglomerative hierarchical clustering.

Below is a 3D-Movie of a C.elegans brain acquired by WF-TEFO. Activation of pairs of BAX and URG neurons is clearly visible in the mid left and top right, respectively, as oxygen is up and downshifted from 4 to 21 %. Videos like this were used to track activity of individual neurons and produce plots like the one above.

Relevant Publications:
Schrödel, Tina*; Prevedel, Robert*; Aumayr, Karin; Zimmer, Manuel and Vaziri, Alipasha (*Co-First Authors),
Brain-wide 3D imaging of neuronal activity Caenorhabditis elegans with sculpted light
Nature Methods 10, 1013–1020 (2013).
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