2-Photon Calcium Imaging

Principle of 2-Photon imaging in Drosophila. a Fly mounted underneath the objective of a 2- Photon microscope. Visual stimuli are being displayed on an LED arena. b,c: 2-Photon images of lamina L2-neurons (marked in green) expressing the genetically encoded Calcium indicator TN-XXL in different depths. d: Principle of visual stimulation: the LED arena is on only during fly-back of the scan-mirror, i.e. during 400 usec per line.

Since its introduction in 1990 (Denk et al., 1990), 2-Photon microscopy is the standard technique for Calcium imaging. Its superior Z-resolution has proven to be particularly useful for its application in the vertebrate retina and in the fly visual system, since it avoids unwanted out-of-focus excitation of photoreceptors that are in the vicinity of the neurons one records from. As an additional problem, however, stimulus light has to be prevented to invade the sensitive photomultipliers: this can be fixed either by temporal separation of the visual stimulation and the line scanning in the millisecond range (Reiff et al., 2010) or by spectral separation (Maisak et al., 2013). In conjunction with genetically encoded Calcium indicators such as TN-XXL (Mank et al., 2008) or GCaMP5 (Akerboom et al., 2012), 2-Photon Calcium imaging has become the method of choice to record the visual responses of small columnar neurons in the fly visual system that are inaccessible to electrophysiological recording methods.

2-Photon Calcium imaging revealed that lamina neurons L2 show only little responses to light-on but respond with a marked and long-lasting increase of presynaptic Calcium to light-off2. Furthermore, 2-Photon Calcium imaging allowed characterizing, for the first time, the visual response properties of T4 and T5 cells in the fly visual system (Maisak et al., 2013). We found that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. While T4 and T5 cells show the same temporal frequency tuning, they functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviors.

2-Photon imaging in T4/T5 terminals.
Confocal image of the optic lobe of a driver line giving rise to expression in T4 and T5 cells, shown in a horizontal cross section (from Schnell et al, 2012). Neurons are marked in green (Kir2.1-EGFP labeled), while the neuropile is stained in red by an antibody against the postsynaptic protein Dlg. Scale bar = 20 mm. b 2-Photon image of the lobula plate of a fly expressing GCaMP5 under control of the same driver line. Scale bar = 5 mm. The size and orientation of the image approximately corresponds to the yellow square in c. c Relative fluorescence changes (DF/F) obtained during 4 sec grating motion along the four cardinal directions, overlaid on the grey-scale image. Each motion direction leads to activity in a different layer. Minimum and maximum DF/F values were 0.3 and 1.0 (horizontal motion), and 0.15 and 0.6 (vertical motion). d Compound representation of the results obtained from the same set of experiments. Scale bar = 5 mm.


Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

Maisak, M. S. et al. A directional tuning map of Drosophila elementary motion detectors. Nature 500, 212–216 (2013).

Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Meth. 5, 805–811 (2008).

Reiff, D. F., Plett, J., Mank, M., Griesbeck, O. & Borst, A. Visualizing retinotopic half-wave rectified input to the motion detection circuitry of Drosophila.Nat. Neurosci. 13, 973–978 (2010).

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