Simultaneous Two-Photon Imaging and Optical Stimulation

The Prairie Ultima was the first commercial system designed to include two sets of conventional galvanometers based scanners, the standard imaging set and the optional uncaging set, for simultaneous imaging and uncaging using two lasers. Synchronization of these two functions ensures that cellular reactions that occur during or immediately following stimulation are not overpassed.

Examples:

• Noguchi et al.¹ combined two-photon fluorescence imaging of a calcium indicator with two-photon photon-uncaging of glutamate to understand the role of calcium signaling during NMDA receptor activation in hipppocampal slices. Two-photon uncaging of glutamate allowed the precise excitation of NMDA receptors on a single spine head, unlike previous attempts that used electrical stimulation and failed to confine electrical excitation to individual synapses. The combination of optical activation of single synapses with an optical measurement of intracellular calcium made possible to measure calcium changes that were induced by photochemical stimulation of a single synapse and the attached dendritic shaft. Calcium concentration was estimated with the calcium-sensitive fluorescent dye Oregon Green and individual neurons were filled with a mixture of both calcium-sensitive and a calcium-insensitive fluorescent dye. Both probes were excited with 830-nm femtosecond laser light, but their emission spectra were far enough apart to determine changes in calcium concentration via the ratio of emitted light. A second femtosecond laser at 720-nm wavelength was used to uncage glutamate. Electrical current generated by the influx of ion inside the cell was also measured by conventional microelectrode intracellular recording. Combined with pharmacological blockage of various NMDA receptors, these experiments determined that excitation of a single synapse does led to calcium increases in the dendritic shaft, and suggested that the extent of the calcium movement between synapse and dendrite depended on the shape of the spine head and neck.
  
• 2PE microscopy is especially well suited for imaging in the retina. Since the retina’s photoreceptors are exquisitely sensitive to visible light, they are rapidly bleached by conventional fluorescence microscopy methods. In contrast, the IR light used for 2PE microscopy is not absorbed by photopigments and therefore allows simultaneous functional imaging of retinal neurons and visual stimulation of photoreceptors.
• The genetically targeted fluorescent calcium indicator proteins along with optical imaging techniques are expected to become instrumental for the construction of macroscopic and microscopic maps of the function of specific brain circuits.

J. Díez-García et al.² reported the use of transgenic mice expressing the fluorescent calcium indicator protein GCaMP2 in cerebellar granule cells to image parallel fiber activity transcranially in vivo.

References:

1. Noguchi, J., Matsuzaki, M., Ellis-Davies, G.C.R. and Kasai, H. (2005) Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. Neuron 46, 609-622.
2. Javier Díez-García, Walther Akemann and Thomas Knöpfel, In vivo calcium imaging from genetically specified target cells in mouse cerebellum, NeuroImage Volume 34, Issue 3, 1 February 2007, Pages 859-869.