2p Microscopy
2P MICROSCOPY
Top-down modulation of the retinal code via histaminergic neurons in the hypothalamus
The mammalian retina is considered an autonomous neuronal tissue, yet there is evidence that it receives inputs from the brain in the form of retinopetal axons. A sub-population of these axons was suggested to belong to histaminergic neurons located in the tuberomammillarynucleus (TMN) of the hypothalamus. Using viral injections to the TMN, we identified these retinopetal axons and found that although few in number, they extensively branch to cover a large portion of the retina. Using Ca2+ imaging and electrophysiology, we show that histamine application increases spontaneous firing rates and alters the light responses of a significant portion of retinal ganglion cells (RGCs). Direct activation of the histaminergic axons also induced significant changes in RGCs activity. Since activity in the TMN was shown to correlate with arousal state, our data suggest the retinal code may change with the animal's behavioral state through the release of histamine from TMN histaminergic neurons.
Multiphoton imaging with next-generation indicators
Two-photon (2P) in vivo functional imaging of genetically encoded fluorescent Ca2+indicators (GECIs) for neuronal activity has become a broadly applied standard tool in modern neuroscience, because it allows simultaneous imaging of the activity of many neurons at high spatial resolution within living animals. Unfortunately, the most commonly used light-sources – tunable femtosecond pulsed ti:sapphire lasers – can be prohibitively expensive for many labs and fall short of delivering sufficient powers for some new ultra-fast 2P microscopy modalities. Inexpensive homebuilt or industrial light sources such as Ytterbium fiber lasers (YbFLs) show great promise to overcome these limitations as they are becoming widely available at costs orders of magnitude lower and power outputs of up to many times higher than conventional ti:sapphire lasers. However, these lasers are typically bound to emitting a single wavelength (i.e., not tunable) centered around 1020-1060 nm, which fails to efficiently excite state of the art green GECIs such as jGCaMP7 or 8. To this end, we designed and characterized spectral variants (yellow CaMP = YCaMP) of the ultrasensitive genetically encoded calcium indicator jGCaMP7, that allows for efficient 2P-excitation at wavelengths above 1010nm. In this talk I will give a brief overview over some of the reasons why using a fiber laser for 2P excitation might be right for you. I will talk about the development of jYCaMP and some exciting new experimental avenues that it has opened while touching on the prospect that shifting biosensors yellow could have for the 2P imaging community. Please join me for an interesting and fun discussion on whether “yellow is the new green” after the talk!
Non-Telecentric 2P microscopy for 3D random access mesoscale imaging
Ultra-low-cost, easily implemented and flexible two-photon scanning microscopy modification offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. Application of our system for imaging neuronal activity has been demonstrated on mice, zebrafish and fruit flies. Website: https://github.com/BadenLab/nTCscope