Abstract
Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.
Highlights
The development of high-efficiency optical indicators of neural activity has widened the application of fiber photometry (FP) [1,2,3,4], a method employing flat-cleaved step-index optical fibers (OFs) to monitor time-dependent functional fluorescence and/or lifetime variations related to several physiological phenomena, including calcium (Ca2+) levels [5], membrane potential [6], neurotransmitters’ transients [7] and the intracellular biochemical state of neurons [8]
Resulting fluorescence was detected by a photomultiplier tube (“μscope PMT”, μ) in non-descanned epifluorescence configuration, and simultaneously a fraction of the signal was collected by the optical fiber and guided to a second PMT (“fiber PMT”, f )
As fiber photometry is widely employed for collecting functional fluorescence from the mouse brain in free-moving animals, the definition of the collection volume was so far mainly based on (i) the distribution of the functional fluorescence in the targeted subpopulation of cells, (ii) the collection properties of the employed device, (iii) the properties of scattering in brain tissue
Summary
The development of high-efficiency optical indicators of neural activity has widened the application of fiber photometry (FP) [1,2,3,4], a method employing flat-cleaved step-index optical fibers (OFs) to monitor time-dependent functional fluorescence and/or lifetime variations related to several physiological phenomena, including calcium (Ca2+) levels [5], membrane potential [6], neurotransmitters’ transients [7] and the intracellular biochemical state of neurons [8]. The brain volume contributing to the overall signal depends on the constitutive parameters of the OF, including numerical aperture (NA), core/cladding dimensions and refractive index [9], and it is the result of the combination of the three-dimensional excitation and collection fields [9,10,11,12]. Both excitation and fluorescence photons undergo tissue attenuation and scattering, and the generated fluorescence strongly depends on: (i) how the excitation light distributes at the output of the OF, and (ii) how many fluorescence photons generated in a specific point reach the fiber facet. The use of genetically encoded fluorescent indicators of neural activity makes a subpopulation of cells act as source of functional fluorescence, while non-tagged neurons influence the collected signal as a passive optical medium, defining the optical properties of the tissue
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