Fiber photometry
The benefits to researchers are that optical fibers are simpler to implant, less invasive and less expensive than other calcium methods, and there is less weight and stress on the animal, as compared to miniscopes.
[8][9][10][11] In both neurons and astrocytes, cellular activity in the form of action potentials, exocytosis of neurotransmitters, changes in synaptic plasticity and gene transcription is coupled to an influx of Ca2+ ions.
[12] The most commonly used calcium indicator for fiber photometry (and other in vivo imaging method) is GCaMP6, although additional GECIs continue to be developed with unique fluorescence spectra, kinetics, signal-to-noise ratios and calcium-sensitivities.
[15][19][13][3][20][21] Fiber photometry systems are designed to deliver precise excitation wavelengths of light that are specific to a calcium (e.g. GCaMP) or neurotransmitter indicator (e.g.
The calcium indicator is that is expressed in a cell-type specific manner is excited by this light and in turn, emits its own signal that travels back through the same fiber.
[3][4][22] These collected emission signals are spectrally-separated by a dichroic mirror, passed through a filter and focused onto a photodetector, scientific camera, or PMT.
Animal motion and tissue autofluorescence are reflected in this calcium-independent signal and can be subtracted or regressed to reveal the true change in fluorescence.
[26]Optimal expression of genetically encoded calcium indicators (GECIs) can be accomplished in two ways: adeno-associated viral vectors and transgenic rodent lines.
Motion and autofluorescence are reflected in this calcium-independent signal and can be subtracted or regressed to reveal the true change in cellular fluorescence during a behavioral task or experimental manipulation.
To observe simultaneous calcium dynamics in multiple cell types, researchers have combined two or more GECIs in a single brain region.
[3][28] For example, a research group recorded fluorescence from the green and red GECIs, GCaMP6f and jRGECO1a, that were differentially expressed in striatal direct- and indirect-pathway spiny projection neurons in freely behaving mice.
[30] The goal of fiber photometry is to precisely deliver, extract and record bulk calcium signal from specific populations of cells within a brain region of interest.
[1] Additionally, optical fibers allow recording from both deep and shallow brain structures, and minimize tissue damage unlike GRIN lenses or cortical windows.
When collecting signal from a single brain region, it is typical to use a photodetector or PMT due to their fast acquisition and low signal-to-noise ratio (SNR).
For individuals in labs that want to integrate calcium imaging into their experiments but may not have the financial or technical circumstances to do so yet, fiber photometry is a low barrier of entry.
This lack of optical resolution can be attributed to the collection of an aggregation of activity within a field of view, only allowing for 'bulk' changes in fluorescent signal.
[5] Although the size of an optical cannula is much smaller than technology used in other calcium imaging methods, such as one- and two-photon microscopy, animals must be securely tethered to a rigid fiber bundle.
Researchers couple miniscopes with implanted gradient-refractive-index (GRIN) lenses or cortical windows that enable deep and superficial brain imaging.
[37] As compared to fiber photometry, miniscopes allow imaging with high cellular resolution, detecting changes in calcium within individual neurons and non-neuronal cells.
[36][42] As compared to other techniques, two-photon offers higher cellular and sub-cellular spatial resolution, such as within dendrites and axonal boutons, within a well-defined focal plane.
