Journal: bioRxiv

Article Title: A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

doi: 10.1101/2022.03.16.484590

Figure Lengend Snippet: (A) Scheme of a conventional photometry system (CPS) based on excitation and emission filters F as well as dichroic mirrors (diagonal lines). (B) Customized fused fiber coupler for FFP: excitation light is delivered in one branch of the coupler (top left) and split into two branches: ∼ 10 % of the power is used for the excitation of biosensors, while 90% are dissipated (top right). This configuration allows collection of 90% of emission light from the indicator by the photodetector (bottom left) through the same fiber; any backscattered excitation light is attenuated by the optical filter F’ . (C) Sketch of assembled FFP system for in vivo recordings. A mouse with implanted cannula (1) is connected via the fused fiber coupler (2) to a light source (3) and a photodetector (4) with an amplifier (5). Optical filters (detailed in insets) to clean the excitation and emission light are installed in the light source and in the photodetector. The light source and photodetector are controlled by a MicroPython pyboard processor (6) connected to a computer (7) to realize indicator excitation and signal acquisition. (D) Within-animal comparison of median detected GCaMP6s fluorescence [nW] normalized to the excitation power [µW] recorded with CPS and FFP, when excited at 405 (purple) and 470 nm (blue). (E) Comparison of dynamic range, defined as the difference between the 1 st and 99 th percentiles of GCaMP6s fluorescence, normalized to the excitation power, recorded with the CPS and FFP systems. (F) Relationship between detected fluorescence and dynamic range of the systems. ( G) Detected fluorescence with the FFP system, normalized to the fluorescence detected with the CPS system. Grey dashed line: Detection efficiency of the FFP system (see ) normalized to the efficiency of the CPS system. (H) Excitation and emission spectrum of GCaMP6f (top) and filter properties of excitation (405/10 nm, cyan; 470/10 nm, blue) and emission filters (black; bottom) to record fluorescence from hippocampal neurons expressing GCaMP6s. (I) Representative raw data traces of 2 min dura0074 ion: raw traces for 470 and 405 nm excitation, as well as isosbestic-corrected calcium signal (ΔF/F), pupil size, and running speed (from top to bottom). (J) Correlation matrix indicates positive correlations between hippocampal calcium activity, pupil size, and running. (K) Excitation and emission spectrum of jRGECO1a (top), properties of excitation (405/10 nm, cyan; 548/10 nm, green) and emission filters (black; bottom) to record fluorescence from hippocampal neurons expressing jRGECO1a. (L) Representative raw data traces of 2 min duration: raw traces for 548 and 405 nm excitation, as well as the 405 nm-corrected calcium signal (ΔF/F), pupil size, and running speed (from top to bottom). (M) Correlation matrix indicates positive correlations between hippocampal calcium activity, pupil size, and running speed. ****: p < 0.0001.

Article Snippet: To monitor calcium-modulated fluorescence across different wavelengths, we injected either rAAV2/9 encoding GCaMP6s ( ) under control of the CaMKII promoter (1.25 × 10 13 gc/ml; AddGene viral prep #107790-AAV9; kindly gifted by James M. Wilson), rAAV2/9 encoding jRGECO1a under control of the human synapsin promoter (4 × 10 13 gc/ml; customized from AddGene plasmid #61563; kindly gifted by Douglas Kim & GENIE Project ( )), or rAAV2/9 encoding for NIR-GECO2 under control of the synthetic CAG promoter (0.5 × 10 12 -1 × 10 13 gc/ml; AddGene plasmid #159603; kindly provided by Robert Campbell ( )).

Techniques: In Vivo, Comparison, Fluorescence, Expressing, Activity Assay

Journal: bioRxiv

Article Title: A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

doi: 10.1101/2022.03.16.484590

Figure Lengend Snippet: The figure illustrates the propagation of excitation light from the light source to the brain (A – C) and emission light from the brain to the detector (D – F). The transformation of the spectra inside the FFC can be described as follows: The broad excitation light spectrum originating from the LED (A) is cleaned up with a band-pass filter (B) to restrict the excitation light to a narrower spectrum (C) , matching the peak excitation spectrum of the indicator fluorophore, but reduces optical contamination of the signal channel in the emission band. Dashed grey line: excitation spectrum of GCaMP6s. The indicator fluorophore emits light of longer wavelengths (D) , which is collected by the optical fiber at the brain end and propagates through the FFC to the detector. Before reaching the detector, the emission light is cleaned up with an additional band-pass filter to eliminate remaining excitation light and any additional optical noise (potentially originating from ambient light etc.) (E) . Finally, the filtered spectrum (F) is collected by the photodetector (P). Overall modulation of the detected light is strongly coupled to the modulation of light emission by the fluorescent indicator.

Article Snippet: To monitor calcium-modulated fluorescence across different wavelengths, we injected either rAAV2/9 encoding GCaMP6s ( ) under control of the CaMKII promoter (1.25 × 10 13 gc/ml; AddGene viral prep #107790-AAV9; kindly gifted by James M. Wilson), rAAV2/9 encoding jRGECO1a under control of the human synapsin promoter (4 × 10 13 gc/ml; customized from AddGene plasmid #61563; kindly gifted by Douglas Kim & GENIE Project ( )), or rAAV2/9 encoding for NIR-GECO2 under control of the synthetic CAG promoter (0.5 × 10 12 -1 × 10 13 gc/ml; AddGene plasmid #159603; kindly provided by Robert Campbell ( )).

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

doi: 10.1101/2022.03.16.484590

Figure Lengend Snippet: (A) Excitation and emission spectrum of GCaMP6s and activation spectrum of ChrimsonR (top) along with filtered LED excitation and stimulation light (405 nm, cyan; 470 nm, blue; 575 nm, orange; bottom) and emission filter band (530/55 nm, black; bottom). (B) Spectrogram indicating absence of optical contamination caused by optogenetic stimulation. Transmittance of the emission filter is indicated in black, while colored lines indicate the spectrum of fiber autofluorescence with (magenta) and without simultaneous illumination with orange light for optogenetic stimulation (green). The overlapping lines indicate the absence of signal contributions originating from optogenetic stimulation as the emission filter fully blocks backscattered stimulation light. (C) Recording of fiber autofluorescence in response to optogenetic stimulation (300 μW for 1 second: magenta line on top). Stimulation artefacts were not detectable with a gain of 1 (top), and only mildly pronounced when the detector operated at a gain of 100 (bottom). Average (solid line) of 20 individual traces (transparent lines). (D) Histological verification of fiber position and expression of ChrimsonR (magenta) as well as GCaMP6s (green) in the hippocampus, overlaid with an inverted bright-field image. Dashed line: lesion caused by the optical fiber. (E) ΔF/F (green) in response to optogenetic stimulation (300 μW for 1 second, 0.1 Hz, 20 trials), indicated by the magenta bars. (F) Individual traces and (F’) mean ± standard deviation of ΔF/F of 20 trials of optogenetic stimulation (starting at t = 0), normalized to stimulus onset. Note the occurrence of pronounced calcium transients in every trial. (G) Average calcium transients in response to optogenetic stimulation with increasing stimulus intensity (20 trials per conditions). (H) Amplitude of average calcium traces in response to optogenetic stimulation as a function of stimulus intensity.

Article Snippet: To monitor calcium-modulated fluorescence across different wavelengths, we injected either rAAV2/9 encoding GCaMP6s ( ) under control of the CaMKII promoter (1.25 × 10 13 gc/ml; AddGene viral prep #107790-AAV9; kindly gifted by James M. Wilson), rAAV2/9 encoding jRGECO1a under control of the human synapsin promoter (4 × 10 13 gc/ml; customized from AddGene plasmid #61563; kindly gifted by Douglas Kim & GENIE Project ( )), or rAAV2/9 encoding for NIR-GECO2 under control of the synthetic CAG promoter (0.5 × 10 12 -1 × 10 13 gc/ml; AddGene plasmid #159603; kindly provided by Robert Campbell ( )).

Techniques: Activation Assay, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

doi: 10.1101/2022.03.16.484590

Figure Lengend Snippet: (A) Illustration of the composition of the detected fluorescence signal. (B) Autofluorescence of the fiber measured by the photodetector as a function of excitation power at the brain port before (solid line) and after 12-hours of photobleaching (dashed line). (C, D) Signal contributions for excitation of GCaMP6s with light of 405 (C) and 470 nm (D): Fluorescence (F) of the optical fiber, brain tissue (fixed or in vivo ) and upper and bottom percentile of the calcium transients.

Article Snippet: To monitor calcium-modulated fluorescence across different wavelengths, we injected either rAAV2/9 encoding GCaMP6s ( ) under control of the CaMKII promoter (1.25 × 10 13 gc/ml; AddGene viral prep #107790-AAV9; kindly gifted by James M. Wilson), rAAV2/9 encoding jRGECO1a under control of the human synapsin promoter (4 × 10 13 gc/ml; customized from AddGene plasmid #61563; kindly gifted by Douglas Kim & GENIE Project ( )), or rAAV2/9 encoding for NIR-GECO2 under control of the synthetic CAG promoter (0.5 × 10 12 -1 × 10 13 gc/ml; AddGene plasmid #159603; kindly provided by Robert Campbell ( )).

Techniques: Fluorescence, In Vivo