! 1 2 A Fluorescence!(A.U.)! 1! 0.8! 0.6! 0.4! 0.2! 0! 400! pH! ExcitaCon! B Emission! + 9.0! 10.2! 10.9! - Wavelength!(nm)! pH! 500! 600! 700! ΔF/F!(%)! F40! F20! 0! 20! C Fluorescence!(A.U.)! 6! 5! 4! 3! 2! 1! -0.2! -0.1! 0! 0.1! 0.2! Fluorescence!(A.U.)! 11! 7! 10! 9! 8! 7! D 1.8! 1.4! 1! 0.6! 0.2! 0! Voltage!on! 0.1! Membrane voltage (V)! Time!(s)! 0.2! 0.3! 0.4! 0.5! λ A B Fluorescence (A.U.)! 1! 0.5! 0! 3! 2! 1! 0! pH!7! pH!9! pH!7! PROPS! pHlourin! 10! 30! 50! 70! 90! 200 % ΔF/F Fluorescence (A.U.)! 60 s 10! 20! 30! Time (s)! 40! 50! 200 % ΔF/F Rotation 2π" θ" 0" C 1s ≈ A violet light B oxygen removal 200% ΔF/F 1 pH unit 30 s C sodium azide D CCCP E Fluorescence$ Blinking$frac / θ θ a)# b)# PLL#Flow#Cell# Agarose#Pad# + = + × ( ) ( ) = σ σ σ A Periplasm pHo Cytoplasm pHi B Voltage Vm < 0 Vloc + Voltage C + Vloc Vm > 0 pHo#8.0# 10#%#ΔF/F# 200#%#ΔF/F# 30#s# pHo#8.5# A" 400%$ΔF/F$ oxygen$removal$ 40%$ΔF/F$ B" 200%$ΔF/F$ 30$s$ 10$mM$azide$ 30%$ΔF/F$ 30$s$ λ Fluorescence!(A.U.)! 1.8! 1.6! 1.4! 1.2! 1! 0.8! 0! 10! 20! 30! 40! Time!(s)! λ 50! 60! 70! 80! 90! λ 4.5" Fluorescence"(A.U.)" 4" 3.5" 3" 2.5" 2" 1.5" 1" 0" 5" 10" 15" 20" 25" Time"(s)" 30" 35" 40" fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 BW25113 (Coli Genome Stock ∆(araD-araB)567, ∆lacZ4787(::rrnB-3), lambda-, rph-1, ∆(rhaDCenter) rhaB)568, hsdR514 BL21 (DE3) UT5600 F- ara-14 leuB6 secA6 lacY1 proC14 tsx-67 Δ(ompT-fepC)266 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1 Thr-1 araC14 leuB6(Am) fhuA31 lacY1 tsx-78 λ- eda-50 hisG4(Oc) rfbC1 rpsL136 xylA5 mtl-1 metF159 thi-1 ∆fliC, sticky fliC allele was cloned into pACYC184 (CmR) under the native promoter of fliC fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 ∆lacZY JY29 (Gift from Howard Berg) Tuner™ (Novagen) Strain Temperature Growth Medium Inducer Antibiotic resistance Amp, Strep (for pHluorin) BL21 33 ºC LB IPTG 0.5 mM BW25113 UT5600 JY29 Tuner™ 33 ºC 33 ºC 30 ºC 33 ºC LB LB Tryptone broth LB Ara 0.0005% Ara 0.02%, IPTG 0.2 mM IPTG 0.5 mM Amp Amp, Strep Amp, Cm Amp Wavelength 633 nm 532 nm 488 nm Power 17 mW 50 mW 60 mW Type HeNe Solid-state Solid-state Manufacturer SpectraPhysics Coherent 215-M Omicron PhoxX Blue-ray DVD player 407 nm White light, wavelength selectable via AOTF 460 – 490 nm 80 mW Solid-state 6W Supercontinuum Fianium SC-450-6-AOTF 3W Luxeon III Philips Plasmid pADD161 pADD173 pADD191 pADD192 pADD193 pADD194 pADD195 pADD206 pADD210 pADD211 pADD221 pADD223 pADD224 pADD226 pADD227 pADD222 pADD228 Gene ss(A) – GPR ss(A) – hGPR ss(A) – hGPR – Venus ss(B) – hGPR – Venus ss(B) – hGPR – Venus – ER export ss(C) – hGPR – Venus ss(C) – hGPR – Venus – ER export ss(A) – hGPR – TS – Venus – ER export ss(B) – hGPR – TS – Venus – ER export ss(C) – hGPR – TS – Venus – ER export ss(A) – hGPR – TS – ER export ss(A) – hGPR – TS – G export – ER export ss(A) – hGPR – TS – Venus – G export – ER export XC(HR) – hGPR – TS – Venus – ER export XC(BR) – hGPR – TS – Venus – ER export SS(C) – Venus – hGPR – TS – ER export SS(C) – Venus – hGPR – TS – G Export – ER export Targeting to eukaryotic plasma membrane No No No No No No No No No No No No No No No No No Cell Line Plasmid Temperature Targeting to eukaryotic plasma membrane No No No No HEK 293 3T3 HeLa COS7 All plasmids pADD223, pADD227 pADD223, pADD227 pADD223, pADD227 30 and 37 °C 30 and 37 °C 30 and 37 °C 30 and 37 °C Voltage Sensitive Dye Di – 4 – ANEPPS Di – 8 – ANEPPS TMRM Excitation wavelength (nm) 488 488 532 Labeled blinking cells No No Yes, but effluxed during blinks λ λ λ λ λ λ λ 3 λ λ λ − − λ ˆ ≥ λmax absorbance (nm)" Wild-type Arch Arch(D95N) 558 585 λmax emissiona (nm)" 687 687 ε633 (M-1 cm-1)b" 6,300 37,500 QY c 9 × 10-4 4 × 10-4 Photostability relative to eGFPd 0.25 0.1 pKa of Schiff base e 10.1 8.9 τresponse f (ms)! < 0.5 41 Noise in VFL (µV Hz-1/2) g 625 260 Photo-current yes no λ τ τ a pH 7.3 Positive Vm pH 11.1 Negative Vm b 1" 0.8" Signal" 0.6" 0.4" 0.2" 0" AbsorpBon" Emission" pH"7.3" pH"11.1" +" + Vm" + +" Vm" 500" 550" 600" 650" 700" 750" Wavelength"(nm)" c Fluorescence"(A.U.)" 2" 1.8" 1.6" 1.4" 1.2" 1" H150" H50" 50" Voltage"(mV)" 150" d Fluorescence"(A.U.)" +30 mV Fluorescence"(A.U.)" 1.2" 1.2" e 1.1" 1.1" 1" H5" 0" 5" Time"(ms)" 10" 1" 0" -70 mV 0.2" 0.4" 0.6" 0.8" 1" ˆ ˆ / σˆ σˆ σˆ a b e σ Vˆ = 7.3 mV! 40"mV" 100"ms" c σ Vˆ = 4.2 mV" d σ Vˆ f 20 mV" ="4.0"mV" 5"ms" g V V 4 ms" 40"mV" 100"ms" Fluorescence" Voltage" λ / λ a" 10"pA" b 3" Fluorescence"(A.U.)" 2.5" 2" WT" D95N" Laser" 1.5" 1" 5150" 0" 1" 2" 3" Time"(s)" 4" 5" 550" 50" Voltage"(mV)" 150" c 1.6" d 60" +30 mV Fluorescence"(A.U.)" Fluorescence"(A.U.)" Voltage"(mV)" 1.4" 1.2" 1" 0" 20" 40" 60" Time"(ms)" Fluorescence 1.4" 1.2" 1" 0" -70 mV 40" 20" 0" Voltage 0.2" 0.4" 0.6" Time"(s)" 0.8" 1" 0" 5" Time"(s)" 10" λ a% b% Arch%WT% Arch%D95N% Voltage%(mV)% 40% 0% -40% -80% 0% Time%(s)% 0.5% 1% 1.5% d% 2% 0% Time%(s)% 0.4% 0.8% 1.2% Fluorescence$ c Voltage$ 40 mV 200 pA 100 ms 1000# PROPS# %'ΔF/F'per'100'mV# 100# Arch'D95N# Arch'WT# Organic#dyes# 10# VSFP#2.x,# Mermaid# FlaSh# VSFP#3.1# FLARE# SPARC# 1# 0.1# 10# 100# Response'speed'(1/s)# 1000# 10000# + β − − − − − − − − − − = × ϵ ϵ × × ϵ λ λ ˆ () ˆ () () ˆ () () ˆ () () ()= + ( )+ϵ( ) () ϵ( ) ⟨ϵ ( )ϵ ( )⟩ = σ δ , δ( − ) ⟨⟩ ϵ( ) () δ ()= ( ) − ⟨ ( )⟩ δ ( ) = ( ) − ⟨ ( )⟩ ˆ ˆ = ⟨δ δ ⟩ δ ˆ = ⟨ ⟩−ˆ⟨ ⟩ ˆ()= () ˆ − ˆ ˆ ζ = ⟨( ˆ ( ) − ( )) ⟩ = /ζ /ζ ˆ ()= ˆ() () () () () ˆ () Absorbance/Fluorescence a Arch(D95N) 0.4 0.3 0.2 0.1 0 400 500 600 700 Wavelength (nm) pH 7.3 pH 11.1 Emission pH 7.3 b Fraction protonated 1 0.8 0.6 0.4 0.2 0 6 7 8 9 10 pH pKa = 8.9 Arch Arch(D95N) pKa = 10.1 11 12 λ Θ Θ Θ Θ Θ Θ Θ + + ! 1" 0.8" /V 0.6" 0.4" 0.2" 0" 0" 10" 10"*2" 10" *4" 10" 0" 10" *6" 10" 10" Freq."(Hz)" 1" 2" 10" 3" 10" 1" 10" Frequency (Hz) 2" 10" 3" ˆ ˆ / ˆ 60" 40" 20" 0" 0" 5" Time"(s)" 10" V Voltage"(mV)" ˆ 100 ms A V 40 mV B 1.4 Fluorescence (A.U.) 1.3 1.2 1.1 1 0.9 0 20 40 60 Time (minutes) 80 50 ms 1" 0.8" /V 0.6" 0.4" 0.2" 0" 0 10 " 10" 10" 22" 24" 26" 10" 100" 101" 102" Freq."(Hz)" 103" 101" 102" Frequency (Hz) 103" Molecule VSFP 2.3 VSFP 2.4 VSFP 3.1 Mermaid SPARC FlaSh Flare PROPS di-4-ANEPPS di-8-ANEPPS RH237 RH421 ANNINE-6plus hVOS DiO/DPA Approx ΔF/F per 100 mV 9.50% 8.90% 3% 9.20% 0.50% 5.10% 0.50% 150% 8% 10% 11% 21% 30% 34% 56% Approx response time 78 ms 72 ms 1-20 ms 76 0.8 ms 2.8 – 85 ms 10 – 100 ms 5 ms < 1 ms < 1 ms < 1 ms < 1 ms < 1 ms < 1 ms < 1 ms Comments Ratiometric (ΔR/R) Ratiometric (ΔR/R) Protein Ratiometric (ΔR/R) Protein Protein Protein Protein Dye Dye Dye Dye Dye hybrid hybrid Arch D95N forward primer Arch D95N reverse primer Arch to pet28 forward primer Arch to pet28 reverse primer pet28 sequence primer FCK sequence primer TTATGCCAGGTACGCCAACTGGC TGTTTACCAC GTGGTAAACAGCCAGTTGGCGTA CCTGGCATAA GATTAGCCATGGACCCCATCGCTC TG CATATCGAATTCGCCTTGTACAGC TC TAA TAC GAC TCA CTA TAG GG GCTCGTCAATCAAGCTGGTTC 4 Rel. Fluorescence (A. U.) a Arch mO2 b 20 10 8 6 4 2 1 QuasAr1 QuasAr2 Arch 100 101 102 103 Illum. Intensity (W/cm2) c 2.5 Fluorescence (A.U.) 2.0 1.5 1.0 –100 –50 0 50 Voltage (mV) QuasAr2 Arch QuasAr1 d –70 mV +30 mV 50 ms 100% ∆F 0% QuasAr1 QuasAr2 200 μs 10% ∆F/F F e f Voltage (mV) 40 20 0 –20 –40 –60 0 λ V(t) V QuasAr1 Voltage 50 mV F 1s t t 10 20 Time (ms) 30 20% ∆F/F g h Voltage (mV) 40 20 0 –20 –40 –60 QuasAr2 Voltage 50 mV 1s 0 10 20 Time (ms) 30 Photocurrent (A.U.) a 1.0 b 0.8 0.6 0.4 0.2 0 350 400 450 500 550 600 650 λ exc (nm) c Peak photocurrent (nA) 2.5 2.0 1.5 1.0 CheRiff CheRiff soma ChR2 H134R ChR2 H134R soma d 1.0 Spike fidelity 0.8 0.6 Stim. intens. (mW/cm2) 230 140 65 0.4 0.2 0 0 20 40 60 80 0.5 0 10-1 100 101 102 103 Intensity (mW/cm2) Stimulation frequency (Hz) α α a CheRiff-eGFP QuasAr2 b 20% ∆F/F 50 mV CheRiff-eGFP QuasAr2 1s 50 mV 20% ∆F/F 100 ms c -3 ms 0 ms 3 ms 6 ms d ∆F/Fcell 30% 20% 10% 0 i ii + synaptic blockers 20% ∆F/F 20% ∆F/F 20 ms 100 ms e 10% ∆F/F f 20% ∆F/F * ROI ii ROI i * 0 ms 0.2 ms 0.8 ms 1 ms 1.2 ms AnkG eGFP − α Threshold intensity (mW/cm2) a 11.1 mW/cm2 b 2.5 2.0 1.5 1.0 0.5 0 ** c Time to first spike (s) 10 0 d 25 TTX 20 15 10 5 Control Frequency at onset (Hz) e Spikes per 500 ms 8 6 4 2 0 6.0 mW/cm2 1.7 mW/cm2 20% ∆F/F 10 -1 500 ms Control +TTX Ctrl TTX 0 5 10 15 Intensity (mW/cm2) 0 0 5 10 Intensity (mW/cm2) 15 0 5 10 Intensity (mW/cm2) 15 Threshold intensity (mW/cm2) f 13.3 mW/cm2 g 10 Spikes per 500 ms 10 8 0 Frequency at onset (Hz) 12 h *** Time to first spike (s) Control KCl i 25 j 6 20 15 10 8.7 mW/cm2 4 6 4 2 0 Ctrl KCl 10 -1 2 3.7 mW/cm2 20% ∆F/F 5 0 0 5 10 Intensity (mW/cm2) 15 500 ms 0 Control +KCl 5 10 15 Intensity (mW/cm2) 0 0 5 10 Intensity (mW/cm2) 15 − a eGFP eGFP QuasAr2 c 20% ∆F/F 20% ∆F/F b 2s 200 ms 20 % ∆F/F 200 ms 20% ∆F/F 20 ms β Φ Φ =Φ ( / ) α τ τ + α β − α α + + τ τ () () { } () () { } { } () () σ σ A B E E C ΔF/F( D Depolarizing end Hyperpolarizing end 10%(ΔF/F( Depolarizing end Hyperpolarizing end 10%(ΔF/F( A" B WT##QuasAr1#QuasAr2 C Normalized#absorbance# 1.0 0.8 0.6 0.4 0.2 0.0 500 *A rc h *Q ua s A r1 *Q ua s A r2 D 1.0 Excita1on# Emission# Normalized#absorbance# 0.8 0.6 0.4 0.2 0.0 500 600 700 *Q ua s A r1 *Q ua s A r2 Wavelength#(nm)# 600 700 Wavelength#(nm)# 800 λ λ A" 250 Photocurrent (pA) 200 150 100 50 0 WT QuasAr2 QuasAr1 640 nm (300 W/cm2) 488 nm (0.5 W/cm2) B" 4 Relative fluorescence 3.5 3 2.5 2 1.5 1 0.5 0 QuasAr2 D95Q D106H QuasAr1 D95H D106H λ λ Manual ROI Mask Average Image 20 % ΔF/F SNR = 73.4 σv!= 1.4 mV Fluorescence Weighted Mask 40 mV SNR = 86.4 20 % ΔF/F σv!= 1.2 mV 100 ms σ A" Fluorescence (A.U.) 4000 3500 3000 2500 1 s 2000 1500 1000 500 0 0 5 10 15 Illumination time (min) Illumination time 29 minutes 15 minutes 1 minute 20 25 30 B" Fluorescence (A.U.) C" 5.5 AP width (ms) 4.5 5 4 AP50 AP30 3.5 3 2.5 2 1.5 0 0 20 40 60 80 100 120 140 160 Time (ms) 5 10 15 20 25 Illumination time (min) 30 A$ 2.5 B$ QuasAr2 QuasAr1 Arclight A242 100% ΔF +30$mV$ !70$mV$ 23 °C C$ !70$mV$ 100% +30$mV$ +30$mV$ !70$mV$ 23 °C Fluorescence (A.U.) 1.5 0% ΔF 0% 50 ms 100% 34 °C 2.0 1.0 100% ΔF 0.5 -100 -50 0 50 0% ΔF 34 °C Voltage (mV) 200 ms 0% 10 ms D$ 10% ΔF/F 34 °C 50 mV 10 s E$ 20% ΔF/F Arclight A242 QuasAr2 50 ms 40 V (mV) 0 -40 0 -40 -80 A" Fluorescence QuasAr1 QuasAr2 4% ΔF/F B" 0.07 0.06 0.05 ΔF/F 0.04 0.03 QuasAr1 QuasAr2 Illumination 488 nm 1 W/cm2 0.02 0.01 400 ms 1 0 0.5 1 1.5 2 640 nm 300 W/cm2 488 nm Intensity (W/cm2) sdChR&eGFP+ CheRiff+ A+ B+ D+ sdChR&TS&eGFP+ C+ τ α τ τ τ A" 1s 1 nA B" Iss$ Ipk$ CheRiff ChR2 H134R ChIEF C" photocurrent (nA) 2.5 2.0 1.5 1.0 0.5 CheRiff ChR2 H134R peak steady-state τoff$ τdes$ ton$ D" ton (ms) 25 20 15 10 5 CheRiff ChR2 H134R E" τdes (ms) 500 400 300 200 100 CheRiff ChR2 H134R F" 30 25 τoff (ms) 20 15 10 5 CheRiff n.s. 0 ChIEF ChR2 H134R 0 ChIEF Vm (mV) G" CheRiff -56 -58 -60 20 pA 500 ms 640 nm 900 W/cm2 ChIEF 0 ChIEF 0 A" CheRiff' QuasAr' B" QuasAr mOr2 TS ER2 P2A CheRiff TS eGFP QuasAr mOr2 TS ER2 …NPG CheRiff TS eGFP 450" 100" Membrane resistance (MΩ) Membrane capacitane (pF) 400" 350" 300" 250" 200" 150" 100" 50" 0" Optopatch2" 900" eGFP" 90" 75" Resting potential (mV) 80" 70" 60" 50" 40" 30" 20" 10" 0" Optopatch2" eGFP" 0" 75" 715" 725" 735" 745" 755" 765" Optopatch2" eGFP" Threshold current (pA) 800" 700" 600" 500" 400" 300" 200" 100" 0" Optopatch2" eGFP" Threshold potential (mV) 710" 715" 720" 725" 730" 735" 740" Optopatch2" eGFP" Vm (mV) 2% ΔF/F -40 -50 -60 -50 -60 100 200 300 400 500 600 700 800 900 +synap'c)blockers) Time (ms) A" Δt"="1"ms" B" Eigenvector number 12 10 8 6 4 2 00 10 20 30 40 Time (ms) 50 60 C" 1 Cumulative variance 0.995 0.99 0.985 5 6 7 8 1 2 3 4 D" before filter after filter 0.98 0.975 0 10 20 30 40 50 Eigenvector number 60 10 20 30 40 Time (ms) 50 60 E" Ac+on"poten+al"delay" τ (ms) 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 F" 7 6 τoptical (ms) 5 4 3 2 1 1 G" eGFP" 2 3 4 τpatch (ms) 5 6 7 H" Δt"="0.1"ms" 0.5$mm$ A" B" Optically stimulated Synaptically coupled 25% ΔF/F 25% ΔF/F 500 ms 500 ms Threshold Intensity (mW/cm2) A" 6 5 4 3 2 1 0 TTX *** B" Time to first spike (s) 100 Control TTX 10-1 Control 10-2 0 2 4 6 8 10 Intensity (mW/cm2) 12 14 C" Frequency at onset (Hz) 80 70 60 Control TTX D" 12 10 8 Spikes 6 4 2 0 Control TTX 50 40 30 20 10 0 0 2 4 6 8 10 Intensity (mW/cm2) 12 14 0 2 4 6 8 10 12 14 Intensity (mW/cm2) − A" 20 mW/cm2 1s 1s 48 hrs 48 hrs 20 mW/cm2 1s 50% ΔF/F 1s 50% ΔF/F 150 mW/cm2 50% ΔF/F B" 150 mW/cm2 50% ΔF/F A" Membrane Resistance (GΩ) 3 2.5 2 1.5 1 0.5 0 Optopatch2 GFP B" Membrane Capacitance (pF) 25 20 15 10 5 0 Optopatch2 GFP C" Resting Potential (mV) 0 -10 -20 -30 -40 -50 -60 Optopatch2 GFP D" Threshold Current (pA) 40 30 20 10 0 E" Threshold Potential (mV) 0 -5 -10 -15 -20 -25 -30 Optopatch2 GFP Optopatch2 GFP α A Control. B Tetraethylammonium. C Lidocaine. 25.mM. 5.mM. 1.mM. 25%.ΔF/F# 25%.ΔF/F# 30.µM. 10.µM. 3.µM. 25%.ΔF/F# 0.mM. 1.s. 0.µM. 1.s. 1.s. A" DAPI hNuc GFP Overlay B" GFP hNuc Overlay A n.s. 80 70 60 50 40 30 20 10 0 B n.s. 1600 1400 1200 1000 800 600 400 200 0 Peak photocurrent (pA) n.s. C Steady-state photocurrent (pA) n.s. 700 600 500 400 300 200 100 0 n.s. n.s. On-cell GFP intensity (A.U.) Ctrl TTX Ctrl KCl Ctrl TTX Ctrl KCl Ctrl TTX Ctrl KCl ROI 1 1 ROI 2 3 2 ROI 3 10% ΔF/F 200 ms A" B" 10% ΔF/F 10% ΔF/F mW/cm2 10 0 200 ms! 200#ms# 5% ΔF/F C" 5% ΔF/F D" 2s E" 20 ms 40 30 20 10 0 QuasAr2 ArcLight 40 V (mV) 0 V (mV) 40 0 -40 -80 -40 -80 ΔF/F (%) SNR Protein Name Arch Arch D95N QuasAr1 QuasAr2 Arch D95H/D106H Arch D95H/D106H/P60S Arch D95H/D106H/F161V Quantum yield N/A* 4×10 8×10 4×10 -4 -3 -3 Quantum yield relative to Arch D95N N/A* 1 19 10 4.2 12 13 2×10-3 5×10-3 5×10-3 Mutant Brightness (λexc = 640 nm) τup (ms, -70 mV to +30 mV) τdown (ms, +30 mV to -70 mV) Sensitivity (ΔF/F per 100 mV) 0.7 W/ cm2 23 °C Arch(WT) QuasAr1 QuasAr2 Arclight A242 34 °C QuasAr2 Arclight A242 1 15.2 3.4 800 W/cm2 τ1 τ2 % τ1 τ1 τ2 % τ1 4.0 10.3 3.4 0.6 0.05 1.2 17.4 NA 3.2 11.8 123 NA 94% 68% 39% 0.25 0.07 1.0 68 1.9 1.9 15.9 121 67% 88% 80% 24% 40% 32% 90% -32% 0.3 12 3.2 72 62% 78% 0.3 21.5 4.0 NA 73% 100% Mutant Trafficking Blue photocurrent (pA; peak, 0.5 W/cm2) Red photocurrent (pA; 640 nm, 300 W/cm2) toff (ms) sdChR-eGFP sdChR-TS-eGFP × 2470±170 ! 38±4 × 26±2.9 × ! CheRiff (sdChR-E154A-TSeGFP) ! 2030 ± 100 ! 10.5±2.8 ! 16±0.5 ! ChR variant Imax (nA; 488 nm, 0.5 W/cm2) Peak Steady state 1.33±0.08 0.81±0.10 0.65±0.09 ton (ms) τdes (ms) τoff (ms) EPD50 (mW/cm2) Red photocurrent (pA; 640 nm, 300 W/cm2) Red light depolarization (mV), 300 W/cm2 CheRiff ChIEF ChR2 H134R 2.0±0.1 0.9±0.1 1.1±0.1 4.5±0.3 18±1.8 9.1±0.7 400±40 51±10 40±5 16±0.8 15±2 25±4 22±4 10.5 ± 2.8 15.0 ± 2.5 2.3 ± 0.3 2.1 ± 0.15 1.0 43±4 2.2 ± 0.9 Peak CheRiff photocurrent (pA) Fluorescence QuasAr1 QuasAr2 0.017 1 Blue (500 mW/cm2) Red (300 W/cm2) 2030±100 10.5±2.8 0.02 3 5