Resources

Microbial rhodopsin-based Voltage-Indicating Proteins

We are happy to share the constructs we developed. On this page you will find information on microscopy, expression of microbial rhodopsins, and available plasmids and available cell lines.

Microscopy

The endogenous fluorescence of microbial rhodopsins is very dim, 100 - 1,000 times dimmer than GFP; but these proteins are very photostable. The voltage sensitivity of wild-type Arch increases with increasing laser illumination intensity. Imaging on a conventional cell biology epifluorescence microscope is not likely to work. Our microscope is adapted from a system designed for single-molecule fluorescence. The key ingredients are:
  1. Inverted microscope. We have a homemade microscope, but a commercial inverted microscope should work too. For single-cell work we often image in through-the-objective TIRF or "pseudo-TIRF", i.e. glancing angle of incidence. Doing so decreases the background from autofluorescence of the imaging medium.
  2. A fairly intense laser configured for wide-field illumination. A 10 mW 632 nm HeNe is barely adequate, though a 100 mW diode at 594 or 635 is preferable. We use a 100 mW 638 nm source from CrystaLaser, part # DL638-100-O. Typical imaging intensities are between 300 - 1500 W/cm2.
  3. A high NA objective. We use either a NA 1.2 60x water immersion objective or a NA 1.45 60x oil objective.
  4. A good filter set. A Cy5 filter set appropriate for single-molecule imaging will work. The emission filter should pass approximately from 650 - 750 nm.
  5. A highly sensitive camera. We use an iXon+ 897 EMCCD from Andor for observing slow dynamics, or an iXon+ 860 EMCCD for watching single action potentials from neurons. Other manfucaturers' EMCCD cameras should work too. We also sometimes use a Hamamatsu sCMOS camera. Due to the noise properties of the sCMOS chip, it is better to demagnify the image, so photons are collected from as few pixels as possible.

Imaging voltage in eukaryotic cells

Archaerhodopsin 3 and its mutants show voltage-sensitive fluorescence in eukaryotic cells. Our current-generation best voltage indicators are QuasAr1 and QuasAr2. QuasAr1 is faster (< 50 microsecond response time) compared to QuasAr2 (~1.2 ms response time). QuasAr1 is also ~3-fold brighter than QuasAr2. But QuasAr2 shows greater voltage sensitivity (90% per 100 mV) compared to QuasAr1 (30% per 100 mV). We use QuasAr2 for most applications, except when extreme temporal precision is necessary. Neither mutant generates a photocurrent. Please remember that all of the microbial rhodopsin proteins require added retinal to function properly, although in many systems (e.g. mammalian neurons or cardiomyocytes) ambient retinal is sufficient to produce good fluorescence, and it is not strictly necessary to supplement with retinal.

Both proteins are *extremely* dim, and require high intensity red laser illumination, a high NA objective, and an EMCCD camera for detection. 

Here is an annotated list of our plasmids on Addgene:

Addgene ID
Plasmid
Gene/Insert
Vector Type
Comments
Publication
Bacterial expression
33780 pJMK001: PROPS Proteorhodopsin Optical Proton Sensor Bacterial Expression Original voltage sensor for bacteria.  Does not traffick to plasma membrane in eukaryotic cells  Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science. 2011 Jul 15;333(6040):345-8. 
58487 VV001: WT Arch-3 in pET28b Archaerhodopsin-3 (with C-terminal His tag) Bacterial Expression For purification of Arch Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin. Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):5939-44. doi: 10.1073/pnas.1215595110. Epub 2013 Mar 25. 
58488 VV020: WT Arch-3 - eGFP in pET28b Archaerhodopsin-3  Bacterial Expression For imaging Arch fluorescence in E. coli Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin. Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):5939-44. doi: 10.1073/pnas.1215595110. Epub 2013 Mar 25. 
Voltage imaging in neurons and cardiomyocytes
51629 DRH229: FCK-QuasAr1-mO2 QuasAr1-mOrange2 Mammalian Expression, Lentiviral CamKII promoter favors excitatory neurons.  mOrange2 fusion as expression marker. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51692 DRH334: FCK-QuasAr2-mO2 QuasAr2-mOrange2 Mammalian Expression, Lentiviral CamKII promoter favors excitatory neurons.  mOrange2 fusion as expression marker. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51695 DRH335: FCK-Optopatch1 Optopatch1 (Synthetic) Mammalian Expression, Lentiviral CamKII promoter favors excitatory neurons.  For single-vector co-expression of QuasAr2-mOrange2 and CheRiff-eGFP. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51694 DRH296: FCK-Optopatch2 Optopatch2 Mammalian Expression, Lentiviral CamKII promoter favors excitatory neurons.  For single-vector co-expression of QuasAr2-mOrange2 and CheRiff-eGFP. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51693 DRH313: FCK-CheRiff-eGFP CheRiff-eGFP Mammalian Expression, Lentiviral Ultra-sensitive, fast, and very well trafficking channelrhodopsin variant. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51696 DRH322: AAV-CaMKIIa-QuasAr2-mO2 QuasAr2-mOrange2  AAV AAV for QuasAr2-mOrange2 All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
51697 DRH337: AAV-hsyn-CheRiff-eGFP CheRiff-eGFP AAV AAV for CheRiff-eGFP. Ultra-sensitive, fast, and very well trafficking channelrhodopsin variant. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Nature Methods (2014) doi:10.1038/nmeth.3000 
34616 pJMK004 Arch D95N Mammalian Expression, Lentiviral Non-pumping Arch voltage indicator.  Obsolete. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods. 2011 Nov 27. doi: 10.1038/nmeth.1782. 
42168 pJMK019 CaViar Mammalian Expression, Lentiviral Fusion of Arch(D95N) to GCaMP5.  Obsolete. Cohen Lab VIP plasmids (unpublished)
Photophysics
51081 Arch(D95H)-eGFP Archaerhodopsin-3 w/ D95H point mutation fused to eGFP  Mammalian Expression, Lentiviral Ultrasensitive, but slow Arch voltage indicator.  Also functions as a light-gated sample-and-hold (i.e. a Flash Memory protein).   Screening fluorescent voltage indicators with spontaneously spiking HEK cells. PLoS One. 2013 Dec 31;8(12):e85221. doi: 10.1371/journal.pone.0085221. eCollection 2013. 
58490 VV017: Archaerhodopsin-3 w/ D95Q point mutation fused to eGFP Archaerhodopsin-3 w/ D95Q point mutation fused to eGFP  Mammalian Expression, Lentiviral Ultrasensitive, but slow Arch voltage indicator.  Also functions as a light-gated sample-and-hold (i.e. a Flash Memory protein).   Flash memory: photochemical imprinting of neuronal action potentials onto a microbial rhodopsin. J Am Chem Soc. 2014 Feb 12;136(6):2529-37. doi: 10.1021/ja411338t. Epub 2014 Jan 27. 
Stoplight channelrhodopsins
58510 VV201: ChR2(C128S)-eGFP in fubi ChR2(C128S)-eGFP Mammalian Expression, Lentiviral Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58513 VV221: sdChR(C138S)-TS-eGFP-ER in fck sdChR(C138S)-TS-eGFP-ER  Mammalian Expression, Lentiviral Parent gene of CheRiff (Scherfellia dubia rhodopsin) Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58511 VV208: sdChR(C138S,E154A)-TS-eGFP-ER in fck sdChR(C138S, E154A)-TS-eGFP-ER Mammalian Expression, Lentiviral CheRiff converted into a bistable opsin Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58523 VV228: sdChR(C138S E154A)-TS in fck sdChR(C138S, E154A)-TS  Mammalian Expression, Lentiviral No GFP, to allow GFP spectrum to be used for a different reporter Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58512 VV220: CoChR(C108S)-eGFP in fck CoChR(C108S)-eGFP  Mammalian Expression, Lentiviral Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58521 VV246: CoChR(C108S)-GCaMP6f-ER in fck CoChR(C108S)-GCaMP6f-ER  Mammalian Expression, Lentiviral Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58522 VV247: sdChR(C138S)-TS-GCaMP6f-ER in fck sdChR(C138S)-TS-GCaMP6f-ER  Mammalian Expression, Lentiviral Fusion with GCaMP6f for calcium imaging with optogenetic stimulation. Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58524 VV209: sdChR(C138S E154A)-TS-GCaMP6f-ER in fck sdChR(C138S, E154A)-TS-GCaMP6f-ER Mammalian Expression, Lentiviral CheRiff stoplight fused with GCaMP6f for calcium imaging with optogenetic stimulation. Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
Electrochromic FRET rhodopsins
59172 FCK-QuasAr2-Citrine eFRET QuasAr2-Citrine eFRET  Mammalian Expression, Lentiviral Yellow eFRET voltage indicator Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat Commun. 2014 Aug 13;5:4625. doi: 10.1038/ncomms5625. 
59173 FCK-QuasAr2-mOrange2 eFRET QuasAr2-mOrange2 eFRET  Mammalian Expression, Lentiviral Orange eFRET voltage indicator Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat Commun. 2014 Aug 13;5:4625. doi: 10.1038/ncomms5625. 
59174 FCK-QuasAr2-mRuby2 eFRET QuasAr2-mRuby2 eFRET  Mammalian Expression, Lentiviral Red eFRET voltage indicator Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat Commun. 2014 Aug 13;5:4625. doi: 10.1038/ncomms5625. 
Misc non-rhodopsin plasmids
58514 VV229: GCaMP6f in fck GCaMP6f  Mammalian Expression, Lentiviral For expressing GCaMP6f under the CamKII promoter Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58500 VV063: 1xCox8 - superecliptic pHluorin in fck 1xCox8 - Superecliptic pHluorin (Synthetic) Mammalian Expression, Lentiviral Mitochondrially targeted superecliptic pHluorin Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 
58502 VV064: 1xCox8 - ratiometric pHluorin in fck 1xCox8 - Ratiometric pHluorin (Synthetic) Mammalian Expression, Lentiviral Mitochondrially targeted ratiometric pHluorin Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020. 

 

Cell culture for PROPS

Grow E. coli to early-log phase (OD600 = 0.3 0.4) in 50 mL of LB medium in a shaking incubator at 33 C. Add inducer along with all-trans retinal (5 microM from a 20 mM stock in ethanol) and conduct further growth in the dark. Harvest cells after 3.5 hours and wash with 30 mL of minimal medium (1x M9 salts, 0.4% glucose, pH 7). Resuspend cells in 5 mL minimal medium and use immediately or store at 4 C for later use.

PROPS does not localize to the plasma membrane in eukaryotic cells.


Cell lines

We have a, E. coli cell line for simultaneous imaging of PROPS and flagellar torque. Cell culture and preparation conditions described in the Supporting Material of our paper on Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein.
Strain Genotype PROPS plasmid Antibiotic
JY29

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

JK2 Cm, Amp

We also have a line of HEK cells stably expressing NaV 1.3 and Kir 2.1. These cells generate spontaneous electrical spikes when cultured to confluence. Here are instructions on culturing spiking HEKs. See: J. Park , C.A. Werley, V. Venkatachalam, J.M. Kralj, S.D. Dib-Hajj, S.G. Waxman, A.E. Cohen, Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells, PLoS ONE, 8, e85221, 2013.

©2012 Adam E. Cohen