1. The document discusses combining optogenetics techniques with electrophysiology recording using Andor Mosaic and Axon pCLAMP. 2. Optogenetics allows control of neuronal firing and silencing using light-sensitive ion channels inserted into neurons via genetic targeting. 3. Andor Mosaic enables precise spatial and temporal light patterning for optogenetic stimulation while Axon pCLAMP allows electrophysiology recording. 4. Combining these techniques allows controlling and monitoring neuronal activity at single cell level for studying neural circuits.

1. Electrophysiology meets Optogenetics
combining Axon pCLAMP™ with Andor Mosaic®
Mark Browne PhD,
Director, Systems Division
December 13, 2012 www.andor.com

2. Optogenetics meets Electrophysiology
Andor Mosaic ® with Axon pCLAMP™
Mark Browne PhD,
Director, Systems Division
December 13, 2012 www.andor.com

3. 1979: Francis Crick noted that a key challenge to neuroscientists is to
improve ability to control single cells among a network/population of cells.
1999: UCSD – Kuffler Lecture Series
• Crick stressed combining techniques in molecular biology and neuroscience
“Basically the argument is that to understand a complex biological system one
must be able to interfere with it both precisely and delicately, probably at all
levels, but especially at the cellular and molecular levels.”
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4. Optogenetics
Nature Methods
Method of Year 2010
Deisseroth, Boyden and others
exploit opsins
(bacterial photo-proteins)
in mammalian systems
Optogenetics comes of age
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5. Opsins are essentially optically controlled ion pumps. This means they can be
used to polarize and depolarize neurons – “fire” and “silence” – enabling
control of brain circuitry.
Optogenetic control can be paired with genetically
encoded fluorescent biosensors (GEBs). Targets
include ions, metabolites, enzyme activities,
protein conformation, membrane voltage.
© Riken Institute
Just as Francis Crick suggested we can manipulate
at molecular (DNA) and cellular level precisely and
delicately (optical patterns) and monitor response
– via optical, electrical, behavioural…
In combination, Opsins and GEBs can be used to perturb and monitor many
aspects of neuronal and glial biology, to visualize synaptic transmission and
activity-dependent signalling in vitro and in vivo.
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6. Is a neuron a linear integrator?
Soma SS ~ -70 mV
Soma SS
S=ΣD?
IF S > TAH (+40 mV)
IF S > TAH
THEN Fire (AP)
1. Neurons are the basic building blocks of the nervous system. These specialized cells are the information-processing
units of the brain responsible for receiving and transmitting information. Each part of the neuron plays a role in the
communication of information throughout the body.
2. Dendrites are treelike extensions at the beginning of a neuron. They increase surface area of the cell body and are
covered with synapses. Synapses receive information from other neurons and transmit electrical stimulation to the
soma.
3. The soma is where the signals from the dendrites are integrated and passed on. The soma and nucleus do not play an
active role in the transmission of the neural signal. Instead, they serve to maintain the cell and keep the neuron
functional.
4. The axon hillock is located at the end of the soma and controls the firing of the neuron. If the total strength of the
signal exceeds the threshold limit of the axon hillock, the structure will fire a signal (known as action potential AP)
down the axon.
5. The axon is the elongated fiber that extends from the cell body to the terminal endings and transmits the neural
signal. The larger the axon, the faster it transmits information. Some axons are covered with myelin that acts as an
insulator. Myelinated axons transmit information much faster than other neurons. Most neurons have only one axon.
6. The terminal buttons (sometimes called synaptic boutons) are located at the end of the neuron and are responsible
for sending the signal on to other neurons. At the end of the terminal button is a gap known as a synapse.
Neurotransmitters are used to carry the signal across the synapse to other neurons.
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7. Opsins and trans-membrane potential

8. Opsins - transgenic mouse
Opsin Wavelength Action Opsin systems
ChR2 470 nm Cation channel, depolarization of membrane ChAT-ChR2 transgenic mouse,
(Chlamydomonas potential - “Fire” Thy1-ChR2 transgenic mouse
channelrhodopsin-2) Lenti-ChR2 virus infected slices
VChR1 (Volvox 535-589 nm Cation channel, depolarization of membrane Thy1-VChR1 transgenic mouse
channelrhodopsin-1) potential - “Fire”
NpHR (Natronomonas 589 nm Chloride pump, hyperpolarization of Under consideration
halorhodopsin ) membrane potential – “Silence”
opto-α1AR 500 nm Light-activated Gq protein, activation of PLC Lenti-OptoGq virus under
pathway – Phospholipase C – G-coupled construction
receptors
opto-β2AR 500 nm Light-activated Gs protein, activation of AC Lenti-OptoGs virus under
pathway construction

9. Precise and delicate control needs targeted illumination
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10. MOSAIC® Patented Digital Mirror Device (DMD).
DMD developed by Texas Instruments
and is widely used in display devices.
Features for Optogenetics
Epi lamp
adapter • Simultaneous illumination of multiple
DMD head complex ROIs for FRAP and photo-
activation
Laser zoom
• Simultaneous Photo-activation/
collimator
combined with Electrophysiolgy
• Constrained illumination – limit
illumination to measurement areas
DMD WF
e.g. LED

11. MOSAIC® Spectral Performance
M osaic Spectral Throughput
45
40
35
30
Throughput %
25
20
15
10
5
0
300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)

12. MOSAIC® On Fixed Stage Microscopes

13. MOSAIC® Features
Simultaneous illumination of multiple ROIs in real time with zero delta time
Diffraction limited resolution
Spectral range (T > 10%) (365 to 800 nm).
Extinction ratio: ~1000:1
Minimum pulse width/exposure time for a single pattern = 75 µs.
System frame to frame refresh (a new pattern) is 200 µs or 5kHz frame rate.
Unlike projector-based systems Mosaic is NOT controlled by graphics technology…
Custom drive electronics delivers precise illumination timing and synchronization
Asynchronous external trigger and sync output
Custom optical interfaces for most scopes, optical fill and resolution
Multi –port optical input available for various illumination sources including:
- Lasers and laser diodes – 375, 405, 445, 488 nm
- LED e.g. XLED1 – 375-750 nm
- Mercury Lamp – 365 nm Hg line - uncaging
- Sutter DG4, EXFO X-Cite, Andor AMH-
- Others available upon request

14. Mosaic - Fields of Illumination
ZCX2 0.4 mm
04
01 3x fields of ZCX1 0.4 mm
Zoom laser
06
illumination Optics
ZCX2 0.2 mm ZCX1 0.2 mm
Power density, Р ≈ (Ps(λ)*T(λ)*M2)/(AF))
Where Ps(λ) = source power at λ
T((λ) = Mosaic throughput at λ
M = objective lens magnification
AF = Area of FOI at primary image plane
Optical power density required at specimen :
•1 to a few 10’s mW/mm2 for Optogenetics
• Mosaic delivers this with Lasers, LEDs and
lamps.
e.g. Channelrhodopsin 2 response vs power density
(Augustine lab, Duke Uni)

15. Mosaic - modes of operation
On time (ms)
1. Individual 500
Binary or Grey Mask Off time (ms)
100
Upload each and expose
Ext Trigger Start or “Bulb”
2. Sequence
Binary or Grey Masks
Upload to head
Software start (timed)
Ext Trigger (start/advance)
3. Sequence “Bulb” mode
Upload Binary sequence to head
Expose each mask for duration of Ext Trigger pulse width
Dark Mask n Dark Mask n+1 Dark
Illuminate Illuminate
4. All modes provide “Exposure” output to Gate Source
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16. Optogenetics –
Axon pCLAMP with Andor Mosaic
Case #1
o Mosaic used for photo-stimulation only.
o Detection – Axon pCLAMP Electrophysiology
o Andor Virtual camera enables Mosaic targeting
 Capture video or
 “image window copy” from 3rd party application
Case #2
o Mosaic provides imaging and photo-stimulation (MetaMorph)
o The same light source can be shared for both

17. Use Case#1 Optogenetics:
selective photo-stimulation at 1-20 cell level (granule cell is
about 8-10 um diameter) for electrophysiological recording
Selective photo-
Axon pCLAMPTM and stimulation with
AxoPatch 200B Mosaic®
Electrophysiology
recording
Courtesy Yakel Lab, NIEHS

18. What’s wrong with open field stimulation?
Open field light stimulation using
configuration from Wang et al
Multi-peak
response!!
Mosaic enables targeted stimulation Want a single
– single synaptic response. peak response
Courtesy Yakel Lab, NIEHS

19. Use Case#1 Optogenetics:
Mosaic + Axon pCLAMP
Andor iQ or
Axon AxoPatch 200B
MetaMorph +
and/or Digidata
Axon pCLAMP

20. Stimulating Action Potentials optogenetics
Wavelength encoding
Optical Excite different ion pumps
stimulus Silence with NpHR
Electrical Fire with ChRh2
response Image with Ca++ or other

21. Andor iQ + Axon pCLAMP Protocols
Target Illum- VC
Sequence – Mosaic SW or SDK TTL sequencing
Trigger– PClamp TTL
Select LED – PClamp TTL
Intensity – USB2
Optical
stimulus
Electrical
response

22. Use Case#2 Optogenetics:
selective photo-stimulation at 1-20 cell level (granule cell is
about 8-10 um diameter) for calcium imaging
Optional Axon pCLAMP Selective photo-
Electrophysiology stimulation with
recording Mosaic®
Mossy fiber giant boutons
Calcium Imaging Courtesy Yakel Lab, NIEHS
FM dye Imaging
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Aug 22

23. Use Case #2 – Photo-stimulation and Imaging
Video monitor
Mosaic
Andor iQ or MetaMorph
+ pCLAMP
Andor iXon EMCCD –
Fast and sensitive
Baseline Response
Stimulus

24. Optogenetics and Mosaic – Case #2
• Mosaic “Static” exposure mode – iQ 2.7.1 onwards
• Triggering may be Internal or External – option needed
Baseline Response
Stimulus
iQ Adapter needs
• External trigger option (IQ 2.8)
• Static mode – (IQ 2.7.1)
• Asynchronous mode (IQ 2.3 onwards)
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Aug 22

25. Optogenetics and Mosaic – Case #2
iQ Protocol example
• Shows a series of Static FRAPPA actions
• Mask is Static until the next FRAPPA action
• At the moment the static action cannot be
invoked
Static regions for imaging Baseline
Timed or Static regions for Stimulus
Static regions for imaging Response
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Aug 22

26. Andor iQ + Axon pCLAMP Protocols
Target Illum- VC
Sequence – Mosaic SW or SDK TTL sequencing
Trigger– PClamp TTL
Select LED – PClamp TTL
Intensity – USB2
Optical
stimulus
Electrical
response

27. Opsins not the only game in town - Hylighter optically active protein
Figure 1. An ionotropic glutamate receptor (top left) and a synthetic photoswitch called Figure 2. MIP image of overlayed confocal stacks HyLighter:GFP
Maleimide-Azobenzene-Glutamate (MAG; bottom) is the basis for the light-controlled with non-specific tdTomatoe. Note the cell shows widespread
HyLighter ion channel (right). Images courtesy of Dr.Harald Janovjak. (yellow) presence of HyLighter GFP in the plasma membrane, which
can be used to silence the neuron when pulse illuminated with
390nm. Pulse illumination at 500nm inactivates the K+ channel.
Hylighter is exciting because it is pulse activated and reversible.
Mosaic @ 390nm silences action potentials, @ 500 nm removes inhibition
– switching off the K+ pump.
Recorded with Axon AxoPatch 200B and pCLAMP
Janovjak et al (Nature Neuro 2009)
Figure 3. Action potentials triggered by current injections are robustly silenced when
HyLighter is activated by 390 nm light (violet bar) and no longer inhibited when
deactivated with 500 nm light (green bar).

28. SEXUAL
physiology of C. elegans
• Definition: Gubernaculum – “a schlerotic structure that aids in the extension of the male
copulatory spicules through the cloaca”.
• The spicule is the nematode version of a penis and the cloaca is the single posterior opening
which serves the reproductive and excretory tracts.
• The gubernaculum muscles control movements of the gubernaculum.
• The movements of the copulatory spicules are controlled by the protractor muscles,
which help to move the spicule into the cloaca.
http://www.wormbook.org/chapters/www_malematingbehavior/malematingbehavior.html
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29. Calcium sensor (GCaMP) combined with ChRh2
Intra-vital “opto-physiology “ in C. elegans
Transgenic nematode - ChRh2 expressed in gubernaculum and GCaMP expressed in protractor muscles
Red channel – co-expression of mCherry
Optical stimulation of ChRh2 in C. elegans gubernaculum muscles (red) modulates
calcium levels (GcaMP fluorescence emission) in the protractor muscles (blue)
Total duration 21 seconds
Courtesy Luis Rene Garcia, Texas A&M Uni
Liu Y. et al PLOS Genetics March 2011
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30. Intra-vital “opto-physiology “ in C. elegans
MetaMorph running Andor iXon EMCCD, Andor Mosaic with illumination at 470 and 560 nm,
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31. Closing remarks
Optogenetics is a powerful tool for neuro-physiology
Axon pCLAMP integrates readily with Optogenetics
Optical patterning with intensity and wavelength control provides new
possibilities in the study of neuronal circuits.
Mosaic delivers precise optical patterning and sequencing
Mosaic integrates easily with pCLAMP and MetaMorph
Andor Technology – Booth # 505
Molecular Devices/Axon Instruments – Booth # 2213
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