optical neurophysiology in freely moving c....
TRANSCRIPT
Optical neurophysiology in freely moving C. elegans
Ce Neuro ConferenceUniversity of Wisconsin, Madison9 July 2014
Andrew LeiferLewis-Sigler Institutehttp://leiferlab.princeton.edu
Thursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014mec-4::ChR2, rig-3::GCaMP3::sl2::mCherryThursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014
• Measure Behavior
mec-4::ChR2, rig-3::GCaMP3::sl2::mCherryThursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014
• Measure Behavior
• Record calcium activity
mec-4::ChR2, rig-3::GCaMP3::sl2::mCherryThursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014
• Measure Behavior
• Record calcium activity
• Optogenetically stimulate
mec-4::ChR2, rig-3::GCaMP3::sl2::mCherryThursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014
• Measure Behavior
• Record calcium activity
• Optogenetically stimulate
• In an awake behaving animal
mec-4::ChR2, rig-3::GCaMP3::sl2::mCherryThursday, July 10, 14
Why study neural activity in free moving worms?
Thursday, July 10, 14
Why study neural activity in free moving worms?
• Working in a behaving animal is the only way to directly probe neural coding of behavior
Thursday, July 10, 14
Why study neural activity in free moving worms?
• Working in a behaving animal is the only way to directly probe neural coding of behavior
• Genetic or laser ablation studies lack insights into neural dynamics
Thursday, July 10, 14
Why study neural activity in free moving worms?
• Working in a behaving animal is the only way to directly probe neural coding of behavior
• Genetic or laser ablation studies lack insights into neural dynamics
• .. and the tools are finally available
Thursday, July 10, 14
Outline• Give a broad overview of existing methods
in freely moving worms for
• Optogenetics
• Calcium imaging
• Discuss practical matters for adopting these techniques in your lab
• Thoughts about the future
Note: no discussion of scientific results
Thursday, July 10, 14
Optical neurophysiology in moving worms requires real-time tracking
Clark et al, J. of Neuroscience 2007.
Chameleon in AFD
• At a minimum feedback is needed to control a stage to keep the worm in the field of view
• A human can provide feedback: steady hands and patience
Thursday, July 10, 14
Computer vision software can track the worm and control stage position
Leifer et al, Nature Methods 2012.
• Real-time computer vision software based on worm outline.
• First implemented by Ben Arous et al., 2010; (Schafer lab)
• Same strategy can be used for tracking or for generating targeted illumination patterns
Thursday, July 10, 14
Computer vision software can track the worm and control stage position
Leifer et al, Nature Methods 2012.
• Real-time computer vision software based on worm outline.
• First implemented by Ben Arous et al., 2010; (Schafer lab)
• Same strategy can be used for tracking or for generating targeted illumination patterns
Thursday, July 10, 14
Leifer et al, Nature Methods 2012.
Code: http://git.io/colbert
Computer vision software can track the worm and control stage position
• Real-time computer vision software based on worm outline.
• First implemented by Ben Arous et al., 2010; (Schafer lab)
• Same strategy can be used for tracking or for generating targeted illumination patterns
Thursday, July 10, 14
Leifer et al, Nature Methods 2012.
Code: http://git.io/colbert
Computer vision software can track the worm and control stage position
• Real-time computer vision software based on worm outline.
• First implemented by Ben Arous et al., 2010; (Schafer lab)
• Same strategy can be used for tracking or for generating targeted illumination patterns
Thursday, July 10, 14
Computer vision based feedback keeps the worm centered over a high magnification objective
Sped-up 10x
10x
40x
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Choosing a real-time tracking methods
Useful Review: Husson et al., “Keeping Track of Trackers,” WormBook, 2012..
Quad PMT
Image processing of bright or darkfield
imagesAnalog fluorescence Image processing of
fluorescence images Wide-field
Pro
• Most widely adopted• Can track any point• Worm body is bright• Open source software solutions
• Commercial system available (ASI)• Tracks neurons directly
• Tracks neurons directly• Has advantages for targeted illumination
• No tracking at all because worms never leave the field of view• Multiple worms simultaneously
Con• Neural locations are only inferred from worm outline
• Poorly suited for targeted illumination
• No open source software• No commercial system
• Incompatible with targeted illumination
Ref• Ben Arous et al., 2010• Leifer et al., 2011• Stirman et al., 2011
• Faumont et al., 2011 • Kowano et al., 2011• Kocabas et al., 2012 • Larsch et al., 2013
Larsch et al., 2013
Thursday, July 10, 14
Example of freely moving calcium imaging setup
Excitation
Emission
Dark Field
X-Y Motorized Stage
IR Mirror
Long Pass
High MagnifcationCalcium Imaging
Low MagnificationTracking & Behavior
Telescope
Objective
IR Illuminator
Spinning Disk
445 nm
Lasers
561 nm
USBCamera
EMCCDCamera
20x
mCherryGCaMP3
Leifer (thesis) 2012; Leifer & Clark, in prepThursday, July 10, 14
Simultaneously measure calcium
transients and behavior
GCaMP3 mCherry Behavior
Stimulus
Leifer & Clark, in prep
Calcium imaging in freely moving worm
Thursday, July 10, 14
Common challenges with calcium imaging in freely moving worms
Challenge Strategy Solution
Keeping up with the target
Optimize for automated tracking Large Field of View (Low magnification)
Calcium signal is
weak and noisy
Increase signal Use brightest indicators like GCaMP5k or GCaMP6s
Calcium signal is
weak and noisy
Collect more photons
Use high NA objectives (high magnification)Calcium
signal is weak and
noisyDetect more
photons
Use high sensitivity cameraCCD: Andor iXon or Photometrics EvolveCMOS: Hamamatsu Orca Flash or Andor
Zyla
Calcium signal is
weak and noisy
Eliminate background fluorescence
• Agarose not agar• Spinning disk confocal
Motion Artifacts Obscure
Signal
Use fiducial references
co-express mCherry(or consider true ratiometric indicators)
How to validate signal?
Use controls Record from GFP instead of GCaMP and ensure your signal is flat
Useful reviews: Kerr, Wormbook 2006; Chung et al., 2013;Thursday, July 10, 14
Common challenges with calcium imaging in freely moving worms
Challenge Strategy Solution
Keeping up with the target
Optimize for automated tracking Large Field of View (Low magnification)
Calcium signal is
weak and noisy
Increase signal Use brightest indicators like GCaMP5k or GCaMP6s
Calcium signal is
weak and noisy
Collect more photons
Use high NA objectives (high magnification)Calcium
signal is weak and
noisyDetect more
photons
Use high sensitivity cameraCCD: Andor iXon or Photometrics EvolveCMOS: Hamamatsu Orca Flash or Andor
Zyla
Calcium signal is
weak and noisy
Eliminate background fluorescence
• Agarose not agar• Spinning disk confocal
Motion Artifacts Obscure
Signal
Use fiducial references
co-express mCherry(or consider true ratiometric indicators)
How to validate signal?
Use controls Record from GFP instead of GCaMP and ensure your signal is flat
Useful reviews: Kerr, Wormbook 2006; Chung et al., 2013;
xy-motion artifacts
time
ΔF/F
0
Thursday, July 10, 14
Common challenges with calcium imaging in freely moving worms
Challenge Strategy Solution
Keeping up with the target
Optimize for automated tracking Large Field of View (Low magnification)
Calcium signal is
weak and noisy
Increase signal Use brightest indicators like GCaMP5k or GCaMP6s
Calcium signal is
weak and noisy
Collect more photons
Use high NA objectives (high magnification)Calcium
signal is weak and
noisyDetect more
photons
Use high sensitivity cameraCCD: Andor iXon or Photometrics EvolveCMOS: Hamamatsu Orca Flash or Andor
Zyla
Calcium signal is
weak and noisy
Eliminate background fluorescence
• Agarose not agar• Spinning disk confocal
Motion Artifacts Obscure
Signal
Use fiducial references
co-express mCherry(or consider true ratiometric indicators)
How to validate signal?
Use controls Record from GFP instead of GCaMP and ensure your signal is flat
Useful reviews: Kerr, Wormbook 2006; Chung et al., 2013;
40x
z-motion artifacts
xy-motion artifacts
time
ΔF/F
0
Thursday, July 10, 14
Common challenges with calcium imaging in freely moving worms
Challenge Strategy Solution
Keeping up with the target
Optimize for automated tracking Large Field of View (Low magnification)
Calcium signal is
weak and noisy
Increase signal Use brightest indicators like GCaMP5k or GCaMP6s
Calcium signal is
weak and noisy
Collect more photons
Use high NA objectives (high magnification)Calcium
signal is weak and
noisyDetect more
photons
Use high sensitivity cameraCCD: Andor iXon or Photometrics EvolveCMOS: Hamamatsu Orca Flash or Andor
Zyla
Calcium signal is
weak and noisy
Eliminate background fluorescence
• Agarose not agar• Spinning disk confocal
Motion Artifacts Obscure
Signal
Use fiducial references
co-express mCherry(or consider true ratiometric indicators)
How to validate signal?
Use controls Record from GFP instead of GCaMP and ensure your signal is flat
Useful reviews: Kerr, Wormbook 2006; Chung et al., 2013;
40xtime
ΔF/F
0
z-motion artifacts
xy-motion artifacts
time
ΔF/F
0
Thursday, July 10, 14
Optogenetic manipulation of behavior in the moving worm
• First optogenetic manipulations of behavior by ChR2 in any animal occurred in free moving C. elegans (Nagel et al., 2005)
• There is an ever-growing toolbox of optogenetic proteins tested in worms
• Previously, ability to target individual neurons was limited by genetic promotor
• Targeted illumination systems first in immobilized worms ( Guo et al., 2009 ) and now in moving worms can provide single cell specificity
Nagel et al, 2005
Halorhodopsin
yellow-green light
Channelrhodopsin-2
blue light
Thursday, July 10, 14
Optogenetic manipulation of behavior in the moving worm
• First optogenetic manipulations of behavior by ChR2 in any animal occurred in free moving C. elegans (Nagel et al., 2005)
• There is an ever-growing toolbox of optogenetic proteins tested in worms
• Previously, ability to target individual neurons was limited by genetic promotor
• Targeted illumination systems first in immobilized worms ( Guo et al., 2009 ) and now in moving worms can provide single cell specificity
Nagel et al, 2005
Review: Husson et al., 2013
Halorhodopsin
yellow-green light
Channelrhodopsin-2
blue light
Thursday, July 10, 14
Optogenetic manipulation of behavior in the moving worm
• First optogenetic manipulations of behavior by ChR2 in any animal occurred in free moving C. elegans (Nagel et al., 2005)
• There is an ever-growing toolbox of optogenetic proteins tested in worms
• Previously, ability to target individual neurons was limited by genetic promotor
• Targeted illumination systems first in immobilized worms ( Guo et al., 2009 ) and now in moving worms can provide single cell specificity
Nagel et al, 2005
Review: Husson et al., 2013
Halorhodopsin
yellow-green light
Channelrhodopsin-2
blue light
Thursday, July 10, 14
Optogenetic manipulation of behavior in the moving worm
• First optogenetic manipulations of behavior by ChR2 in any animal occurred in free moving C. elegans (Nagel et al., 2005)
• There is an ever-growing toolbox of optogenetic proteins tested in worms
• Previously, ability to target individual neurons was limited by genetic promotor
• Targeted illumination systems first in immobilized worms ( Guo et al., 2009 ) and now in moving worms can provide single cell specificity
Nagel et al, 2005
Review: Husson et al., 2013
Halorhodopsin
yellow-green light
Channelrhodopsin-2
blue light
Thursday, July 10, 14
Optogenetic manipulation of behavior in the moving worm
• First optogenetic manipulations of behavior by ChR2 in any animal occurred in free moving C. elegans (Nagel et al., 2005)
• There is an ever-growing toolbox of optogenetic proteins tested in worms
• Previously, ability to target individual neurons was limited by genetic promotor
• Targeted illumination systems first in immobilized worms ( Guo et al., 2009 ) and now in moving worms can provide single cell specificity
Nagel et al, 2005
Review: Husson et al., 2013
Leifer et al., 2011mec-4::ChR2
ChR2 in touch neurons
Halorhodopsin
yellow-green light
Channelrhodopsin-2
blue light
Thursday, July 10, 14
Targeted illumination systems in freely moving worm
• One of two systems developed independently (Leifer et al, 2011; Stirman et al 2011)
• 80 Hz
• Round trip latency of 28 ms
CoLBeRT: Controlling Locomotion and Behavior in Real-Time
700,000 mirrors
0.55 inches
Leifer et al, Nature Methods, 2011
Thursday, July 10, 14
Targeted illumination systems in freely moving worm
• One of two systems developed independently (Leifer et al, 2011; Stirman et al 2011)
• 80 Hz
• Round trip latency of 28 ms
CoLBeRT: Controlling Locomotion and Behavior in Real-Time
700,000 mirrors
0.55 inches
Leifer et al, Nature Methods, 2011
Digital micromirror device Outline-based targeting
Thursday, July 10, 14
Targeted illumination systems in freely moving worm
• One of two systems developed independently (Leifer et al, 2011; Stirman et al 2011)
• 80 Hz
• Round trip latency of 28 ms
CoLBeRT: Controlling Locomotion and Behavior in Real-Time
700,000 mirrors
0.55 inches
Leifer et al, Nature Methods, 2011
Digital micromirror device Outline-based targeting Fluorescence targetingKocabas et al., 2012
Thursday, July 10, 14
Targeted illumination systems in freely moving worm
• One of two systems developed independently (Leifer et al, 2011; Stirman et al 2011)
• 80 Hz
• Round trip latency of 28 ms
CoLBeRT: Controlling Locomotion and Behavior in Real-Time
700,000 mirrors
0.55 inches
Leifer et al, Nature Methods, 2011
Digital micromirror device Outline-based targeting Fluorescence targetingKocabas et al., 2012
Thursday, July 10, 14
HeadTail
CoLBeRT has anterior-posterior accuracy
Head
Tail
Ventral
Dorsal
Leifer et al, Nature Methods, 2011
n=14 worms
~30 micron reproducibility
CoLBeRT can reproducibly target a single motor neuron
Pegl-6::ChR2::YFPGift of N. Ringstad
Channelrhodopsin in egg-laying motorneuron
Thursday, July 10, 14
Patterned illumination systems
• Projector is well documented and cost effective but latency can be problematic
• Commercial systems will require software development
Vendor / ModelSoftware for real-
time C. elegans targeting
References
Off the Shelf
ProjectorHitachi
open source(LabView)
Stirman et al., 2011Stirman et al., 2012
Build from components
open source (C)http://git.io/colbert
Leifer et al., 2011
Pre-built none publicly availableKocabas et al, 2012;
N/AN/A
Thursday, July 10, 14
Shipley et al., Front Neural Circuits, 2014mec-4::ChR2, rig-3::GCaMP3::sl2::mCherry
Combining calcium imaging and optogenetics in the moving worm
Thursday, July 10, 14
AVA activates during reversals in response to touch stimuli
Shipley et al, Front Neural Circuits, 2014
WormX-Y Motorized Stage
Objective
IR Light
Dichroic
Digital Micromirror Device
10x
Spatio-temporally Patterned Illumination
473 nm
561 nm
siCMOSDichroic
Fluorescence Imaging
Behavior Imaging
Hi-SpeedCMOS
b.
a.
v
a
d
pAVM
ALMAVA
GCaMP3 & mCherry ChR2
Thursday, July 10, 14
�ǻR
/ R
)A
VA
Act
ivity
0
-10 0 10 20 30
0
0.5
1
1.5
Wav
e V
eloc
ity(b
ody
leng
ths
* s
-1)
Time (s)-10 10
-0.5
0
0.5
0 20 30
b.v
AVA activates during reversals in response to touch stimuli
Shipley et al, Front Neural Circuits, 2014
WormX-Y Motorized Stage
Objective
IR Light
Dichroic
Digital Micromirror Device
10x
Spatio-temporally Patterned Illumination
473 nm
561 nm
siCMOSDichroic
Fluorescence Imaging
Behavior Imaging
Hi-SpeedCMOS
b.
a.
v
a
d
pAVM
ALMAVA
GCaMP3 & mCherry ChR2
Thursday, July 10, 14
Time (s)
ǻR
/ R
0
Velocity (w
orm lengths / s)
1
0
-1
0
-15 0 15 30
0
5
10
15
20
VelocityCalcium ActivityCalcium Fit
Supplementary Figure 1
�ǻR
/ R
)A
VA
Act
ivity
0
-10 0 10 20 30
0
0.5
1
1.5
Wav
e V
eloc
ity(b
ody
leng
ths
* s
-1)
Time (s)-10 10
-0.5
0
0.5
0 20 30
b.v
AVA activates during reversals in response to touch stimuli
Shipley et al, Front Neural Circuits, 2014
WormX-Y Motorized Stage
Objective
IR Light
Dichroic
Digital Micromirror Device
10x
Spatio-temporally Patterned Illumination
473 nm
561 nm
siCMOSDichroic
Fluorescence Imaging
Behavior Imaging
Hi-SpeedCMOS
b.
a.
v
a
d
pAVM
ALMAVA
GCaMP3 & mCherry ChR2
Thursday, July 10, 14
Time (s)
ǻR
/ R
0
Velocity (w
orm lengths / s)
1
0
-1
0
-15 0 15 30
0
5
10
15
20
VelocityCalcium ActivityCalcium Fit
Supplementary Figure 1
�ǻR
/ R
)A
VA
Act
ivity
0
-10 0 10 20 30
0
0.5
1
1.5
Wav
e V
eloc
ity(b
ody
leng
ths
* s
-1)
Time (s)-10 10
-0.5
0
0.5
0 20 30
b.v
�ǻ)
R /
RA
ppar
ent A
VA
Act
ivity
0
-30 -20 -10 0 10 20 30
0
0.5
1
1.5
Wav
e V
eloc
ity(b
ody
leng
ths
s-1)
-30 -20 -10 0 10 20 30-0.5
0
0.5
Time (s)
GFP Control
AVA activates during reversals in response to touch stimuli
Shipley et al, Front Neural Circuits, 2014
WormX-Y Motorized Stage
Objective
IR Light
Dichroic
Digital Micromirror Device
10x
Spatio-temporally Patterned Illumination
473 nm
561 nm
siCMOSDichroic
Fluorescence Imaging
Behavior Imaging
Hi-SpeedCMOS
b.
a.
v
a
d
pAVM
ALMAVA
GCaMP3 & mCherry ChR2
Thursday, July 10, 14
Current limitations of calcium imaging & optogenetics in freely moving worms
• A few neurons at a time from worms with sparse expression
• No z-sectioning
• Opsins and indicators must be on separate promotors and separate cells
• Simple descriptions of behavior
Thursday, July 10, 14
Future directions
• Expanded optogenetic toolbox (R-GECI; voltage indicators, brighter GCaMPs etc)
• Richer behavioral descriptions (Stevens et al., 2010)
• Bringing 3D imaging (Schroedel et al., 2013) and 3D stimulation to the moving worm
“Eigenworm analysis” Posture Mode 1Posture Mode 2
Post
ure
Mod
e 3
0 200100 300Whole brain imaging in immobilized worms
Thursday, July 10, 14
Future directions
• Expanded optogenetic toolbox (R-GECI; voltage indicators, brighter GCaMPs etc)
• Richer behavioral descriptions (Stevens et al., 2010)
• Bringing 3D imaging (Schroedel et al., 2013) and 3D stimulation to the moving worm
“Eigenworm analysis” Posture Mode 1Posture Mode 2
Post
ure
Mod
e 3
0 200100 300Whole brain imaging in immobilized worms
Thursday, July 10, 14
Future directions
• Expanded optogenetic toolbox (R-GECI; voltage indicators, brighter GCaMPs etc)
• Richer behavioral descriptions (Stevens et al., 2010)
• Bringing 3D imaging (Schroedel et al., 2013) and 3D stimulation to the moving worm
“Eigenworm analysis” Posture Mode 1Posture Mode 2
Post
ure
Mod
e 3
0 200100 300Whole brain imaging in immobilized worms
Thursday, July 10, 14
Ashley Linder
George Plummer
Fred Shipley
Kevin Mizes
https://www.zotero.org/groups/CeNeuroWorkshop2014
Bibliography:
Slides will be posted at leiferlab.princeton.edu
The Leifer Lab
Chris ClarkAlkema Lab
UMass Worcester
Mark AlkemaUMass Worcester
Aravi SamuelHarvard
Chris Fang-Yen
UPenn
Collaborators
Funding:
Poster 65
Thursday, July 10, 14