Course: Systems Neuroscience 1. Func4onal neuroanatomy d. Func4onal imaging techniques II (two-‐photon imaging) Shin Nagayama (MSE R438)
Goal of this lecture Understand the basic principle of two-‐photon imaging technique.
Understand how the two-‐photon imaging system has been contributed to Neuroscience.
Understand the fronDer of two-‐photon opDcal imaging technique.
1. Principle of two-‐photon microscopy
Principle Merit/Demerit
2. Applica4on (in vivo) Single cell imaging (DendriDc integraDon)
PopulaDon imaging (Different cell types) Neurons associated with same network module
3. Fron4er of New techniques Increase the speed of imaging: AOD scanning 4D imaging
Two-‐Photon microscope Two-‐photon excitaDon 1. Principle of two-‐photon microscopy
Principle
E=hc/λ
Energy of photon (E) and Wave length of the light (λ) are given by the equaDon
hc=1.99 x 10-‐25 h is Planck’s constant c is the speed of light
Two-‐Photon microscope Two-‐photon can excite probe just in the focus area
1. Principle of two-‐photon microscopy Principle
Single-‐Pho
ton
Two-‐Ph
oton
Two-‐Photon microscope
Two-‐photon excitaDon breaches fluorescence just in the focus plane
1. Principle of two-‐photon microscopy Principle
Two-‐Photon microscope Two-‐photon imaging system efficiently collect emission light
1. Principle of two-‐photon microscopy Principle
• Merit: deep layer imaging, lower photo damage, efficient photon collecDon – >> fit to in vivo funcDonal imaging
• Demerit: lower z-‐resoluDon, slow scanning speed
1. Principle of two-‐photon microscopy Merit/demerit
HB Jia et al. Nature 464, 1307-‐1312 (2010) doi:10.1038/nature08947
Visually evoked acDon potenDals, subthreshold depolarizaDons and global dendriDc calcium signals.
2. Applica4on (in vivo) Single cell imaging
(DendriDc integraDon)
Subthreshold local dendriDc calcium signals
HB Jia et al. Nature 464, 1307-‐1312 (2010) doi:10.1038/nature08947
2. Applica4on (in vivo) DendriDc integraDon
Spike associated dendriDc calcium signals
Heterogeneity and distribuDon paeern of orientaDon-‐tuned dendriDc hotspots.
HB Jia et al. Nature 464, 1307-‐1312 (2010) doi:10.1038/nature08947
2. Applica4on (in vivo) DendriDc integraDon
Kato et al. (2013) Neuron
2. Applica4on (in vivo) PopulaDon imaging
Figure 1Olfactory sensory neurons
EPL
GL
MCL
GCLIPL
Granule cell
Mitral cell
Tufted cell
PG cell
ONL
PV cell
GlomerulusSA cell
Kato et al. (2013) Neuron
2. Applica4on (in vivo) PopulaDon imaging
Figure 1Olfactory sensory neurons
EPL
GL
MCL
GCLIPL
Granule cell
Mitral cell
Tufted cell
PG cell
ONL
PV cell
GlomerulusSA cell
Labeled glomerular neurons 2. Applica4on (in vivo)
Neurons associated with same network module (kikuta)
a
c
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cd
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inter-neuronal� distance� (μm)similarity� of� the� odorant� selectivity� (%
)
0 100 200 300 4000
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n=48 pairs of mitral cells
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Lat.Ant.
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0
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0
inter-neuronal� distance� (μm)similarity� of� the� odorant� selectivity� (%
)
0 100 200 300 4000
20
40
60
80
100
p=0.49R=0.11
n=40 pairs of JG cells
inter-neuronal� distance� (μm)similarity� of� the� odorant� selectivity� (%
)
0 100 200 300 4000
20
40
60
80
100 R= -0.76p<0.001
n=48 pairs of mitral cells
Mitral cells in a given glomerulus display diverse odorant selec4vity
Kikuta S. 2013 Neuron
Olfactory sensory neurons
EPL
GL
MCL
GCLIPL
Granule cell
Mitral cell
Tufted cell
PG cell
ONL
PV cell
GlomerulusSA cell
Distance dependence of mitral cells odor selec4vity Glom.#1
Cell#2Cell#110%
9CHO
5CHO6CHO7CHO8CHO
3CHO4CHO
Cell#3-10%
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7
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Kikuta S. 2013 Neuron
Glom.
OSN
※
EPL
GL
MCL
GCL
IPL
granule cell
mitral cell
tufted cell
JG cell
ONL
Odor information
Ran
AOD Crystal
Incomming
Beam
Deflected
Beam
Acoustic wave
Acousto-‐opDc deflectors (AOD) two-‐photon imaging
3. New techniques Increase imaging speed: AOD scanning
Scanning modes 3. New techniques
Increase imaging speed: AOD scanning
Galvano-‐mirror Scanner
AOD Scanner (random access mode)
Recording conDnuously while moving the posiDon
Scanning 200 x 200 pixel filed 5 µs/pixel for recording
~1 ms/line ~200 ms/frame (~5 Hz)
Recording only from deliberately selected pixels
Scanning 40 selected pixels 5 µs/pixel + 5 µs for every movement
0.4 ms/cycle (2.5 kHz)
3. New techniques Increase imaging speed: AOD scanning
Respiration
Odor Stimulus
4
3
5
6
2
1
7
1 0
8
9
0.5 sec
50% DF/F
Thy1-‐GCaMP3 mouse 1kHz sampling rate
Odorant responses of presumed external tuked cells
near glomerular layer.
3. New techniques 4D imaging
Coeon et al, (2013) Front-‐Neural-‐Circuits
3. New techniques 4D imaging
Coeon et al, (2013) Front-‐Neural-‐Circuits
• Two-‐photon imaging system is powerful tools to understand system neuroscience.
Goal of this lecture Understand the basic principle of two-‐photon imaging technique.
Understand how the two-‐photon imaging system has been contributed to Neuroscience.
Understand the fronDer of two-‐photon imaging technique.
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