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

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Kato et al. (2013) Neuron

2.  Applica4on  (in  vivo)      PopulaDon  imaging  

Figure 1Olfactory sensory neurons

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MCL

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ONL

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Labeled  glomerular  neurons  2.  Applica4on  (in  vivo)    

 Neurons  associated  with  same  network  module  (kikuta)  

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Mitral  cells  in  a  given  glomerulus  display  diverse  odorant  selec4vity  

Kikuta S. 2013 Neuron

Olfactory sensory neurons

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Odor information

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AOD Crystal

Incomming

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Deflected

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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

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1

7

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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.