Download Stimulated Raman Scattering Microscopy

Stimulated Raman Scattering Microscopy
Wei Min
Department of Chemistry
Columbia University
Raman scattering
C. V. Raman
Stimulated emission
A. Einstein
Stimulated Raman scattering microscopy
Freudiger*, Min*, … Xie. Science (2008)
Min et al, Annu. Rev. Phys. Chem (2011)
Stimulated Raman scattering (SRS)
Ω
vibrational level
Stokes
rate Stim .
 nStokes  1  10 8
rate Spon .
Pump
Stokes
Pump
virtual state
Ω
pump
Stokes
Beating at
pump– Stokes
Min et al, Annu. Rev. Phys. Chem (2011)
Bose statistics of photons
If N photons occupy a given state, the transition rates into
that state are proportional to (N+1).
Matrix
element:
n 1 a n  n 1
The more photons, the merrier!
Stimulated Raman gain and Stimulated Raman loss
Stokes
Pump
Light-molecule
interaction
Ω
Stimulated Stimulated Raman gain Raman loss
Ω
SRS micro-spectroscopy
--- Non-resonant background
~3000 molecules
Freudiger*, Min*, et al, Science (2008)
High frequency modulation
Noise spectrum in frequency domain
1/f noise
log(Vnoise)
Shot noise
0
0.1 1kHz 10 100 1MHz
~100ns
log( f )
Label-free chemical imaging with SRS
Raman
spectra
Drug distributions in skin tissue
30 μm
Freudiger*, Min*, … Xie. Science (2008)
Label-free 3D tissue imaging
skin tissue
brain tissue
Label-free lipid imaging of C. elegans
SRS imaging of different mumants
B0252: fibroblast/platelet-derived growth
factor receptor
Daf-2: insulin receptor
F59F5.3: related receptor tyrosine kinases
Control
Fold Change in SRS Intensity (%)
Mutant 1
200
150
100
50
0
Mutant 2
control
Daf-2
B0252.1
F59F5.3
Wang*, Min*, et al, Nature Methods (2011)
Coherent anti-Stokes Raman scattering (CARS)
Stokes
energy
Pump
virtual states
Ω
vibrational level
Spectroscopy problem:
Distorted spectrum due to the interference
Non-resonant
background
Detection sensitivity problem:
Limited sensitivity due to the associated noise
virtual state
Microscopy problem
Imaging artifact
CARS vs. SRS
C-H onresonance
CARS
SRS
C-H offresonance
CARS vs. SRS microscopy
CARS
SRS
Parametric process (molecules left
unchanged after the interaction)
Distorted complex spectra
 
( 3) 2
Energy transfer between light and matter
 
Identical spectra to Raman  Im
Suffering from laser intensity noise
Shot noise limited sensitivity
Quadratic concentration dependence
Linear concentration dependence
Contamination from 2-p fluorescence
Immune to background fluorescence
Non-existence of point spread function
Existence of point spread function
( 3)
Min et al, Annu. Rev. Phys. Chem (2011)
Molecule of
interest
Insufficient specificity
Molecule of
interest
Molecule of
interest
Vibrational tag
Fluorescent
probe
Too bulky for small bio-molecules
Bioorthogonal nonlinear vibrational imaging
Label free
spectroscopic imaging
Alkyne tags
DNA replication
RNA synthesis
Lipid metabolism
glucose uptake
drug tracking
Isotope labels
protein synthesis
protein degradation
Bioorthogonal chemical imaging
First SRS detection of alkyne
10,000 alkyne within 100μs
Wei, Hu, Shen, … and Min, Nature Methods, 2014
Metabolic incorporation of alkyne-tagged
small precursor molecules
Wei, Hu, Shen, … and Min, Nature Methods, 2014
SRS imaging of EdU for DNA synthesis
Live HeLa cells incubated with 100 μM EdU for 15 hrs Live HeLa cells incubated with 100 μM EdU + 10 mM hydroxyurea
Wei, Hu, Shen, … and Min, Nature Methods, 2014
Tracking dynamics
A dividing cell during mitosis
Wei, Hu, Shen, … and Min, Nature Methods, 2014
SRS imaging of EU for RNA synthesis
Live HeLa cells incubated with 2 mM EU for 7 hrs
Live HeLa cells incubated with 2 mM EU + 200 nM Actinomycin D for 7 hrs
Wei, Hu, Shen, … and Min, Nature Methods, 2014
Tracking RNA turnover dynamics in live cells
Pulse-chase imaging of turnover dynamics of EU labeled RNA
Wei, Hu, Shen, … and Min, Nature Methods, 2014
SRS imaging of alkyne tagged choline for
phospholipid synthesis
Live neurons incubated with 0.5 mM propargyl‐
choline 24 hrs
Wei, Hu, Shen, … and Min, Nature Methods, 2014
SRS imaging of metabolic process of fatty acids
Macrophages
17-octadecynoic acid
Worms
Wei, Hu, Shen, … and Min, Nature Methods, 2014
Imaging delivery of alkyne-tagged drug
Terbinafine
Allylamine antifungal proved by FDA
Drug solution is topically applied
to the ear tissue of a live mouse
SRS @ 2230 cm‐1 Wei, Hu, Shen, … and Min, Nature Methods, 2014
Glucose metabolism
Glucose PET probe
Fluorodeoxyglucose
(18F-FDG )
Glucose Raman probe
Synthetic Scheme
3‐propargylglucose
O
O
O
O
O
O
OH
O
Br
O
OH
O
O
O
TFA
O
OH
O
OH
OH
Hu, Chen, … and Min. in preparation
Glucose-on
Glucose-off
1655 cm-1
2129 cm-1
2003 cm-1
Imaging glucose uptake by live mammalian cells
Amide
Incubation HeLa cells with 25 mM 3-propargylglucose for 4 hours
Hu, Chen, … and Min. in preparation
Fluorescent proteins
Quantum dots
Can we create different vibrational colors?
Synthetic route
Et
C
O Mo
O2 N
N
O
O
NO2
Zhang's Catalyst
5 eq.
n-C8H17 Si
C
C
Si C8H17-n
alkyne cross-metathesis
100 eq.
CCl4, 70°C
NO2
Si
O
13
C
C
O
C8H17-n
13
C
HN
HN
O
AcO
N
CH
TBAF, K2CO3
O
O
HO
N
O
MeOH-H2O
AcO
HO
2
Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)
Isotope effect to shift vibrational color
1655 cm-1 (0.1X)
2000 cm-1
2048 cm-1
2077 cm-1
2125 cm-1
Merge
50 m
Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)
Simultaneous three-color chemical imaging
a
b
2123
2120
2126
2000
2050
2100
Raman Shift (cm-1)
2053 2077
2000
2150
(
2050
2100
2150
Raman Shift (cm-1)
)
c
2120
(
2053 cm-1
2077 cm-1
)
2125 cm-1
Merge
EU-13C2
EdU-13C
1655 cm-1
amide
17-ODYA
2000 cm-1
off
25 m
Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)
Bioorthogonal nonlinear vibrational imaging
• DNA replication
• RNA synthesis
• Lipid metabolism
Alkyne tags
• glucose uptake
• drug tracking
• multicolor chemical imaging
• protein synthesis
Isotope labels
• protein degradation
Stable isotopes
Deuterium has been used for SRS
SRS image of d6-DMSO
penetrating the human skin
SRS imaging of deuterated
lipids in live CHO cells
SRL images of dcholesterol crystals
Saar, … Xie. Science, 2010
Zhang, Slipchenko, Cheng. Alfonso-García, … Potma.
J Phys Chem Lett, 2011
J Biomed Opt, 2014
Imaging protein synthesis by metabolic incorporation
of deuterium-labeled leucine
d10-leucine
Wei, Yu, Shen, Wang and Min, PNAS, 2013
Metabolic labeling of deuterium-labeled
all essential amino acids
D
Deuterium-labeled
D Amino Acids D
AA
D D
D
D
H
H
Ribosome
D
D
AA
H
H
H
D
AA
D
New Protein
Synthesis
D
D
Ribosome
me
D
H
H
H
H
H
Live Cell
Drug
inhibition
Wei, Yu, Shen, Wang and Min, PNAS, 2013
Time-dependent protein synthesis
5 hr
10 min
12 hr
1 hr
20 hr
3 hr
5 hr
Wei, Yu, Shen, Wang and Min, PNAS, 2013
Protein synthesis during cell differentiation
New protein
Total protein
Neuron-like N2A cells
Merged image
Wei, Yu, Shen, Wang and Min, PNAS, 2013
Monitoring protein synthesis in neurons
8 day neurons in CD-NBM medium for 20 h
8 day neurons in CD-NBM medium
+ 1 μM anisomycin for 20 h
What about protein degradation?
Shen, Xu, Wei, Hu and Min. Angew Chem 2014
Imaging protein degradation in live cells
Reactive Oxygen Species
Shen, Xu, Wei, Hu and Min. Angew Chem 2014
Neurodegenerative diseases:
hungtingtin aggregation
Shen, Xu, Wei, Hu and Min. Angew Chem 2014
Label free
spectroscopic imaging
Alkyne tags
DNA replication
RNA synthesis
Lipid metabolism
glucose uptake
drug tracking
Isotope labels
protein synthesis
protein degradation
Bioorthogonal chemical imaging
The sensitivity comparison between
stimulated Raman scattering microscopy
and spontaneous Raman microscopy
Spontaneous Raman scattering signal
The number of Pump photons spontaneously scattered into the
Stokes wavelength within τ
S spon. Raman  CVN A
 Ppump
A hv pump
C: the concentration of vibrational oscillator
V: the confocal detection volume
σ : the Raman scattering cross section of the vibrational oscillator
A: the area of the laser focus
τ:
the acquisition time period per pixel
Ppump: the incident average power of the pump beam
Sensitivity of spontaneous Raman microscopy
Shot-noise-limited S/N
 Ppump
S
 CVN A
 
A hv pump
 N  spon. Raman
Assuming
100% of photon signal collection efficiency
there is no other noise source such as autofluorescence or detector noise
Signal size of stimulated Raman imaging
The number of stimulated Raman Loss (SRL) photons experienced by
the pump beam within τ
S SRL  nStokes CVN A
rate Stim .
 nStokes  1
rate Spon .
 Ppump
A hv pump
Estimation of the amplification factor
The original report using 40 mW of average power of the Stokes
beam (which is a 76 MHz pulse train with 6 ps pulse width)
Freudiger*, Min*, … Xie. Science (2008)
•A 5 mM methanol solution (~ 3105 C-H bonds within the focal volume) gives
a measured SRL signal of about ΔISRS/Ip ~ 710-8.
•With a known ~ 10-29 cm2 for one C-H bond, the total spontaneous Raman
scattering cross sections of 3105 bonds will add up to a cross section of 310-24
cm2. Given a laser waist area of 10-9 cm2, one would expect to produce a relative
spontaneous Raman signal of ΔIspon.Raman/Ip = (310-24cm2)/(10-9cm2) ~ 310-15.
•Therefore, the amplification nStokes is estimated to be (710-8)/(310-15) ~ 107
Estimation of the amplification factor
“Given our typical probe photon flux of 1012
photons/cm2/s/Hz at the sample, we estimate the relative
ratio of SRS to spontaneous Raman to be ∼107 ”
McCamant, D. W.; Kukura, P.; Mathies, R. A. Femtosecond
Broadband Stimulated Raman: a New Approach for HighPerformance Vibrational Spectroscopy. Appl. Spectrosc.
2003, 57, 1317.
“The experimentally obtained stimulated gain is
estimated at 109, which is in reasonable agreement
with the theoretically predicted value”
Sensitivity of SRS microscopy
NoiseSRL 
Ppump
hv pump
Shot-noise-limited S/N
 Ppump
S
   nStokesCVN A
A hv pump
 N  SRL
Assuming
100% of photon signal collection efficiency
there is no other noise source such as detector noise
Sensitivity comparison
Under the same Pump beam excitation and acquisition time
 Ppump
S
   nStokesCVN A
A hv pump
 N  SRL
 Ppump
S

CVN
 
A
A hv pump
 N  spon. Raman
S N SRL
S N spon.Raman
S N SRL
S N spon.Raman
 nStokes CVN A

A
nStokes C  1016 liter  N A

1010
V=0.1 femto liter, A=10-9 cm2, σ =10-29 cm2
Sensitivity comparison
Power of Stokes beam (mW)
10-1
100
101
102
103
104
108
100
Stimulated
Raman
10-2
10-4
Spontaneous
Raman
106
104
102
10-6
1
10-8
105
106
107
nStokes
108
109
1010
Number of oscillators
Concentration (M)
102
Acknowledgements
Raman subgroup
Fluorescence subgroup
Lu Wei
Xinxin Zhu
Yihui Shen
Dr. Ya-Ting Kao
Fanghao Hu
Lu Wei
Zhixing Chen
Dr. Luyuan Zhang
Fang Xu
Zhixing Chen
Collaborators
Prof. Meng Wang
Prof. Rafael Yuste
Prof. Colin Nuckolls
Prof. Virginia Cornish
Prof. Kimara Targoff
Dr. Luyuan Zhang
NIH Director's New
Innovator Award
MURI of Department of Defense
Kavli Institute for Brain Science
Alfred P. Sloan foundation
Blavatnik Awards
for Young Scientists
RISE program of
Columbia University