Stimulated Raman Scattering Microscopy Wei Min Department of - - PowerPoint PPT Presentation

Wei Min Department of Chemistry Columbia University

Stimulated Raman Scattering Microscopy

• C. V. Raman
• A. Einstein

Stimulated Raman scattering microscopy

Raman scattering Stimulated emission

Freudiger*, Min*, … Xie. Science (2008) Min et al, Annu. Rev. Phys. Chem (2011)

Stimulated Raman scattering (SRS)

vibrational level virtual state

Pump Stokes

Ω

8 . .

10 1   

Stokes Spon Stim

n rate rate

Beating at pump– Stokes

pump Stokes

Min et al, Annu. Rev. Phys. Chem (2011)

Pump Stokes

Ω

Bose statistics of photons

If N photons occupy a given state, the transition rates into that state are proportional to (N+1).

1 1   



n n a n

Matrix element:

The more photons, the merrier!

Pump

Ω Ω

Stimulated Raman loss Stimulated Raman gain

Stokes

Light-molecule interaction

Stimulated Raman gain and Stimulated Raman loss

Freudiger*, Min*, et al, Science (2008)

SRS micro-spectroscopy

~3000 molecules

• Non-resonant background

High frequency modulation

~100ns

log(Vnoise) log( f )

Noise spectrum in frequency domain

1/f noise Shot noise

0.1 1kHz 10 100 1MHz

Label-free chemical imaging with SRS

Raman spectra 30 μm

Drug distributions in skin tissue

Freudiger*, Min*, … Xie. Science (2008)

Label-free 3D tissue imaging

skin tissue brain tissue

Mutant 1 Control Mutant 2

Label-free lipid imaging of C. elegans

SRS imaging of different mumants

Wang*, Min*, et al, Nature Methods (2011)

B0252: fibroblast/platelet-derived growth factor receptor Daf-2: insulin receptor F59F5.3: related receptor tyrosine kinases

50 100 150 200

control Daf-2 B0252.1 F59F5.3 Fold Change in SRS Intensity (%)

Coherent anti-Stokes Raman scattering (CARS)

vibrational level virtual states

Pump Stokes

Ω energy

virtual state

Non-resonant background Spectroscopy problem:

Distorted spectrum due to the interference

Detection sensitivity problem:

Limited sensitivity due to the associated noise

Microscopy problem

Imaging artifact

C-H on- resonance C-H off- resonance

CARS vs. SRS SRS CARS

CARS SRS

Parametric process (molecules left unchanged after the interaction) Energy transfer between light and matter Distorted complex spectra Suffering from laser intensity noise Identical spectra to Raman Shot noise limited sensitivity Quadratic concentration dependence Linear concentration dependence

CARS vs. SRS microscopy

Contamination from 2-p fluorescence Immune to background fluorescence Non-existence of point spread function Existence of point spread function

2 ) 3 (

 

 

) 3 (

Im  

Min et al, Annu. Rev. Phys. Chem (2011)

Fluorescent probe Molecule of interest Molecule of interest Molecule of interest Molecule of interest Molecule of interest Molecule of interest Vibrational tag

Too bulky for small bio-molecules Insufficient specificity

Bioorthogonal nonlinear vibrational imaging

Label free spectroscopic imaging

Alkyne tags Isotope labels

DNA replication RNA synthesis Lipid metabolism glucose uptake drug tracking 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

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

SRS imaging of EU for RNA synthesis

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

17-octadecynoic acid

Macrophages Worms

Wei, Hu, Shen, … and Min, Nature Methods, 2014

SRS @ 2230 cm‐1

Allylamine antifungal proved by FDA

Imaging delivery of alkyne-tagged drug

Terbinafine

Drug solution is topically applied to the ear tissue of a live mouse

Wei, Hu, Shen, … and Min, Nature Methods, 2014

Glucose Raman probe

Synthetic Scheme

3‐propargylglucose

O OH OH O OH OH O O O OH O O O O O O O O Br TFA

Fluorodeoxyglucose (18F-FDG )

Glucose metabolism

Glucose PET probe Hu, Chen, … and Min. in preparation

Incubation HeLa cells with 25 mM 3-propargylglucose for 4 hours

2003 cm-1 2129 cm-1 1655 cm-1

Amide Glucose-on Glucose-off

Imaging glucose uptake by live mammalian cells

Hu, Chen, … and Min. in preparation

Can we create different vibrational colors?

Fluorescent proteins Quantum dots

Synthetic route

alkyne cross-metathesis

Mo C Et O O O NO2 N O2N NO2 Zhang's Catalyst 5 eq. C C Si Si n-C8H17 C8H17-n O N HN AcO AcO O O

13C C

Si C8H17-n 100 eq. CCl4, 70°C O N HN HO HO O O

13C CH

TBAF, K2CO3 MeOH-H2O 2

Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)

Isotope effect to shift vibrational color

2048 cm-1 2077 cm-1 2125 cm-1 2000 cm-1 1655 cm-1 (0.1X) Merge

50 m

Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)

Simultaneous three-color chemical imaging

2000 2050 2100 2150

Raman Shift (cm-1) 21202123 2126

a

2000 cm-1 1655 cm-1

EU-13C2 EdU-13C 17-ODYA

• ff

amide

25 m

Merge

2053 cm-1 2077 cm-1 2125 cm-1 ( ) ( )

c

2000 2050 2100 2150

Raman Shift (cm-1) 2053 2077 2120

b

Chen, … Nuckolls and Min, J. Am. Chem. Soc. (2014)

• DNA replication
• RNA synthesis
• Lipid metabolism
• glucose uptake
• drug tracking
• multicolor chemical imaging
• protein synthesis
• protein degradation

Alkyne tags Isotope labels

Bioorthogonal nonlinear vibrational imaging

Stable isotopes

SRS image of d6-DMSO penetrating the human skin

Saar, … Xie. Science, 2010

SRS imaging of deuterated lipids in live CHO cells

Zhang, Slipchenko, Cheng. J Phys Chem Lett, 2011

Deuterium has been used for SRS

SRL images of d- cholesterol crystals

Alfonso-García, … Potma. J Biomed Opt, 2014

Wei, Yu, Shen, Wang and Min, PNAS, 2013

Imaging protein synthesis by metabolic incorporation

• f deuterium-labeled leucine

d10-leucine

Metabolic labeling of deuterium-labeled all essential amino acids

AA D D D D

Live Cell

Deuterium-labeled Amino Acids

Ribosome

New Protein Synthesis

Ribosome

AA D D AA D D D D D

D D D H H H H H H H H H H H H H H

me

D D D H H H H H H

Wei, Yu, Shen, Wang and Min, PNAS, 2013

Drug inhibition

Time-dependent protein synthesis

5 hr 12 hr 20 hr

10 min 1 hr 3 hr 5 hr Wei, Yu, Shen, Wang and Min, PNAS, 2013

Protein synthesis during cell differentiation

Wei, Yu, Shen, Wang and Min, PNAS, 2013

Merged image Neuron-like N2A cells

New protein Total protein

Monitoring protein synthesis in neurons

8 day neurons in CD-NBM medium + 1 μM anisomycin for 20 h 8 day neurons in CD-NBM medium 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 Isotope labels

DNA replication RNA synthesis Lipid metabolism glucose uptake drug tracking protein synthesis protein degradation

Bioorthogonal chemical imaging

The sensitivity comparison between stimulated Raman scattering microscopy and spontaneous Raman microscopy

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

The number of Pump photons spontaneously scattered into the Stokes wavelength within τ

pump pump A Raman spon

hv P A CVN S   

.

Spontaneous Raman scattering signal

pump pump A Raman spon

hv P A CVN N S         

.

Assuming 100% of photon signal collection efficiency there is no other noise source such as autofluorescence or detector noise

Sensitivity of spontaneous Raman microscopy

Shot-noise-limited S/N

The number of stimulated Raman Loss (SRL) photons experienced by the pump beam within τ

pump pump A Stokes SRL

hv P A CVN n S   

Signal size of stimulated Raman imaging

1

. .

 

Stokes Spon Stim

n rate rate

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)

• 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

Freudiger*, Min*, … Xie. Science (2008)

McCamant, D. W.; Kukura, P.; Mathies, R. A. Femtosecond Broadband Stimulated Raman: a New Approach for High- Performance Vibrational Spectroscopy. Appl. Spectrosc. 2003, 57, 1317.

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 ” “The experimentally obtained stimulated gain is estimated at 109, which is in reasonable agreement with the theoretically predicted value”

Assuming 100% of photon signal collection efficiency there is no other noise source such as detector noise

Sensitivity of SRS microscopy

Shot-noise-limited S/N

pump pump SRL

hv P Noise  

pump pump A Stokes SRL

hv P A CVN n N S         

Sensitivity comparison

Under the same Pump beam excitation and acquisition time

   

A CVN n N S N S

A Stokes Raman spon SRL

 

.

pump pump A Raman spon

hv P A CVN N S         

.

pump pump A Stokes SRL

hv P A CVN n N S         

V=0.1 femto liter, A=10-9 cm2, σ =10-29 cm2

   

10 16 .

10 10

A Stokes Raman spon SRL

N liter C n N S N S   



Stokes

n

105 1010

Concentration (M)

106 107 108 109 102 10-8 10-2 100 10-4 10-6

Number of oscillators

1 106 108 104 102

Power of Stokes beam (mW)

10-1 104 100 101 102 103

Spontaneous Raman Stimulated Raman

Sensitivity comparison

Acknowledgements

RISE program of Columbia University NIH Director's New Innovator Award MURI of Department of Defense Kavli Institute for Brain Science Blavatnik Awards for Young Scientists Alfred P. Sloan foundation Lu Wei Yihui Shen Fanghao Hu Zhixing Chen Fang Xu

• Dr. Luyuan Zhang

Xinxin Zhu

• Dr. Ya-Ting Kao

Lu Wei

• Dr. Luyuan Zhang

Zhixing Chen

• Prof. Meng Wang
• Prof. Rafael Yuste
• Prof. Colin Nuckolls
• Prof. Virginia Cornish
• Prof. Kimara Targoff

Raman subgroup Fluorescence subgroup Collaborators