Stimulated Raman Scattering Microscopy Wei Min Department of Chemistry Columbia University
Raman scattering Stimulated emission C. V. Raman A. Einstein Stimulated Raman scattering microscopy Freudiger*, Min*, … Xie . Science (2008) Min et al , Annu. Rev. Phys. Chem (2011)
Stimulated Raman scattering (SRS) virtual state rate Stokes Stokes 1 8 Stim . n 10 Pump Pump Stokes rate Spon . vibrational level Ω 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 n 1 a n n 1 element: The more photons, the merrier!
Stimulated Raman gain and Stimulated Raman loss Stimulated Stimulated Raman loss Raman gain Stokes Pump Light-molecule interaction Ω 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(V noise ) Shot noise 0 log( f ) 0.1 1kHz 10 100 1MHz ~100ns
Label-free chemical imaging with SRS Raman spectra Drug distributions in skin tissue 30 μ m Freudiger*, Min*, … Xie . Science (2008)
Label-free 3D tissue imaging skin tissue brain tissue
Label-free lipid imaging of C. elegans B0252 : fibroblast/platelet-derived growth SRS imaging of different mumants factor receptor Daf-2 : insulin receptor F59F5.3 : related receptor tyrosine kinases Control 200 Fold Change in SRS Intensity (%) 150 100 Mutant 1 50 0 control Daf-2 B0252.1 F59F5.3 Mutant 2 Wang*, Min*, et al , Nature Methods (2011)
Coherent anti-Stokes Raman scattering (CARS) virtual states Stokes Pump energy vibrational level Ω Spectroscopy problem: Distorted spectrum due to the interference Non-resonant Detection sensitivity problem: background Limited sensitivity due to the associated noise virtual state Microscopy problem Imaging artifact
CARS vs. SRS C-H on- C-H off- resonance resonance CARS SRS
CARS vs. SRS microscopy CARS SRS Parametric process (molecules left Energy transfer between light and matter unchanged after the interaction) 2 Im ( 3 ) ( 3 ) Distorted complex spectra Identical spectra to Raman 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 Min et al , Annu. Rev. Phys. Chem (2011)
Molecule of Molecule of interest interest Insufficient specificity Molecule of Molecule of Vibrational tag interest interest Molecule of Molecule of Fluorescent interest interest probe Too bulky for small bio-molecules Bioorthogonal nonlinear vibrational imaging
Label free spectroscopic imaging Alkyne tags Isotope labels DNA replication protein synthesis RNA synthesis Lipid metabolism protein degradation glucose uptake drug tracking Bioorthogonal chemical imaging
First SRS detection of alkyne 10,000 alkyne within 100 μ s 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 μ M EdU for 15 hrs Live HeLa cells incubated with 100 μ M 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 SRS @ 2230 cm ‐ 1 Drug solution is topically applied to the ear tissue of a live mouse
Wei, Hu, Shen, … and Min, Nature Methods , 2014
Glucose metabolism Glucose PET probe Fluorodeoxyglucose ( 18 F-FDG ) Glucose Raman probe Synthetic Scheme 3 ‐ propargylglucose O OH O O Br O TFA O O O OH O O O O O O OH OH OH Hu, Chen, … and Min. in preparation
Imaging glucose uptake by live mammalian cells 2003 cm -1 1655 cm -1 2129 cm -1 Glucose-on Glucose-off 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 C 8 H 17 - n O C C Si O N alkyne cross-metathesis Si NO 2 n -C 8 H 17 O 2 N 100 eq. Zhang's Catalyst CCl 4 , 70°C 5 eq. NO 2 Si C 8 H 17 - n O O 13 C CH 13 C C HN HN O N O N TBAF, K 2 CO 3 HO AcO O O MeOH-H 2 O HO AcO 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 21202123 2053 2077 2120 2126 2000 2050 2100 2150 2000 2050 2100 2150 Raman Shift (cm -1 ) Raman Shift (cm -1 ) ( ) ( ) c 2053 cm -1 2077 cm -1 2125 cm -1 Merge EU- 13 C 2 EdU- 13 C 17-ODYA 1655 cm -1 2000 cm -1 amide 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 imaging of deuterated SRL images of d- SRS image of d 6 -DMSO lipids in live CHO cells cholesterol crystals penetrating the human skin Zhang, Slipchenko, Cheng. Alfonso-García, … Potma. Saar, … Xie. Science , 2010 J Phys Chem Lett , 2011 J Biomed Opt , 2014
Imaging protein synthesis by metabolic incorporation of deuterium-labeled leucine d 10 -leucine Wei, Yu, Shen, Wang and Min, PNAS , 2013
Metabolic labeling of deuterium-labeled all essential amino acids Deuterium-labeled D D D Amino Acids D D AA AA D D D D D H H AA H H H H Ribosome D H H H H New Protein Synthesis D D D D H H D D Ribosome me H H H H H H H H Live Cell Drug inhibition Wei, Yu, Shen, Wang and Min, PNAS , 2013
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 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 μ M 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 Isotope labels DNA replication protein synthesis RNA synthesis Lipid metabolism protein degradation glucose uptake drug tracking 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 P pump S CVN spon . Raman A 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 P pump : the incident average power of the pump beam
Sensitivity of spontaneous Raman microscopy Shot-noise-limited S/N P S pump CVN A N A hv spon . Raman pump 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 P pump S n CVN SRL Stokes A A hv pump rate Stim . n 1 Stokes rate Spon .
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