3/30/2017
Stimulated Raman Imaging of
Biomolecules
Anthony Sotelo
Burke Group
Chem 575
March 2nd 2017
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Fluorescence Imaging
Advantages
Specificity
nM concentration
Video Rate Imaging
Great method for protein visualization
Terai, T.; et al., Eur. J. Physiol., 2013, 465, 347
Disadvantages
Photobleaching
Perturbation small molecule
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Stimulated Raman Imaging (SRI) of Biomolecules
Bio-orthogonal Tag
Isotopologues
Native Imaging
Chlorothalonil
Glyphosate-d-2
Lipids
Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Rayleigh Raman Scattering
Incident Photon
ωo
Virtual State
Rayleigh Scattering
ωo
Virtual State is an
unobservable and
short-lived quantum
state
Ground State
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Anti-Stokes Raman Scattering
Incident Photon
ωo
Rayleigh Scattering
ω o+ ω Ω
Virtual State
Vibrational Level
Ground State
Ω
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Stokes Raman Scattering
Incident Photon
ωo
Stokes Scattering
ω o- ω Ω
Virtual State
Ω
Ground State
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Spontaneous Raman Scattering
Stokes
ω0 - ωΩ
Incident Photon ω0
Rayleigh
ω0
Antistokes
ω0 + ω Ω
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Stimulated Raman Scattering
Pump Beam ωpump
Stokes Beam ωstokes
Virtual State
Ω
Ground State
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Stimulated Raman Scattering
Pump Beam ωpump
Stokes Beam ωstokes
-No non-resident
background
-Linear concentration
dependence
-Video Rate Imaging
-Match spontaneous
Raman spectrum
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Freudiger, C.; et al., Science, 2008, 1857
Stimulated Raman Instrumentation
Tipping, W.J.; Chem. Soc. Rev., 2016, 45, 2075-2089
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Stimulated Raman Imaging of Biomolecules
Bio-orthogonal Tag
Chlorothalonil
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Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
Imaging of Fungicides in Maize Leafs
O
H3CO
N
O
OCH3
N
O
N
Chlorothalonil
2234cm-1
Azoxystrobin
2225cm-1
Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
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Bioorthogonal nitrile tag – Agrochemical uptake
O
H3CO
N
O
OCH3
N
2234cm-1
O
Chlorothalonil
N
2225cm-1
Azoxystrobin
Wavenumber cm-1
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Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
Chlorothalonil uptake in Maize Leaf
C≡N 2234cm-1
CH3 2930cm-1
Chlorothalonil
Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
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Azoxystrobin uptake in Maize Leaf
C≡N 2225cm-1
CH3 2930cm-1
O
H3CO
N
O
OCH3
N
O
N
Azoxystrobin
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Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
SRI of Isotopolgoues
Isotopologues
Glyphosate-d-2
Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
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Isotopologue-Deuterated glyphosate
2850cm-1
Formulation
Crystal
2164cm-1
Deuterated glyphosate
Mansfield, et al., Anal. Chem., 2013, 85, 5055-5063
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Isotopologues- Deuterated Amino Acids for Imaging New Proteins
Image at 2133cm-1
Wei, Lu; et al., ACS Chem.Bio., 2015, 10, 901-908
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Newly Synthesized Proteins in Liver
2133cm-1 Visualizes Deuterated Amino Acids
1655cm-1 Visualizes Proteins
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Wei, Lu; et al., ACS Chem.Bio., 2015, 10, 901-908
Newly Synthesized Proteins in Intestine
10µm
2133cm-1 Visualizes Deuterated Amino Acids
Wei, Lu; et al., ACS Chem.Bio., 2015, 10, 901-908
1655cm-1 Visualizes Proteins
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Brain Slice from dentate gyrus to show newly synthesized proteins
500µm
2133cm-1 Visualizes
Deuterated Amino Acids
2940cm-1 Images CH3
exclusively from old proteins
2845cm-1 Images CH2
exclusively from lipids
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Wei, Lu; et al., ACS Chem.Bio., 2015, 10, 901-908
Large field overlay of Newly Synthesized Proteins in Rat Brain Slice
Newly synthesized proteins
from deuterated amino acids
CH3 exclusively from old proteins
CH2 exclusively from lipids
Wei, Lu; et al., ACS Chem.Bio., 2015, 10, 901-908
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Stimulated Raman Imaging of Biomolecules
Native Imaging
Lipids
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Lu, et al., Cancer Research, 2016, 76, 3451-3462
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Hematoxylin and Eosin (H&E) Stain
Hematoxylin
Stains DNA and RNA
Stained Violet
hematoxylin
Eosin
Stains cytoplasmic
filaments and proteins
pink
Eosin
100µm
Lu, et al., Cancer Research, 2016, 76, 3451-3462
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Native SRI of Glioblastomas Show Lipid/Protein Contrast
2940 cm-1
2854 cm-1
Overlay
100µm
Proteins
Lipids
Protein + Lipids
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Lu, et al., Cancer Research, 2016, 76, 3451-3462
SRI Differentiates White from Grey Matter
500µm
Lu, et al., Cancer Research, 2016, 76, 3451-3462
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SRI can distinguish blood vessels not show by H&E stain
500µm
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Lu, et al., Cancer Research, 2016, 76, 3451-3462
Methods and Timeline of Amyotrophic Lateral Sclerosis
Electromyography (EMG)
Immunohistochemistry (IHC)
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Ex vivo Imaging of Disease Model Sciatic Nerve by Stimulated Raman
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Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Native Imaging- CH2 Lipid Vibrations ex vivo
Week 4 WT
50µm
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Native Imaging- CH2 Lipid Vibrations ex vivo
Week 4 Disease Model
50µm
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Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Monitoring Sciatic Nerve Lipids by SRI
Week 4 WT
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Week 4 Disease Model
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Monitoring Sciatic Nerve Lipids by SRI
Week 16 WT
50µm
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Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Monitoring Sciatic Nerve Lipids by SRI
Week 16 Disease Model
50µm
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Monitoring Sciatic Nerve Lipids by SRI
Week 16 - WT
Week 16 Disease Model
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Tian, F.; et al., Nat. Comm., 2016, 7, 13283
CH2 Lipid Vibrations reveal Ovoid Formation
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Direct Stimulated Raman Imaging of Mouse Nerve
1cm
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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SRI in comparison to Electromyography (EMG)
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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ALS drug monitoring by lipid ovoid formation
8 weeks
Vehicle
SODG193A
50µm
minocycline
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Minocycline slows lipid ovoid formation
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Stimulated Raman reveals early signs of myopathy in ALS models
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Tian, F.; et al., Nat. Comm., 2016, 7, 13283
Outlooks and Challenges
Nitrile
Alkyne
Deuterium
Fluorine
Azide
Challenges
200µM to mM concentration requirements
Not specific to individual lipids or amino
acids in native imaging
Outlooks
Extended Conjugation of Raman-active Tags
Isotopologue + Nitrile/Alkyne
Improvement in instrument to detect changes in intensity
Multicolor Imaging Techniques
Wang, P.; et al., Angew. Chem. Int. Ed., 2014, 53, 11787-11792
Chen, Z.; et al., J. Am. Chem. Soc., 2014, 136, 8027-8033
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Stimulated Raman Imaging of Biomolecules
No fixing or staining
Specific for Ω of interest
Video-rate Imaging
Minimal to no perturbation
Spatiotemporal Imaging
Mansfield, et al., Analytical Chemistry, 2013, 85, 5055-5063
Tian, F.; et al., Nat. Comm., 2016, 7, 13283
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Acknowledgements
PI
Prof. Martin Burke
Committee Members
Prof. Doug Mitchell
Prof. Paul Hergenrother
Burke Group (Image)
Chem 575
Adam DiCarpio
Christopher Reinhardt
Effie Zhou
Meng Wu
Martin McLaughin
Mark Ryan Gancayco
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