Stimulated Raman Scattering Microscopy

Ji-Xin Cheng Biomedical Engineering, Chemistry,

Center for Cancer Research

Purdue University

jcheng@purdue.edu

Where’s Wally?

We know the presence of target molecules, but we don’t know where are the molecules, what is their dynamics, and how they interact with others inside the cell.

Cell lysis

Homogenizing

Centrifuge

Biochemical assays HPLC, LC-MS, NMR.

Data analysis

Cell culture

Current Bioanalytical Assay

Super-resolution Fluorescence Imaging Review by Xiaowei Zhuang et al, Cell, 2010, 143: 1047

Raman spectroscopy cell-based biosensors, Sensors 2007, 7, 1343-1358

A cell is not a bag of molecules

wp

ws was=2wp-ws

wp ws 2wp-ws

W

wp c(3)

Coherent Anti-Stokes Raman Scattering (CARS)

Single-Frequency Coherent Raman Scattering

CARS: coherent anti-Stokes Raman scattering SRG: stimulated Raman gain SRL: stimulated Raman loss

W

wp wS

v = 0 v = 1

Vibrational excitation by wp - wS

SRS and CARS on the same Microscope

Spectrometer

Pump

Stokes

OM

PD1

F1

Scan DM

L

PH

F3

PMT1

F2

PMT2

F5

PD2

F4

Y

X

OM: optical modulator PD: photodiode LIA: Lock-in amplifier

Lock-in amplifier

Stokes

Pump

Stokes

Stimulated Raman gain

SRS: stimulated Raman scattering

Ploetz et al. Appl. Phys. B 2007 Freudiger et al. Science 2008 Nandakumar et al. New J. Phys. 2009 Ozeki et al. Opt Express 2009

Axon

Axon

Axon

Axon

Axon

2600 2800 30000

10

20

30

40

50

CA

RS

In

ten

sity (

a.u

.)Raman shift (cm

-1)

CARS spectrum of myelin

Biophys. J. 2005, 89: 581-591.

Myelin Sheath

Label-free Imaging of Lipid Bodies C. elegans Small Intestine Tissue

Red: SRS Green: TPEF

Developmental Cell 2013, 24: 384-399 In collaboration with Robert Farese, UCSF

CARS, Hellerer et al, PNAS 2007 SRS, Wang et al, Nat Methods 2011 SRS, Zhang et al. J Phys Chem Lett 2011, 2, 1248-53

Hyperspectral Stimulated Raman Scattering Microscopy Scanning the Fingerprint Region

ωp1 ωs ω

Ω1

Is(SRG)

Ip(SRL)

Wavelength scanning

ωp2 ωp3

Ω2 Ω3

Ozeki et al, Nat Photon 2012, 6, 844 Potma et al, Biophys J 2012, 102, 1988 Delong Zhang & Ping Wang et al, Anal Chem 2013, 85:98-106 Ping Wang et al, Angew Chem Int Ed, 2013, 52: 13042-46. Fu et al, J Phys Chem B 2013, 117, 4634−4640 Mansfield et al, J Biophotonics 2013, 6: 803

From C-H region to Fingerprint region

0 500 1000 1500 2000 2500 3000 3500

500

1000

1500

2000

2500

S

ignal [a

.u.]

Raman shift [cm-1]

C-H stretching Fingerprint Window

spectrum of cholesterol

……

……

X

Y

λ

λ

Pix

els

D C

C1 C2 Cq

Unfold

Refold

Spectral profiles

……

ST

S1T

S2T

SqT

λ

……

Channel 1

Channel 2

Channel q

*

Concentrations Data matrix

Initial estimation

Constraints

ALS optimization

Multivariate Curve Resolution (MCR) Analysis of Hyperspectral SRS Image Produces Chemical Maps

Delong Zhang et al, Anal Chem 2013, 85:98-106

TD C S E= MCR model:

Spectroscopic Imaging with Sub-micron Resolution: Chemical Histology of Intact Artery Tissue

Ping Wang et al, Angew Chem Int Ed, 2013, 52: 13042-46.

Scalar bars: 20 µm

Sterol C=C

Triglyceride (TG)

Acyl C=C

Cholesterol

1620 1650 1680 1710 1740 1770

Inte

nsity

Raman shift (cm-1)

Acyl C=C

Sterol C=C

EsterC=O

Ping Wang et al, 2014, under review

Cholesterol; fat droplets; oxidized lipid; protein

b Daf-2, L2 stage

Intestinal cells

a WT N2, L2 stage

Bar: 50 µm

Imaging Lipid Metabolism in live C.elegans by hyperspectral SRS

Hyperspectral image 1620 cm-1 to 1800 cm-1

Hyperspectral SRS image analysis

k-means Clustering

1 2 3 4

1620 1640 1660 1680 1700 1720 1740 1760 1780 1800

0

5

10

15

20

25

30

Inte

nsity (

vo

lt)

Raman shift (cm-1)

Site 1

Site 2

Site 3

Site 4 (Manully selected)

• Oxidized lipid: featured by the peak at 1680 cm-1 for aldehyde • Fat droplet: featured by the peak at 1655 cm-1 for acyl C=C and at 1745 cm-1 for ester C=O • Cholesterol-rich organelles: featured by the peak at 1669 cm-1 for sterol C=C band • Protein: featured by the broad amide I band around 1650 cm-1

1640 1680 1720 1760 18000

5

10

15

20

Inte

nsity (

Vo

lts)

Raman shift (cm-1)

Oxidized lipid

Fat droplets

Cholesterol

Protein

MCR-ALS analysis produces concentration maps

Coherent Raman Scattering, Broadband

W0 =

1 =

pw

Sw

Pump Stokes

CARS

w

SRL

SRG

Anti-Stokes

CARS: coherent anti-Stokes Raman scattering; SRG: stimulated Raman gain; SRL: stimulated Raman loss

Multiplex CARS microscopy reached ~30 ms per pixel: M. Bonn and coworkers, Biophysical Journal, 2008, 95: 4908-14 M. Cicerone and coworkers, Analytical Chemistry, 2011, 83: 2733-2739

µs Spectral Imaging of Living Systems by Parallel Excitation/Detection of SRS

ω ωs ωp1 ωp2 ωp3

Is(SRG)

Ω2 Ω3

Ω1

Ip(SRL)

Parallel excitation/detection

Purdue BME305: Lab 9 -- RLC model of cochlea

Resonant Detector for Lock-in-free SRS Imaging Slipchenko et al. J. Biophotonics 2012. DOI 10.1002/jbio.201200005, PCT filed Jan 2013

Imaging Single Cell Metabolism

Raman tags of large cross scattering

section

High-speed Raman or

SRS microscopy

C-D bond, Zhang et al, JPC Lett, 2011, 2, 1248-53 C-D bond, Wei et al, PNAS 2013, 110, 11226-31 CC bond, Wei et al, Nature Methods 2014 CC bond, Hong et al, Angew Chem Int Ed 2014

Raman spectrum of d31 palmitic fatty acid

C-D

C-H

2000 2200 2400In

tensity (

a.u

.)Wavenumber (cm

-1)

SRL images of CHO cells treated with d31 palmitic fatty acids

J Phys Chem Lett 2011, 2, 1248-53

Clinical Examination by Spectroscopic Imaging

Normal prostate (n = 19)

Benign prostatic hyperplasia (BPH, n = 10)

Prostatic intraepithelial neoplasia (PIN, n = 3)

Low-grade prostate cancer (Gleason grade 3, n = 12)

High-grade prostate cancer (Gleason grade 4/5, n = 12)

Prostate cancer metastases (n = 9)

We examined a spectrum of human prostate pathologies from a total of 64 healthy donors and prostate cancer patients

Coherent Raman

Spontaneous Raman

Speed & Spatial Resolution

Bandwidth Delong Zhang et al. Fast vibrational imaging of single cells and tissues by stimulated Raman scattering microscopy, Accounts of Chemical Research, 2014, online

Raman Spectromicroscopy: High-speed imaging and spectral analysis using ps Laser

Spectrometer

PD F1

SU DM

L

PH

F3

PMT

F2

wp

Combiner

wS

LSM SRS or pump-probe imaging

CARS imaging

Raman spectral analysis

Slipchenko et al., J. Phys. Chem. B 2009, 113: 7681-86.

SRS image of metastase

Identifying Cholesteryl Ester by Spectral Profiling

Raman spectrum of cholesteryl palmitate shows bands from 400 to 1200 cm−1 with the most intensive ones at 428, 538, 614 and 701 cm−1.

Krafft et al. Spectrochimica Acta Part A 61 (2005) 1529–1535

Raman shift (cm-1)

Inte

nsity (

a.u

.)

43

0 c

m−1

54

8 c

m−1

6

14

cm

−1

70

2 c

m−1

Prostate cancer cell/tissue data: C-H

C=C

CE Depletion Impairs Prostate Cancer Growth by Limiting Uptake of Essential Fatty Acids

Avasimibe

LDLr

Lysosome

CE-rich LD

FC

ACAT-1

Proliferation Tumor growth

ω-6 PUFA (e.g. AA)

CE in LDL

CE in LD

FC PI3K/AKT/mTOR

PTEN loss

SREBP

FC: free cholesterol; PUFA: polyunsaturated fatty acid AA: arachidonic acid

Prostate Cancer Progression

Debes and Tindall. N Engl J Med 2004, 351;15.

CE Depletion Impairs Prostate Cancer Aggressiveness

0 1 2 3 4 50

200

400

600

800

1000

1200

1400

***

***

***

Via

bili

ty (

% o

f d

ay 0

)

Time (day)

Control

Avasimibe

**

Avasimibe

PI fluorescence

G2/M

S

G1 Sub

-G1

0 400 800

150

100

50

0

Control

PI fluorescence

G2/M

S

G1

Su

b-G

1

500

400

300

200

100

0 0 400 800

Ctl Ava0

20

40

60

Mig

rate

d c

ells

***

Migration & Invasion

Avasimibe Control

Ctl Ava0

40

80

120

Inva

de

d c

ells

***

**: p < 0.005; ***: p < 0.0005 Scalar bar, 50 µm

Proliferation Cell Cycle

Veh Ava0

20

40

60

80

% K

i67

positiv

e c

ells

**

Veh Ava0

1

2

3

4

5

6

**

% T

UN

EL p

ositiv

e c

ells

0 10 20 3020

25

30

35

Bo

dy w

eig

ht (g

ram

)

Time of treatment (day)

Vehicle

Avasimibe

CE Depletion Suppresses Tumor Growth in vivo with Negligible Toxicity

*: p < 0.05; **: p < 0.005; ***: p < 0.0005 Scalar bar, 100 µm

0 5 10 15 20 25 30

2

4

6

8

10

12

14

*

**

****

****

**

Re

lative

tum

or

volu

me

Time of treatment (day)

Vehicle

Avasimibe

***

Ava Veh

Vehicle

Hea

rt

Kid

ney

Li

ver

Lun

g Sp

leen

Avasimibe

CE: Potential Target for Treatment of Aggressive Prostate Cancer

Post from American Society of Clinical Oncology

Cheng Lab: Spectroscopic Imaging & Nanomedicine

Dr. Ji-Xin Cheng (PI) Dr. Mikhail Slipchenko Dr. Ping Wang Dr. Jesse Zhang Dr. Bing Song Shuhua Yue (PhD, BME) Pu Wang (PhD, BME) Delong Zhang (PhD, Chem)

Seung-Young Lee (PhD, BME) Junjie Li (PhD, Biology) Rui Li (PhD, BME) Chien-Sheng Liao (PhD, BME) Brittani Bungart (MD PhD) Wei Wu (PhD, Visiting) Hui Jie (PhD, Phys) Evan Philipps (PhD, BME)

Bin Liu (PhD, Visiting) Yen Bui (PhD, Chem) Undergraduate students: Jien Nee Tai Forrest Oberhelman Scott Vicenzi Clara Suh Zhouyang Lou

Acknowledgements

Current funding: • NIH R21CA182608 • NIH R21EB015901 • NIH R01CA129287 • NIH R21GM10681 • NIH R01HL117990 • DOD Prostate Cancer

Program • DOD Spinal Cord Injury • Coulter Foundation • American Heart Association • State of Indiana • Purdue Research

Foundation

Collaborators: IUSOM: Michael Sturek, Xiaoming Xu, Liang Cheng, Ghassan S. Kassab, Northwestern: Stephen Miller, Washington U: Lihong Wang, Georgia Tech: Younan Xia, Cornell: Chris Xu, UC Irvine: Zhongping Chen, Eric Potma. USC: Qifa Zhou; Purdue University: Kinam Park, Ignacio Camarillo, Kimberly Buhman, Philip Low, Tere Carvajal, Chen Yang, Graham Cooks, Chang-Deng Hu, Sophie Lelievre; Julia Kirshner, Yeo Yoon, Donald Bergstrom, Garth Simpson, Kee-Hong Kim, David Umulis, Andy Weiner, Timothy Ratliff, Xiaoqi Liu, Paul Robinson, Robert Lucht.

4th Summer Workshop Label-free Spectroscopic Imaging July 10-11, 2014 @Purdue Univ, West Lafayette, IN 47907 Organizer: Ji-Xin Cheng, jcheng@purdue.edu

Prof. Ji-Xin Cheng (Purdue University), “Spectroscopic imaging: Linear versus nonlinear” Prof. Paola Borri (Cardiff School of Biosciences, United Kingdom) “CARS hyperspectral imaging of cells using a single 5fs Ti:Sa laser: Quantitative chemical analysis” Prof. Frank Wise (Cornell University), “Low-noise fiber OPO for coherent Raman scattering imaging” Prof. Stephen Wong (Houston Methodist Research Institute) “Intra-operative molecular vibrational imaging via stimulated Raman techniques” Prof. Heidi Tissenbaum (University of Massachusetts) “Label-free spectroscopic imaging of metabolism in live c. elegans” Prof. Song Hu (University of Virginia) “Photoacoustic microscopy: Auscultating cancer and ischemia” Prof. Charles Lin (Harvard Medical School) “In vivo sensing of the bone marrow microenvironment”