Coherent	  Anti-­‐Stokes	  Raman	  Scattering	  Imaging:	  A	  Basic	  Overview	  with	  an	  Emphasis	  on	  the	  Optics	  Required	  	  Coherent	   anti-­‐Stokes	   Raman	   scattering	   (CARS)	   imaging	   is	   a	   nonlinear	   optical	   technique	   that	  captures	   intrinsic	   molecular	   vibrations	   to	   create	   optical	   contrast.	   It	   offers	   submicron	   level	   spatial	  resolution	   and	   video-­‐speed	   imaging	   rate	   without	   the	   needs	   for	   extrinsic	   labels	   (e.g.	   fluorochromes).	   In	   a	  CARS	   process,	   a	   pump	   laser,	   a	   Stokes	   laser	   and	   a	   probe	   laser	   (usually	   same	   as	   the	   Pump	   laser)	   are	  involved.	  When	  the	  frequency	  difference	  (beating	  frequency)	  between	  the	  pump	  and	  the	  Stokes	  lasers	  is	  in	  resonance	  with	   the	  frequency	  of	  a	  molecular	  vibration,	  an	  enhanced	  optical	  signal	  is	  generated	  at	  the	  anti-­‐Stokes	  frequency.	  The	  major	  advantage	  of	  CARS	  is	  that	  the	  signal	  yield	  is	  much	  higher,	  typically	  six	  orders	   of	   magnitude,	   than	   the	   signal	   yield	   obtained	   through	   the	   conventional	   spontaneous	   Raman	  scattering	  process.	  By	  tuning	  the	  beating	  frequency	  to	  a	  designated	  vibrational	  mode	  of	  a	  chemical	  bond	  (e.g.	   O-­‐P-­‐O,	   H-­‐C-­‐H),	   CARS	   enables	   chemically	   selective	   imaging	   of	   targeted	   molecules.	   Several	   optical	  designs	   have	   been	   reported	   for	   CARS	   imaging.	   These	   designs	   use	   different	   wavelength	   combinations	   for	  the	   pump	   and	   Stokes	   beams,	   resulting	   in	   variations	   of	   CARS	   emission	   wavelength	   for	   the	   same	   chemical	  bond.	  Consequently,	  different	  dichroic	  mirrors	  and	  emission	  filters	  are	  required.	  In	  this	  application	  note,	  we	  will	  primarily	  focus	  on	  filter	  sets	  for	  CARS	  excitation	  of	  symmetric	  vibration	  of	  the	  H-­‐C-­‐H	  bond,	  which	  is	  the	  most	  popular	  vibrational	  mode	  used	  in	  biomedical	  imaging	  applications.	  
The	   first	   configuration	   utilizes	   a	   pair	   of	  Ti:sapphire	   lasers	   (e.g.	   5-­‐ps,	   Tsunami,	   Spectra-­‐physics)	   as	   the	   pump	   and	   the	   Stokes	   lasers.	   In	  this	   configuration,	   an	   output	   of	   700	   to	   840	   nm	  from	   one	   laser	   is	   used	   as	   the	   pump	   while	   an	  output	   of	   782	   to	   900	   nm	   from	   the	   other	   laser	  system	  is	  used	  as	  the	  Stokes.	  Both	  shortpass	  and	  longpass	  dichroic	  can	  be	  used	  depending	  on	  the	  system	   configuration.	   For	   CH2	   imaging,	   typically	  recommended	  filters	  are	  as	  follows:	  	  Dichroic	   (Fig.	   1):	   Shortpass:	   680dcspxr	   (lot	  203184)/zt700dcspxxr	   (lot	   209488);	   Longpass:	   Figure 1: Transmission diagrams of the dichroic mirrors for the first CARS
670dcxxr	   (lot	   208561)/700dcxxr	   (lot	   212919).	   configuration which utilizes a pair of Ti:sapphire lasers. For wavelengths
visible to NIR, a reflection diagram can be calculated by inversing aEmission	   Filter	   (Fig.	   2):	   ET680sp-­‐2P8	   (lot	   fromtransmission diagram assuming zero absorption.
238150),	  ET700sp-­‐2P8	  (lot	  237176),	  ET605/70m-­‐
2p	  (lot	  202293)	  	  	  Note:	  Dichroic	  mirrors	  are	  designed	  to	  be	  used	  at	  45°	  AOI	  while	  emission	  filters	  are	  designed	  to	  be	  used	   at	   0-­‐5°	   AOI.	   All	   lot	   numbers	   are	   listed	   for	  illustration	  purpose	  only.	  
For	   CARS	   imaging,	   we	   recommend	   the	  use	   of	   a	   serial	   of	   two	   emission	   filters	   before	   the	  detector	   for	   optimal	   blocking	   (theoretically	   to	  OD10)	   of	   the	   excitation	   lasers	   (Fig.	   3).	   A	  combination	   of	   a	   shortpass	   filter	   (ET680sp-­‐2P8	   or	  ET700sp-­‐2P8)	   with	   a	   bandpass	   filter	   (e.g.	  
ET605/70m-­‐2p)	   is	   recommended	   to	   serve	   this	  2: Transmission diagrams of the emission filters for the first CARS
purpose.	   ET680sp-­‐2P8	   and	   ET700sp-­‐2P8	   are	   Figure
configuration.	  
specially	   designed	   filters	   with	   an	   average	   OD8	  100

95
90
85
8075
70

Transmission (%)

65
60
55
50
45
40
3530
2520
15
10

50
400

450

500

550

600

650

203184700
750
Wavelength (nm)209488208561

800

850

900

950

1000

950

1000

212919

100

95
9085
80
75
70

Transmission (%)

65
60
55
50
45
40
3530
25
20
15
10

50
300

350

400

450

500

550

600
650
700
Wavelength (nm)238150	  237176

202293

750

800

850

900

blocking	   up	   to	   1060	   nm.	   Two-­‐photon	   filters	   are	  recommended	   over	   one-­‐photon	   filters	   as	  potential	   bandpass	   filters	   in	   combination	   with	  ET680sp-­‐2P8	   or	   ET700sp-­‐2P8.	   This	   is	   because	   of	  their	   additional	   blocking	   (an	   average	   of	   OD6)	   in	  the	   NIR	   region.	   For	   example,	   both	   ET660/30m	   (lot	  215558)	   and	   ET740/60m	   (lot	   242560)	   might	  satisfy	   the	   spectral	   requirement	   of	   certain	   CARS	  emission.	   However,	   both	   of	   them	   possess	   a	  certain	  level	  of	  spectral	  leakage	  around	  900-­‐1000	  nm	   (Fig.	   4).	   This	   leakage	   might	   allow	   a	   small	  portion	  of	  excitation	  lasers	  to	  enter	  the	  detector,	   Figure 3: OD diagrams of the emission filters for the first CARS
configuration.
resulting	   in	   a	   lower	   S/N	   ratio.	   In	   contrast,	   	  ET660/40m-­‐2p	   (lot	   211696)	   and	   ET750/50m-­‐2p	   (lot	   215534)	   are	   better	   options	   to	   cover	   similar	  bandwidths	  by	  holding	  an	  average	  of	  OD6	  blocking	  across	  NIR	  (Fig.	  5).	  Still,	  under	  certain	  circumstances,	  some	  of	  the	  one	  photon	  filters	  (e.g.	  ET731/30m,	  lot	  19731),	  which	  also	  block	  beyond	  800	  nm,	  could	  be	  utilized,	  especially	  when	  being	  used	  in	  combination	  with	  	   the	  OD8	  blocking	  shortpass	  filters.	  
9

8.5

8

7.5

7

6.5

Optical Density

65.5

5

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0300

400

500

600

700800Wavelength (nm)238150 OD

100237176 OD

900

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9009501000

202293 OD

795

6.5

90

685
80

5.5

70

565

4.560

Optical DensityTransmission (%)

75

55
50
45
40

4
3.5
3
2.5

35
30

225
1.5

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15

1

10
0.5

50600

650

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750
215558800
Wavelength (nm)211696242560

850

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0600

1000

650

215534

Figure 4: Transmission diagrams of representative one-photon and twophoton bandpass emission filters.

700

750

215558 OD

211696 OD

800
Wavelength (nm)850242560 OD

215534 OD

Figure 5: OD diagrams of representative one-photon and two-photon
bandpass emission filters.	  
The	   second	   configuration	   includes	   a	   	  frequency-­‐doubled	   Nd:YVO4	   modelocked	   laser	  (e.g.	   High-­‐Q)	   and	   an	   optical	   parametric	   oscillator	  	  (OPO).	   The	   Nd:YVO4	   laser	   delivers	   7-­‐ps,	   76-­‐MHz	  pulse	   trains	   at	   both	   532	   nm	   and	   1,064	   nm	   	  wavelengths.	   The	   532	   nm	   beam	   is	   used	   to	   pump	  the	   OPO,	   which	   generates	   two	   5-­‐ps	   tunable	  outputs	   (called	   signal	   and	   idler):	   a	   signal	   beam	  from	   670	   nm	   to	   980	   nm	   and	   an	   idler	   beam	   from	  1130	   nm	   to	   1600	   nm.	   A	   zero	   degree	   longpass	   filter	  (e.g.	   ET650lp,	   lot	   215145)	   is	   normally	   used	   right	  after	  the	  OPO	  to	  block	  the	  residual	  532	  nm	  pump	  Figure 6: Transmission diagrams of a representative longpass filter for
laser	  (Fig.	  6).	  	  blocking of the 532 nm pump laser after the OPO.
Two	   types	   of	   strategies	   have	   been	  reported	  in	  using	  these	  beams	  for	  CARS	  excitation.	  (1)	  To	  use	  the	  signal	  beam	  from	  OPO	  as	  the	  pump	  and	  use	  the	  1064	  nm	  output	  from	  the	  Nd:YVO4	  laser	  as	  	   the	  Stokes.	  (2)	  To	  use	  the	  signal	  and	  idler	  beams	  from	   OPO	   as	   the	   pump	   and	   the	   Stokes,	   respectively.	   For	   scenario	   (1),	   an	   additional	   dichroic	   beam	  	  100959085
80
75
70

Transmission (%)

65
60
55
50
45
403530
2520
15
10

50500

600

700

800
900
Wavelength (nm)215145	  1000

1100

1200

combiner	   (BCM)	   is	   needed	   to	   combine	   the	   signal	  beam	   and	   the	   1064nm	   beam.	   Depending	   on	   the	  system	   configuration,	   either	   a	   shortpass	   dichroic	  or	   a	   longpass	   dichroic	   can	   be	   used	   to	   serve	   this	  purpose.	  Bellow	  is	  our	  recommended	  filter	  set.	  
(1)	   Pump:	   Signal	   from	   OPO:	   775-­‐920	   nm,	  Stokes:	  Nd:YVO4	  modelocked	  laser	  (1064	  nm)	  BCM	   (Fig.	   7):	   T970dcspxr	   (lot	   226962),	   q1020lpxr	  (lot	  212733)	  	  Dichroic	   (Fig.	   8):	   Q770dcxxr	   (lot	   211623),	  750dcxrxt	  (lot	  204792)	  	  Figure 7: Transmission diagrams of dichroic mirrors for combination of theEmission	  Filter	  (Fig.	  8):	  ET680sp-­‐2P8	  (lot	  200185),	   pumpand Stokes beams.
ET700sp-­‐2P8	   (lot	   200101),	   ET720sp-­‐2P8	   (lot	  204561),	   ET750sp-­‐2P8	   (lot	   212882),	   ET660/40m-­‐ 	  2p	  (lot	  211696)	  	  (2)	   Pump:	   Signal	   from	   OPO:	   670	   nm	   to	   980,	  Stoke:	  Idler	  from	  OPO:	  1130	  nm	  and	  1600	   nm,	   e.g.	  	  P:	  924	  nm,	  S:	  1254	  nm	  Dichroic	  (Fig.	  9):	  	  	  Q770dcxxr	  (lot	  211623)	  	  Emission	   Filter	   (Fig.	   9):	   HQ750/210m-­‐2p	   (lot	  226179),	  +	  ET731/30m	  (lot	  219534)	  It	   is	   worth	   mentioning	   that	   HQ750/210m-­‐
2p	   is	   a	   very	   useful	   emission	   filter	   for	   CARS	   imaging	  because	   of	   its	   superior	   blocking	   capability	   of	   Figure 8: Transmission diagrams of the microscopy dichroic mirror andexcitation	  lasers	  to	  an	  average	  of	  OD6	  in	  NIR	  (Fig.	   emission filters for scenario (1) in the second configuration for CARS
imaging.
10).	   Although	   the	   transmission	   range	   of	   this	   filter	  has	   extended	   into	   the	   transmission	   range	   of	   the	   dichroic	   mirror,	   HQ750/210m-­‐2p	   mainly	   functions	   to	  	  block	  the	  excitation	  lasers	  instead	  of	  selection	  of	  designated	  emissions.	  	  
100

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Transmission (%)

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Wavelength (nm)226962

1050

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1150

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212733

100

95
9085
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75
70

Transmission (%)

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60
55
50
45
40
35
30
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20
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10

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200185600

200101

700
800
Wavelength (nm)204561212882211696900

1000

211623204792

1100

1200

7

100
95

6.5	  9085680

5.5

75
570

4.5

60

Optical Density

Transmission (%)

65
55
50
45
40

4
3.5
3
2.5

35
30

225
1.5

20
15

1

10

0.5

50300

400

500

600

700
800
Wavelength (nm)211623226179

900

1000

1100

Figure 9: Transmission diagrams of the microscopy dichroic and emission
filters for scenario (2) in the second configuration for CARS imaging.	  	  	  	  	  	  

0
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650

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750
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850
Wavelength (nm)226179 OD

219534

900

950

1000

1050

1100

1150

1200

219534 OD

Figure 10: Transmission diagrams of the microscopy dichroic andemission filters for the third system configuration.	  	  	  The	   third	   configuration	   utilizes	   a	  picosecond	  Nd:Vanadate	  laser	  pumping	  two	  OPOs.	  
Two	   of	   the	   three	   beams	   (The	   two	   signal	   beams	  from	  the	  OPOs	  and	  the	  1064	  nm	  laser)	  can	  be	  used	  as	   the	   pump	   and	   Stokes	   beams,	   enabling	   probing	  any	   molecular	   vibration	   in	   the	   important	   Raman	  fingerprint	  range	  of	  0-­‐3,400	  cm-­‐1.	  For	  CH2	  imaging,	  typically	  recommended	  filters	  are:	  Dichroic	  (Fig.	  11):	  	  	  750dcxrxt	  (lot	  204792)	  Emission	  Filter	  (Fig.	  11):	  ET680sp-­‐2P8	  (lot	  200185),	  ET700sp-­‐2P8	   (lot	   200101),	   ET720sp-­‐2P8	   (lot	  204561),	  ET580/30m-­‐2p	  (lot	  234074),	  ET605/75m-­‐
2p	  (202293)	  or	  ET660/40m-­‐2p	  (lot	  211696)	  	  100
95
90
85
80
75
70
Transmission (%)

65
55
50
45
40
35
30
25
20
15
10
50300

400

500
204792600
200185700
800
Wavelength (nm)2001012045612340749001000202293

211696

1100

1200

Figure 11: Transmission diagrams of the microscopy dichroic mirror andemission filters for scenario (3) in the second configuration for CARS
imaging.	  	  Liang	  (Alex)	  Gao,	  Ph.D.	  
Applications	  Scientist	  agao@chroma.com	  	  Michael	  Stanley,	  Ph.D.	  
Senior	  Applications	  Scientist	  mstanley@chroma.com	  	  Chroma	  Technology	  Corp.	  
10	  Imtec	  Lane	  Bellows	  Falls,	  VT,	  05101	  	  60