350 OPTICS LETTERS / Vol. 7, No.

8 / August 1982

Scanning coherent anti-Stokes Raman microscope

M. D. Duncan, J. Reintjes, and T. J. Manuccia

Received April 26,1982

We have constructed a spatially scanning coherent anti-Stokes Raman spectroscopic (CARS)apparatus that allows
us to image the distribution of distinct chemical species in a microscopic sample region. Images of onion-skin cells
have been obtained by using the CARS signal produced by the 2450-cm-l band of deuterated water. Future appli-
cations will be discussed.

Those working in the fields of biology and applied mi- where X is a nonlinear susceptibility, Ip and I, are the
croscopy have shown substantial interest in having the intensities of the incident pump and Stokes waves, re-
ability to determine the spatial distribution of specific spectively, K is a factor containing the molecular
molecular species in samples on a cellular or subcellular number density and constants, and the integral is over
scale (<1 iAm). Excellent discrimination among distinct the area of overlap of Ip and S. In general, for any
chemical species can be obtained by using infrared ra- average power level of Pp and PS, more signal is pro-
diation tuned to characteristic vibrational frequencies, duced with laser radiation that has a large peak-to-
but the spatial resolution, limited to a few micrometers average-power ratio, such as that produced by pulsed
by the wavelength of the infrared light, is generally in- or cw mode-locked lasers. The useful peak intensity
sufficient to resolve cells or smaller structures. Better is normally limited in biological samples by damage
spatial resolution of chemical species can be obtained mechanisms from a single pulse, such as dielectric
by using stains or fluorescing agents illuminated by breakdown for picosecond pulses or thermally induced
visible or ultraviolet light, but these techniques are damage for longer pulses. Intensities of the order of
limited to certain materials, require elaborate prepa- 1010 W/cm 2 can generally be tolerated with visible-
ration, and introduce foreign agents into the sample. wavelength picosecond pulses. The average power is
Here we describe a technique that permits both mo- limited by bulk sample heating and is affected by
lecular specificity and high spatial resolution by using sample absorption, heat capacity, and thermal con-
coherent anti-Stokes Raman spectroscopy (CARS)with ductivity.
visible lasers. Specificity is achieved by using Raman Our experimental apparatus was designed to take full
scattering from characteristic molecular vibrations, and advantage of the nonlinear properties of the CARS
high resolution is obtained by imaging the visible anti- process. It contains two synchronously pumped cw
Stokes signal. Images have been obtained from a va- mode-locked dye lasers, as shown in Fig. 1. One dye
riety of pure organic liquids and from deuterated water laser is operated with rhodamine 6G dye and provides
in onion-skin cells. This technique has the potential the pump wavelength for the CARS interaction. It is
of permitting the observation of the uptake, metabo-
lism, and ultimate distribution of nutrients, drugs, or
toxins on a cellular or subcellular basis in living organ-
isms.
Previous work in Raman microscopy has mainly in-
volved spontaneous Raman scattering'- 3 and has been
characterized by weak signals, long acquisition times,
and fluorescence interference. Spatial resolution was
limited by the size of the focused probe laser beam for
the case of single-spot analysis1' 2 or by the need to image
through a large monochromator for larger fields of
view.2 '3 The CARS process has been used in flame4 and
jet5 studies to map a specific molecular component, but
the best resolution obtained was limited to 20 /im in a
single dimension.
CARS is a nonlinear Raman process and therefore has SAMPLE FILTERS VIDICON
the potential for producing large signals at high optical
field intensities. Assuming that phase-matching con- Fig. 1. Schematic diagram of the CARS microscope appa-
ditions are met, the power in the CARS signal is given ratus. A conventional microscope is used to collect light
by emerging from the sample and is represented by a single lens
to the right of the sample. Beam separation and focusing
PAS = K IX x 2IPIIhdA, angles are exaggerated for clarity.

0146-9592/82/080350-03$1.00/0 © 1982, Optical Society of America

tunable from 565 to 620 nm, provides an average power interface between the two liquids. In Fig. 2(a) the
of about 250 mW, and produces 6-psec pulses at an frequency difference of the two incident lasers is ad-
80-MHz repetion rate. The second dye laser provides justed to match the frequency of the CeN vibration in
the Stokes wavelength and is tunable from 620 to 700 acetonitrile, and the bottom half of the scanned area is
nm by using the dye 4-(dicyanomethylene)-2-methyl- bright. In Fig. 2(b) the frequency difference is tuned
6-(para-dimethylaminostyryl)-4H-pyran. It provides near the C-H vibration in octane, and the top part of
150 mW of average power and produces 8-psec the picture is bright. The octane C-H vibration is
pulses. centered at 2870cm-' but produces enough anti-Stokes
The light from the dye lasers is brought through a signal at 2500 cm-1 to be seen with the CARS pro-
suitable optical path to allow for temporal synchroni- cess.
zation of the pulses and for proper matching of beam Images of onion-skin cells taken in transmission with
diameters. Both beams are then reflected from a white-light illumination are shown in Figs. 3(a) and 3(c).
scanning mirror and are focused onto the sample. The Images of the same samples taken with the CARS mi-
separation of the beams at the focusing lens is adjusted
to satisfy phase-matching requirements. For the
thinner samples studied, phase-matching requirements
were less critical. After interaction in the sample, both
input beams and the generated anti-Stokes beam are
collected by a conventional microscope. A bank of
spike and dichroic filters is used to block the Stokes and
pump beams while passing the anti-Stokes signal to the
detector. These filters are sufficient to give greater
than 1012 rejection of the unwanted light while passing
-10% of the anti-Stokes light. The filtered anti-Stokes
light is then intensified by a factor of 104 by using a (a) (b)
three-stage image intensifier and is finally imaged onto
a storage vidicon tube. The storage vidicon allows Fig. 2. Demonstration of the molecular selectivity of the
CARS microscope. The area viewed contains an interface
image integration from 0.03 to 20 sec and provides image region between acetonitrile (bottom) and octane (top). (a)
readout to a monitor and a video tape recorder. Signal from the acetonitrile region when the dye lasers are
A large field of view is obtained with high peak in- tuned to 2250 cm-1 . (b) Signal from the octane when the
tensities by scanning the input laser beams in two or- lasers are tuned to 2500cm-'. The scanned area is 300 Am
thogonal directions by using piezoelectric drivers on the X 300 Am.
scanning mirror. The field of view covered is typically
200 gm on a side and is given by
Ad = fAO,
where Ad is the, field of view along one dimension, f is
the focal length of the focusing lens, and AOis the an-
gular tilt of the scanning mirror. The input laser beams
are focused to a spot on the sample approximately 10
gim in diameter, and the length of the region of beam 1Z 200 I' ,
overlap is greater than the typical sample thickness of
30 gAm.
The spatial-resolution and fluorescence problems of nuPM
earlier Raman microscopes are eliminated by using the (a) (b)
CARS technique. The spatial resolution of the CARS
microscope is determined entirely by the imaging sys-
tem rather than by the diameter of the focused laser
beams. No spectrometer is needed, and therefore
spatial resolution is not limited by grating optics. The
imaging lens at present is a conventional microscope
capable of a resolution of 0.7 Am. Improvements in this
microscope should eventually permit a resolution of
approximately 0.35gm. Fluorescence is not a problem
when the CARS technique is used since the detected H-2 200 pm- 1 -2 201
00 bim - *1
signal is at a shorter wavelength than the exciting-laser (c) (d)
wavelengths.
Fig. 3. White-light and CARS images of onion-skin cells that
A demonstration of the molecular specificity of the have been soaked in D20. (a), (c) Transmission white-light
CARS microscope is shown in Fig. 2. Here the CARS images with slightly different focusing conditions. (b), (d)
image is shown from a 100-gm-thick optical cell that CARS images of the same regions as (a) and (c) when lasers
contains an immisciblecombination of octane at the top are tuned to the 2450-cm-1 band of D 2 0. The CARS images
and acetonitrile at the bottom. The laser radiation is were obtained in 2 sec. The onion-skin cells were soaked in
scanned over a 300-gumX 300-gm region containing the D 2 O for 4 h.
352 OPTICS LETTERS / Vol. 7, No. 8 / August 1982

croscope are shown in Figs. 3(b) and 3(d). Onion-skin ficulties that are universal to the CARS technique:
cells were chosen for investigation because of their large nonresonant background and loss of signal at low molar
size and easy availability. Sheets of the cells with concentrations. The nonresonant background is pri-
thicknesses between 20 and 50 Atmwere soaked in D2 0 marily a problem in aqueous samples and is due to the
for 4 h. The cells were then placed between cover slips interference of the long tail of the 3300-cm-1 band of
and placed in the CARS microscope apparatus. water. A variety of approaches, including digital image
White-light pictures were taken of regions by simply manipulation, is being investigated as possible means
blocking the laser beams and substituting a white-light of minimizing this problem. The loss of signal at low
source. Cells and cell boundaries are clearly evident, molar concentrations is a more fundamental problem
as are cell nuclei. Figures 3(a) and 3(b) were focused and can result in an ultimate limit for the sensitivity of
slightly differently from Figs. 3(c) and 3(d) to obtain the system.
different diffraction effects in the white-light and CARS In conclusion, we have developed a scanning CARS
images. microscope that allows us to image specific molecular
Figures 3(b) and 3(d) show the CARS images made species in a thin, transparent sample. Studies to date
by tuning the lasers to the 2450 cm- 1 band of O-D. have demonstrated the chemical selectivity and high
The similarity between these images and the corre- spatial resolution of the device. Future applications
sponding white-light images is evident, and it appears will include the study of dynamic processes that occur
that D20 has infiltrated most regions of the onion-skin in living cells, such as the uptake and distribution of
cells. However, neither Fig. 3(b) nor 3(d) shows sig- glucose.
nificant D2 0 signal from the cell nuclei. Diffraction or
refraction effects, such as perturbation of the phase-
matching angle, could account for the lack of signal from References
the nuclei, but evidence from these pictures and others 1. J. L. Abraham and E. S. Etz, "Molecular microanalysis of
indicate that the lack of signal could be due to a lower pathological specimens in situ with a laser-Raman mi-
concentration of D2 0 in the cell nuclei. croprobe," Science 206, 716-718 (1979).
The present CARS microscope is optimized to view 2. E. S. Etz, "Raman microprobe analysis: principles and
a spectral region corresponding to frequency shifts in applications," in Scanning Electron Microscopy1l979/I
the range from 1900 to 2700 cm-'. This region was (IT Research Institute, Chicago, Ill., 1979), pp. 67-82.
chosen because it is relatively free of Raman lines from 3. P. Dhamelincourt and P. Bisson, "Principe et realisation
naturally occurring molecules but includes a number d'un microscope optique utilisant 1'effet Raman," Microsc.
of Raman lines for deuterated C-H and 0-H bonds. Acta 79, 267-276 (1977).
4. P. R. Regnier and J. P. E. Taran, "On the possibility of
By using deuteration of selected bonds, it is expected measuring gas concentrations by stimulated anti-Stokes
that high-contrast images can be made of chemical scattering," Appl. Phys. Lett. 23, 240-242 (1973).
species of interest. The use of deuterated species also 5. 0. V. Murphy, M. B. Long, R. K. Chang, and A. C. Eck-
has the potential of allowing the uptake of substances breth, "spatially resolved coherent anti-Stokes raman
by an organism to be observed dynamically. spectroscopy from a line across a CH4 jet," Opt. Lett. 4,
Problems encountered in CARS microscopy are dif- 167-169 (1979).