multi-focus excitation coherent anti-stokes raman scattering (cars) microscopy and its applications...

Multi-focus excitation coherentanti-Stokes Raman scattering (CARS)

microscopy and its applications forreal-time imaging

Takeo Minamikawa1, Mamoru Hashimoto1, Katsumasa Fujita2,Satoshi Kawata2,3, and Tsutomu Araki1

1Graduate School of Engineering Science, Osaka University,Toyonaka, Osaka, 560-8531, Japan

2Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan3RIKEN, Wako, Saitama, 351-0198, Japan

[email protected]

Abstract: We developed a multi-focus excitation coherent anti-StokesRaman scattering (CARS) microscope using a microlens array scanner forreal-time molecular imaging. Parallel exposure of a specimen with lightfrom two highly controlled picosecond mode-locked lasers (jitter of 30 fsthrough an electronic low-pass filter with 150 Hz bandwidth, point-by-pointwavelength scan within 300 ms) and parallel detection with an image sensorenabled real-time imaging. We demonstrated real-time CARS imaging ofpolystyrene beads (frame rate of 30 fps), a giant multi-lamellar vesicle ofdipalmitoylphosphatidylcholine (frame rate of 10 fps), and living HeLacells (frame rate of 10 fps).

© 2009 Optical Society of America

OCIS codes: (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering; (110.0180) Mi-croscopy.

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(C) 2009 OSA 8 June 2009 / Vol. 17, No. 12 / OPTICS EXPRESS 9526#106179 - $15.00 USD Received 9 Jan 2009; revised 13 Feb 2009; accepted 2 Mar 2009; published 22 May 2009

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1. Introduction

Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical-sensitive three-dimensional imaging of biological specimens without staining [1, 2]. CARSmeasurement generally uses two highly synchronized picosecond mode-locked lasers of dif-ferent wavelengths (ω1 light and ω2 light). When the frequency difference of the ω1 and ω2

beams coincides with a molecular vibration (Ω = ω1 −ω2), a CARS signal (ωas = 2ω1 −ω2)is resonantly enhanced. CARS microscopy features high spatial resolution, label-free imaging,strong signals compared with conventional Raman scattering microscopy, the absence of Stokesshifted fluorescence, and the ability to detect molecular species. CARS microscopy has beenapplied to biological measurements. Applications include studies of lipid metabolism [3, 4], or-ganelle transport [5], viral disease [6], dividing cell dynamics [7], membrane chemistry [8, 9],brain medicine [10], and so on.

In biological measurement, high-speed imaging is required for the observation of biologicalphenomena that change spatio-temporally. In the case of CARS microscopy, video-rate CARSimaging of tissue in vivo was realized by the use of a high-speed laser scanner with a polygonmirror and a galvanometer mirror [11]. The mirrors quickly scanned a single focal point on aspecimen, enabling the dwell time at each point to be shorten. Consequently, the intensity ofthe incident excitation laser light needs to be high to obtain a sufficiently high CARS signalfor imaging. However, a high intensity might cause photo-damage of the specimen. High-speedimaging of a biological specimen therefore requires a high-speed laser scanning system, a highsignal-to-noise ratio imaging system, and low-intensity excitation laser light.

In the present study, we realized real-time CARS imaging with low-intensity excitation ofeach spot by the use of a multi-focus excitation technique, and we demonstrated real-time imag-ing of polystyrene beads (30 fps), giant multi-lamellar vesicles (MLVs) of dipalmitoylphos-


phatidylcholine (DPPC) lipids (10 fps), and living HeLa cells (10 fps).

2. Multi-focus CARS microscope

The intensity of a CARS signal Ias from a spot on a specimen is written as

Ias ∝∫ τex

0dt{χ(3)}2I1

2I2, (1)

where χ(3), I1, I2, and τex are the third-order nonlinear susceptibility, the intensity of ω1 light,the intensity of ω2 light, and the dwell time at the spot. When performing high-speed CARSimaging with single beam scanning, a sufficient CARS signal is generally obtained by increas-ing the excitation laser intensity to compensate for the short dwell time. However, the laserintensity is limited to the range of a few milliwatts to a few tens of milliwatts. Because a CARSmicroscope generally uses near-infrared lasers (oscillating wavelength of 700–1000 nm) witha pulse duration of a few picoseconds or shorter, nonlinear photochemical damage is the ma-jor problem rather than photothermal damage because of the weak linear absorption of near-infrared light in most cells, except hemoglobin, chlorophyll, and other strong near-infraredabsorbers [12, 13]. The photochemical damage is caused through a second- or higher-orderphoton process in which the photodamage efficiency is proportional to the second- or higher-order power of the intensity of the laser spot, and thus the peak power of the spot is limitedfor non-invasive imaging. In the case of multi-focus excitation, the dwell time is increased inproportion to the number of focal spots without reducing a frame rate. When the beams aresplit into 100 beamlets, the signal-to-noise ratio of the obtained CARS image is improved onehundredfold compared with single-beam scanning with the same spot intensity.

Unfortunately, the total laser intensity of all spots in multi-focus excitation is higher than thatin single-beam excitation, and long exposure with high intensity might increase the temperatureof the specimen. The temperature of the specimen, however, will not increase by more thana few degrees Kelvin during a <10 s exposure of near-infrared light with a power of 100–200 mW [14], and this temperature increase may not cause critical photothermal damage. Ofcourse, in long-exposure imaging, such as high signal-to-noise ratio imaging of a weak Ramanband or real-time imaging over a long duration, the temperature of the specimen might increaseto the critical temperature for photothermal damage. However, in the case of a specimen inan aqueous culture medium, a temperature increase can be prevented by reflowing the culturemedium because water is the major absorber of near-infrared light in a biological specimen.

We used a microlens array scanner for forming of multiple focal spots [15, 16]. The mi-crolens array scanner has many microlenses on a rotating disk. An excitation laser beam inci-dent on the microlens array scanner is split into multiple beamlets to expose multiple pointson a specimen simultaneously. An image is obtained by rotating the microlens array disk. Themulti-focus excitation technique with a microlens array scanner has been applied to fluores-cence microscopy [17, 18] and second harmonic generation (SHG) microscopy [19], to realizereal-time imaging. In this study, we have realized real-time CARS imaging by the use of ahigher efficiency microlens array scanner and a higher sensitive image sensor than the previousstudy [15, 16].

3. Experimental setup

The optical setup of the multi-focus CARS microscopy system is shown in Fig. 1. The systemconsisted of two picosecond mode-locked Ti:sapphire lasers operating at different wavelengths(pulse duration of 5 ps, repetition rate of 80 MHz, Tsunami, Spectra-Physics), a high-precisionpulse synchronization system [20], an automatic pulse duration minimizing system [21], a


microlens array scanner (lens diameter of 0.58 mm, focal length of 11.6 mm, MLA1-DD,Nanophoton), a modified inverted microscope (TE-200, Nikon), and an electron-multiplyingcharge-coupled device camera (EM-CCD, DV-897, Andor).

ps mode-locked

Ti:S laser (ω1)

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Fig. 1. Optical setup of the developed multi-focus CARS microscopy system: L, lenses; TL,tube lens; OL, objective lenses; TPD, two-photon detector; DSP, digital signal processor;GTI, Gires-Tournois interferometer.

The two lasers were synchronized by phase-locked loop (PLL) control using two types oferror signals: the fundamental signal of the repetition frequency of the lasers and the signal of abalanced cross-correlator using two-photon detectors. The lasers were synchronized to within1 ps of timing jitter by using the fundamental signal of the repetition frequency of 80 MHzbeforehand. Then the error signal of the PLL controller was switched to the signal of thebalanced-cross correlator, and the timing jitter between the two lasers was reduced to within30 fs by using an electronic low-pass filter with 150 Hz bandwidth. A digital signal processor(DSP, TMS320C6713, Texas Instruments) with an analog-digital/digital-analog converter in-terface (DSK6713IF-A, Hiratsuka Engineering) was used as a controller for the PLL control.These synchronized laser beams were spatio-temporally overlapped and were made incidenton a microlens array scanner to split them into multiple beamlets. The microlens array scan-ner was high efficiency for the CARS microscope, i.e., the larger diameter of the microlensand antireflection coating for near-infrared light to increase an excitation intensity on each spotcompared with the previous study [15, 16]. The beamlets were collimated with relay lenses andwere focused to multiple spots on the specimen with an objective lens (S Fluor, Nikon, x40,N.A. = 0.85). The system produced seven focal spots on the specimen from laser beams 2 mmin diameter. CARS signals from the multiple focal spots on the specimen were collected by an-other objective lens (UPlanApo, Olympus, x40, N.A. = 0.85), and the fundamental signals werecut with optical filters. The CARS signals were observed in parallel with the EM-CCD camera,and a CARS image was obtained by rotating the microlens array disk. The observed molecularvibration was tuned by adjusting the wavelength of the ω2 light. The pulse duration of the ω2

light was automatically minimized by the use of a two-photon absorption detector. The sig-nal from the two-photon absorption detector was inversely proportional to the pulse duration;therefore, the group delay dispersion of the laser cavity was compensated with a Gires-Tournoisinterferometer to minimize the pulse duration by maximizing the signal of the two-photon ab-sorption detector. The preparation time for CARS measurement, including wavelength tuning


in short wavelength range, pulse duration optimization, and jitter control in the point-by-pointwavelength scan, was within 300 ms.

4. Results

4.1. Real-time spectral-imaging of polystyrene beads

The real-time CARS imaging capability was demonstrated with polystyrene beads. Thepolystyrene beads (3 μm diameter, Polybeads Microspheres 3.00μm, Polysciences) mixed withdeionized water were dispersed on a slide glass and sealed by a coverslip with a 50-μm-thicksilicone spacer.

Figure 2 shows the CARS spectra of the polystyrene beads and the background (water) at1000 cm−1. The spectra were obtained from a pixel of the CARS images of the polystyrenebeads in the water at each Raman shift in which the beads were not moved. The CARS im-ages were obtained with an image acquisition time of 33 ms/image. The total intensities were75.9 mW at 780 nm and 29.7 mW at 846 nm at the focal plane with 7 focal spots. The CARSspectrum of the polystyrene beads had a strong resonance at 1000 cm−1; however, the water didnot have any peaks. As a result, we could selectively observe the polystyrene beads in a shortexposure time with the developed microscope. The 1000 cm−1 resonance of the polystyrenebeads is assigned to the phenyl ring breathing mode [22].

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Fig. 2. CARS spectra of the polystyrene beads (solid line) and background (dashed line)obtained from a pixel of an image (33 ms/image). Strong resonance for the polystyrenebeads was observed at 1000 cm−1, which corresponds to the phenyl ring breathing mode.

A video of real-time CARS images of moving polystyrene beads in water observed at1000 cm−1 is shown in Fig 3 (Media 1). The images were obtained at a frame rate of 30 fps.The flowing polystyrene beads exhibiting Brownian motion in water were clearly observed.

We also demonstrated real-time CARS imaging of polystyrene beads with wavelength scan-ning. Figure 5 shows the temporal behaviors of the signal from the two-photon absorptiondetector that monitored the pulse duration of the ω2 light, the observed Raman shift, and theCARS images during wavelength scanning. A video of the CARS images is shown in Fig 4 (Me-dia 2). When the observed molecular vibration was changed from 998 cm−1 after about 10 s bytuning the oscillating wavelength of the ω2 light by adjusting a birefringence filter driven by acomputer controlled stepping motor, the two-photon signal decreased because the group-delaydispersion in the cavity was not optimized, and thus, the CARS image of the beads disappeared.However, the signal from the two-photon absorption detector spiked immediately, and the ob-served molecular vibration became 1004 cm−1 within 0.3 s. After about 45 s, at which theobserved molecular vibration was changed from 1005 cm−1 to 1000 cm−1, the CARS image of


Fig. 3. Video of real-time CARS images of the polystyrene beads (diameter of 3 μm)in water with a frame rate of 30 fps (Media 1). The observed molecular vibration was1000 cm−1. Total incident laser intensities were 75.9 mW at 780 nm and 29.7 mW at846 nm with 7 focal spots. The image size was 40 μm x 40 μm.

998 cm-1

Fig. 4. Video of CARS spectral images of polystyrene beads (3 μm diameter) during wave-length scanning (Media 2). The image size was 40 μ m x 40 μ m.

the beads appeared within 0.3 s after adjusting the wavelength of the ω2 light. The signal fromthe two-photon absorption detector showed the same behavior at other molecular vibrations.As a result, the pulse duration and the timing jitter of the two pulses were optimized within0.3 s in the point-by-point wavelength scanning in short wavelength range. The system is thuscapable of CARS spectral imaging of polystyrene beads with a frame rate of 1 fps, includinginformation in 2–3 Raman shift signals.

4.2. Real-time imaging of a giant multi-lamellar vesicle

We also demonstrated real-time imaging of a DPPC MLV. The preparation of the DPPC MLVwas as follows: First, DPPC lipids (P0763, Sigma) dissolved in a chloroform/methanol solution(98:2 vol/vol) were evaporated under a flow of nitrogen for 24 hours and in vacuo for at least90 minutes in an Erlenmeyer flask. A HEPES buffer (10 mM, HEPES-NaOH, pH 7.0) was thenadded to the dried lipid films at room temperature, and the lipids formed MLVs. The MLVswere spread on a slide glass and sealed by a coverslip with a 50-μm-thick silicone spacer.

DPPC lipids exhibit CH2 deformation vibrations of the acyl chains at a Raman shift of1442 cm−1 [24]. CARS and Raman spectra of DPPC lipid powder are shown in Fig. 6. TheCARS spectrum of the DPPC lipids was much broader than the Raman spectrum and exhib-ited a dispersive line shape, and so the peak of the CARS spectrum (1442 cm−1) was slightlyshifted from that of the Raman spectrum (1445 cm−1). In this study, we visualized DPPC MLVs


http://www.opticsexpress.org/viewmedia.cfm?URI=oe-17-12-9526-1


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1007 cm-1

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(a)

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Fig. 5. Real-time CARS spectral imaging of polystyrene beads (3 μm diameter) duringwavelength scanning. (a) Temporal behavior of the signal from the two-photon absorptiondetector (TPD) that monitored the pulse duration of the ω2 light, and the observed Ramanshift. (b) CARS images of the polystyrene beads at each Raman shift. Total incident laserintensities were 75.9 mW at 780 nm and 29.7 mW at 846 nm with 7 focal spots. The imageswere obtained with a frame rate of 30 fps. The scale bar represents 10 μm.

through the CH2 deformation vibrations.Figure 7 shows a CARS image of a DPPC MLV observed at 1442 cm−1 and 1486 cm−1.

The image acquisition time was 3 s/image. When the observed Raman shift was set at theCH2 deformation vibrations (1442 cm−1), a cross-sectional image of the DPPC MLV near theequator was clearly observed. In case of a nonresonant image (1486 cm−1), the CARS singnalwas weakened. Because we used a two-dimensional image sensor in our developed systemfor simultaneous observation of multiple focal points, the spatial resolution in the xy planewas limited by the observation optics and was lower than in a conventional point-by-pointone-dimensional CARS system [23]. On the other hand, the spatial resolution along the z-axisdepended on the excitation optics and the third-order nonlinearity of the CARS generation. Thedeveloped multi-focus CARS microscope thus had a high spatial resolution along the z-axis(∼2 μm), and z-section CARS images of the DPPC MLVs were clearly visualized.

A video and snapshots of the real-time imaging of a DPPC MLV at various z-positions isshown in Fig. 8 (Media 3) and Fig. 9. The depth position of the DPPC MLV was set with apiezoelectric transducer stage (PZT stage, 17 ANC 001/MD, NanoBlock xyz Flexure Stage,Melles Griot). The observed molecular vibration was set at 1442 cm−1. Each cross-sectionalCARS image of the DPPC MLV was observed within 100 ms. The images were obtained fromthe equator to the pole of the DPPC MLV, and the cross-sectional images of the DPPC MLV at


each z-position were clearly observed.Three-dimensional reconstruction of the CARS images of a DPPC MLV was also demon-

strated (Fig. 10). The z-section CARS images were obtained by moving the specimen over10 μm (200 nm/step) with the PZT stage. The observed molecular vibration was set at1442 cm−1. The image acquisition time of each cross-sectional image was 100 ms/image. Thethree-dimensional CARS image of the DPPC MLV consisted of 50 slices and was obtainedwithin 7 s, and a hollow-sphere-shaped DPPC MLV with a diameter of about 20 μm was visu-alized.

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Fig. 6. Raman and CARS spectra of the DPPC lipid powder at 1442 cm−1. Strong reso-nance was observed at 1442 cm−1, which corresponds to the CH2 deformation mode.

1442 cm-1

1486 cm-1

Fig. 7. CARS images of the DPPC MLVs at a Raman shift of 1442 cm−1 and 1486 cm−1.The total incident laser intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nmwith 7 focal spots. The image acquisition time was 3 s (exposure time of 100 ms and 30acquisitions). The scale bar represents 10 μm.


Fig. 8. Video of real-time CARS images of the DPPC MLV obtained while varying thez-position (Media 3). The frame rate was 10 fps. The observed molecular vibration was1442 cm−1. The total incident intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nmwith 7 focal spots. The image size was 60 μm x 60 μm.

(a) (b)

(c)

Fig. 9. Real-time CARS image of the DPPC MLV at z-positions of (a) 0 μm, (b) 5 μm, and(c) 10 μm. The observed molecular vibration was 1442 cm−1. The total incident intensitieswere 46.2 mW at 780 nm and 77.4 mW at 879 nm with 7 focal spots. The image acquisitiontime was 100 ms. The scale bar represents 5 μm.



Fig. 10. Three-dimensional reconstruction of the CARS images of a DPPC MLV. The rightbottom, left, and top panels represent a xy, yz, and zx optical slice taken at the equator of theDPPC MLV, respectively. The observed molecular vibration was 1442 cm−1. The incidenttotal laser intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nm with 7 focal spots.The total image acquisition time of the DPPC MLV was 7 s per 50 slices (100 ms percross-sectional image). The scale bar represents 5 μm.

4.3. Real-time imaging of living cells

We also demonstrated real-time imaging of living HeLa cells. The HeLa cells were culturedon a slide glass bottom culture dish immersed in Dulbecco’s modified Eagle’s medium sup-plemented with 10% fetal bovine serum. The culture medium was replaced with a modifiedTyrode’s solution before CARS imaging. A water immersion objective lens (NIR Apo, Nikon,x60, N.A. = 1.0, water immersion) was used for collection of CARS signals.

Figure 11 shows a CARS image of the HeLa cells observed at 2840 cm−1. The image ac-quisition time was 1 s/image. Since the molecular vibration at 2840 cm−1 is assigned to CH2

stretching mode of lipids, lipid rich regions in the HeLa cells were clearly observed. A video ofthe CARS images of the HeLa cells with a frame rate of 10 fps is shown in Fig 12 (Media 4).As a result, we could also visualize the living cells without any treatment in a short exposuretime with the developed microscope.

5. Conclusion

In conclusion, we have developed a multi-focus CARS microscopy system for real-time molec-ular imaging. With multi-focus excitation, the signal-to-noise ratio of a CARS image was im-proved in proportion to the number of focal spots without increasing the intensity of each focalspot. Real-time CARS spectral imaging of polystyrene beads, a DPPC MLV, and living HeLacells was demonstrated. The system was capable of visualizing the polystyrene beads within33 ms, and the DPPC MLV and the HeLa cells within 100 ms, respectively. The preparationtime for point-by-point wavelength scanning was less than 300 ms.

For more detailed visualization of the behavior of biological specimens, analysis of CARSimages at various Raman shifts in the fingerprint region is necessary. The CARS spectrumin the fingerprint region reflects structural changes of biological molecules, and analysis ofCARS images at several Raman shifts gives us information about not only the distribution ofthe molecules, but also the activity of the molecules.

As the number of foci was seven in the present study, the multi-focus effect might be not


1000080006000400020000

Intensity / a. u.Fig. 11. CARS images of the HeLa cells at a Raman shift of 2840 cm−1. The total incidentlaser intensities were 80.1 mW at 712 nm and 40.5 mW at 892 nm with 7 focal spots. Theimage acquisition time was 1 s (exposure time of 100 ms and 10 acquisitions). The scalebar represents 10 μm.

Fig. 12. Video of real-time CARS images of the HeLa cells (Media 4). The frame ratewas 10 fps. The observed molecular vibration was 2840 cm−1. The total incident laserintensities were 80.1 mW at 712 nm and 40.5 mW at 892 nm with 7 focal spots. The imagesize was 40 μm x 40 μm.

so high. A large number of foci will provide a much higher multi-focus effect, however, aphoto-damage risk to live cell or tissue caused by strong laser illumination might be increased.Therefore, more investigations will be required to show the advantage of multi-focus system.

Acknowledgements

This work was partially supported by a Grant-in-Aid for Scientific Research (A) from the Min-istry of Education, Culture, Sports, Science and Technology (MEXT) and the program of De-velopment of Systems and Technology for Advanced Measurement and Analysis (SENTAN)from the Japan Science and Technology Agency (JST). One of the authors (T.M.) acknowledgesthe support by Grant-in-Aid for JSPS Fellows from Japan Society for the Promotion of Science(JSPS).



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