Holographic coherent anti-Stokes Raman scattering bio-imaging.

CARS holography captures both the amplitude and the phase of a complex anti-Stokes field, and can perform three-dimensional imaging by digitally focusing onto different depths inside a specimen. The application of CARS holography for bio-imaging is demonstrated. It is shown that holographic CARS imaging of sub-cellular components in live HeLa cells can be achieved.

1. Introduction

Since its inception, holography has found numerous applications in a wide range of scientific fields [1]. Classically, a hologram captures the change of the amplitude and the phase of an optical wave due to its interaction with a specimen (e.g., reflection, transmission, or scattering) with the complex linear susceptibility (). Consequently, holographic imaging usually relies on the differences in to provide image contrast. The merging of nonlinear optics and holography has created new opportunities to exploit the nonlinear susceptibility as a contrast mechanism. For instance, second harmonic holography utilizes (second-order nonlinear susceptibility) to provide image contrast and can record the second harmonic field generated by nano-crystal markers [2,3] or the endogeneous second harmonic radiation [4,5]. We recently proposed and demonstrated coherent anti-Stokes Raman scattering (CARS) holography [6,7], a special case of four wave mixing (FWM) holography which utilizes (third-order nonlinear susceptibility) as a contrast mechanism. This allows us to probe low-energy material excitations such as molecular vibrations and utilize the spectral response as a molecular “fingerprint” to provide image contrast. In particular, CARS holography fuses the three-dimensional (3D) imaging capability of holography with the high-sensitivity (up to ~five orders of magnitude improvement over the spontaneous Raman scattering [8]) and chemical selectivity of CARS into a new label-free, non-scanning, 3D imaging modality. Here we explore CARS holography for imaging live biological cells. In the following, we first discuss CARS holography by establishing the analogy between the generation of an anti-Stokes field and the diffraction by a volume hologram. This is then followed by experimental demonstration of holographic CARS imaging of endocellular components of HeLa cells.

CARS holography captures a complex anti-Stokes field by interfering it with a frequency-matched reference wave. Interestingly, the physical process of CARS, or more generally, FWM is also analogous to holography [9]. In a typical CARS process, a pump beam with the frequency interferes with a Stokes beam with the frequency to record a moving grating, or hologram, in a specimen of interest. The moving grating can be stored in the form of a molecular vibration if the frequency difference matches a resonant vibrational frequency. A probe beam, which oftentimes is the pump itself, can then read out the molecular vibration hologram to produce an anti-Stokes Raman signal. To further carry out this analogy, let us compare the governing equations of CARS [10] and diffraction of a volume hologram [11]:

For simplicity, a scalar formalism is used here, i.e., the fields involved are assumed to be linearly polarized along the same direction. To arrive at Eq. (1), un-depleted excitation and a homogeneous refractive index n are assumed. , , and are the fields associated with the anti-Stokes, pump, probe, and Stokes beams, is the relevant third-order nonlinear susceptibility distribution, is the angular frequency of the generated anti-Stokes signal, and c is the speed of light in vacuum. In deriving Eq. (2), the Born approximation is utilized [12]. We have also neglected the term involving the dot product between the gradient of the permittivity and the field. represents the average refractive index, Δn is the refractive index modulation resulted from a recorded hologram, and are the incident and the diffracted field respectively, and ω is the angular frequency. Evidently, the molecular vibration plays the role of a hologram. When the molecular vibration is “read out” by a probe beam, an anti-Stokes signal is generated in a way similar to a holographic reconstruction process. Just as in conventional holography the reconstructed signal wave (i.e., ) contains the image information of Δn, the generated anti-Stokes field () carries the image information of . By digitally recording both the amplitude and the phase of the anti-Stokes field holographically, it allows us to digitally focus onto different depths inside a specimen and thereby realize 3D CARS imaging without scanning. We note that this is different from the optically sectioned scanning CARS microscopy [13], which directly images the three-dimensional nonlinear source distribution. For sparse objects, 3D tomographic reconstruction can be accomplished from a single hologram by using the technique of compressive holography [7,14].

2. Experiments and results

The schematic diagram of our experimental system is shown in Fig. 1 . A nanosecond laser (Continuum Surelite III, fundamental wavelength: 1064 nm, second harmonic wavelength: 532 nm, repetition rate: 10 Hz, pulse duration ~5 ns, injection seeded) together with a tunable type II optical parametric oscillator provide the pump (also used as the probe) and Stokes beams for the CARS signal generation as well as the reference beam for recording CARS holograms. The pump/probe and Stokes beams were focused by a pair of lenses respectively (focal lengths: 750 mm and 150 mm), and spatially overlapped at the sample plane at an angle of about 20 degrees. The generated CARS/FWM image was then magnified by an imaging system consisting of an objective (numerical aperture: 0.42, focal length: 10 mm) and a lens (focal length: 500mm) and filtered by a band-pass filter (Chroma D800/30). The reference beam was first coupled into a section of a single-mode fiber (length: ~1 meter) and hence spatially filtered. It was subsequently collimated and combined with the CARS/FWM signal by using a pellicle beam splitter (reflectivity and transmittance: 8% and 92%, respectively). The two beams had a small angle (~2 degrees) between them. Their interferogram, or the resulting CARS/FWM hologram, was detected with a charge coupled device camera (Apogee 32ME).

Fig. 1

Experimental setup of CARS holography. The fundamental beam with angular frequency of from the nanosecond laser was used as the pump and the probe for generating CARS or four wave mixing (FWM) signal. The frequency-doubled laser beam () was used to pump the OPO to produce an idler beam at , which was utilized as the Stokes, and a signal beam at , which was used as the reference for recording CARS holograms. Note that the frequency of the reference beam automatically matches that of the CARS or FWM signal as the Stokes wavelength is tuned.

The CARS holographic imaging system was utilized to image live cervical cancer HeLa cells, which were grown on glass coverslips in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum and 1% penicillin-Streptomycin in a 5% CO2 incubator at 37°C. The Stokes wavelength was tuned to excite the CH aliphatic vibrational mode at 2913cm−1, which was reported to be rich in mitochondria clusters or lipid vesicles in Ref. [13,15]. To verify the CARS resonance, we detuned the wavelength of the Stokes beam from the resonant wavelength to record a sequence of wide-field CARS images as shown in Fig. 2 . Figure 2(b) shows the CARS image when the frequency of the Stokes beam was tuned so that the 2913 cm−1 Raman vibrational mode can be resonantly excited. Figures 2(a) and 2(c) show the images acquired when the Stokes wavelength was detuned with Raman shifts of 2905 cm−1 and 2921 cm−1 respectively. It should be noted that as the wavelength was tuned the power of the Stokes beam may also vary slightly. However, Fig. 2 clearly shows the trend that as the detuning increases the signal strength reduces accordingly, consistent with the existence of a resonant mode.

Fig. 2

Wide-field CARS images of HeLa cells around the resonant mode at 2913 cm−1 recorded by tuning the Stokes wavelength. CARS images (a) at 2905 cm−1, (b) at 2913 cm−1, (c) at 2921 cm−1

We performed CARS holographic imaging of HeLa cells. The pulse energies of the pump and Stokes were adjusted to about 10 mJ and 4 mJ respectively. During the experiments, the cells were tested quickly after they were taken out of an incubator and kept alive during the measurements. Visual microscopic inspection followed to confirm that there was no apparent photo-damage to the cells during the experiment. However, sometimes samples were damaged due to defects or contaminants on the cover glass, which absorbed light and resulted in ablation. This problem may be alleviated by using picosecond laser systems, which can exhibit higher peak power with significantly less pulse energy. Figure 3(a) shows a CARS hologram of three HeLa cells (c.f., Fig. 3(d)) recorded with a single-shot exposure of a pair of pump and Stokes pulses (~5 ns), while Figs. 3(b) and 3(c) are the reconstructed field amplitude and phase distribution respectively. By performing digital back propagation, a sequence of images at different depth positions in the sample volume can be retrieved as shown in Figs. 3(e)-(h), where the sub-cellular components at different depth levels can be brought into focus sequentially.

Fig. 3

3D CARS holographic imaging of live HeLa cells. (a) Recorded CARS hologram. (b) Reconstructed field amplitude. (c) Reconstructed phase. (d) Micrograph of the three HeLa cells that were imaged. (e)-(h) Sequence of reconstructed CARS images at different depth positions in the sample volume, where the sub-cellular components at different depth levels can be brought into focus sequentially

To compare the digital focusing ability of CARS holography with analogue (manual) focusing in wide-field CARS microscopy [16], we captured a series of wide-field CARS images (i.e., direct images without the reference beam) of some HeLa cells by manually focusing onto different depth layers of the sample (i.e., through manual translation of the objective in the axial direction). Figures 4(a)-(e) show a series of holographically reconstructed CARS images of HeLa cells at different depth positions by using digital propagation, while the corresponding manually focused wide-field CARS images are shown in Figs. 4(f)-(j). These two sets of images largely exhibit similar features although small differences exist partly due to the limited precision of the manual focusing. These results clearly demonstrate the equivalence of digital and analogue focusing. Note that multiple-shot exposure (~10 pulses) was employed in the wide-field imaging scheme to increase the signal strength. On the other hand, single shot exposure was used in CARS holography, which promises laser pulse duration limited speed but also results in relatively weak signal.

Fig. 4

Comparison of holographic CARS imaging (upper) and wide-field CARS imaging (bottom, with manual focusing) of HeLa cells at different depth positions (z: relative positions) demonstrating the equivalence of digital and analogue focusing. (a)-(e) Series of holographically reconstructed CARS images of HeLa cells at different depth positions by using digital propagation. (f)-(j) Corresponding manually focused wide-field CARS images. multiple-shot exposure was employed in the wide-field imaging scheme to increase the signal strength while single shot exposure was used in CARS holography.

3. Summary

In summary, we have demonstrated non-scanning chemically selective 3D bio-imaging via holographic CARS microscopy. In addition to reconstructing the intensity distribution at different depth positions CARS holography also captures the phase information, which is not available in the wide-field imaging modality, and can lead to tomographic reconstruction through advanced digital processing algorithm (e.g., compressive holography [14]). Our imaging results on live HeLa cells indicate that CARS holography is useful as a practical tool for 3D cellular imaging. The unique ability of label-free and thus non-toxic imaging allows for long duration studies of sub-cellular components in live specimens. Furthermore, with the added capability of single-shot imaging, CARS holography has the potential to image fast biological processes at laser pulse duration limited speed.