1. Introduction

Biological and medical disciplines have seen a rapid recent increase in the importance of vibrational imaging of living samples such as tissues and cells, to study the distribution of molecular species in cells and to obtain information about their structure and environment. For this purpose, infrared (IR) absorption and Raman scattering microscopes enable in situ measurement of molecular vibrations without labeling and/or physiological damage to targets [1–5]. Furthermore, Raman and CARS microscopes posses the ability to obtain the vibrational image of a single cell through sub-micrometer imaging [2–4]. On the other hand, due to the longer wavelength and larger diffraction limit of IR light, the application of IR microscopes for the vibrational imaging of minute samples, such as single cells, is rather underdeveloped compared to Raman microscopes, which employ shorter wavelength visible light [6,7]. Importantly though, IR spectroscopy is a complementary technique to Raman spectroscopy, and both of them are indispensible for the complete understanding of molecular vibrations of biological molecules in the cell

To overcome the shortcoming of existing IR-based techniques, we have developed a new vibrational sum-frequency generation (VSFG) detected IR super-resolution microscope. VSFG is a nonlinear optical process in which a visible and an IR photon are incident with a molecule, and a new photon, whose energy is equal to the sum-frequency of first two photons (ν_vis + ν_IR) [8], is emitted at interfaces such as cell walls and cytomembranes and/or from chiral molecules through vibrational resonance of the molecule with the IR light [9–13]. The largest benefit of the VSFG method for IR microscopy is the conversion of IR absorption to visible emission. Because the VSFG signal has a visible wavelength, the image is observed at the resolution of visible light, which is about 10 times smaller than that of IR light. Furthermore, it is possible to apply this method to the majority of non-fluorescencing biological molecules.

To date, by using the VSFG detected IR super-resolution microscope, we have succeeded in IR imaging onion root cells at a spatial resolution of 1.1 μm, which exceeds the diffraction limit of IR light. In addition, we have also successfully observed the difference of the distribution of specific molecular species in the cell interior by changing the IR wavelength [14]. The present work aims at the IR imaging of molecular species in cultured cancer cell interiors using this microscope. This ability is indispensible to cell biological and/or medical applications of the VSFG microscope.

2. Experimental methods

The laser setup for our two-color IR super-resolution microscope has been described previously [14,15]. Figure 1 shows optical layout used for IR imaging detection. Both IR and visible light with a spectral bandwidth of 20 cm⁻¹ and a pulse width of 2 ps, generated by a picosecond laser system, were introduced into a home-made laser fluorescence microscope. IR and visible light beams were superposed on a colinear path by a beamcombiner and focused into the sample using a CaF₂ lens (f = 100 mm). The focal spot sizes of IR and visible light beams were adjusted to about 100 μm diameter at the sample position. During the experiment, IR light of < 10 μJ / pulse and visible light of < 10 nJ / pulse were used to irradiate the sample at a repetition rate of 1 kHz. The VSFG light from samples was collected from the opposite side of the sample from the incident light, by a NA = 0.5 objective lens (Newport, M-50X), projected onto a CCD camera with an image-intensifier (Princeton Instruments; PI-MAX-1K filmless) and recorded by a personal computer as a VSFG image. Here, we used the 610 nm wavelength of the visible light. This is because the wavelength of VSFG signal is fixed to the photon detection maximum (~500 - 600 nm) of ICCD detector. To remove the excitation lasers, notch and IR-cut (Y48, HOYA, Tokyo, Japan) filters were placed behind the objective. In addition, we used short (Y48, HOYA, Tokyo, Japan) and long cut (Asahi Spectra, Tokyo, Japan) filters, which acted together as a band-pass filter to eliminate background light and to allow selective detection of the VSFG signal.

 

Fig. 1 Schematic diagram of the experimental setup of the VSFG microscope.

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A549 cells, a lung carcinoma cells were used as samples. The cells were cultured in Dulbecco modified Eagle medium (GIBCO) with 10% fetal bovine serum on a cover glass. Just before VSFG imaging, the sample was covered with another cover glass and sealed using nail varnish to prevent it from drying.

3. Results and Discussions

Figure 2a shows a transmission image of an A549 cultured cell. We applied only visible (Fig. 2b, wavelength = 610 nm), only IR (Fig. 2c, wavelength = 3300 nm), and (d) both IR and visible beams to the cell. No VSFG signal was detected for cases (b) (only visible) and (c) (only IR). However, a VSFG image was clearly observed by simultaneously introducing both IR and visible light (Fig. 2d). VSFG images almost disappeared when the IR wavelength was changed to 2728 cm⁻¹, where the majority of biological molecules do not absorb IR light (Fig. 2e). The difference between Figs. 2d and 2e clearly indicates that stronger intensity VSFG signals correspond to stronger absorption of IR light. This relationship is similar to the previously reported results for onion root cells, and therefore we conclude that the images in Figs. 2c and 2d reflect the IR absorption of the molecule [14]. Furthermore, we measured of the spectrum of the VSFG signal, observing a strong and very sharp peak at a wavelength of 514 nm, which is 3030 cm⁻¹ greater than the visible excitation beam (data not shown). This strongly confirms that the image in Fig. 2(d) originates from VSFG. Therefore, we conclude that IR imaging of cultured A549 cell was successfully demonstrated by the VSFG detected IR microscope.

 

Fig. 2 VSFG images of an A549 cultured cell. (a) Transmission image of the cell. Image obtained by (b) only visible (λ _VIS = 610 nm) and (c) only IR (λ _IR = 3273 nm (ν _IR = 3055 cm⁻¹)) beams, (d) simultaneous introduction of visible and IR beams (λ _VIS = 610 nm, λ _IR = 3273 nm (ν _IR = 3055 cm⁻¹)). (e) As for (d), but with the IR wavelength increased to 3666 nm (ν _IR = 2728 cm⁻¹). The white scale bars in each figure indicate 5 μm.

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Next, we measured the IR wavelength dependence of the A549 cultured cell by changing the IR wavelength. The exciting beams employed were visible light (610 nm) and IR light (3150, 3055, 2925 and 2803 cm⁻¹) corresponding to specific vibrational modes. In each VSFG image, their strength and contrast changed along with the IR wavelength (Figs. 3a , 3b, 3c and 3d). We consider that the changes of the signal intensity reflect the strength of IR absorption by each specific vibrational mode of the cell. 3150 and 3055 cm⁻¹ correspond to the aromatic -C-H mode and may reflect chemical species such as proteins and nucleotides which are expected to be rich in aromatic groups [16]. Figures 3a, 3b show a strong signal in the cell interior where these chemical species exist. Interestingly, we can observe many strong regions in Figs. 3a and 3b. This may reflect the heterogeneous distribution of those species. Such heterogeneous distribution of proteins was reported by Raman microscopy [2] and our finding is consistent with that result. In contrast, 2925 and 2803 cm⁻¹ correspond to the aliphatic -C-H mode and this is especially strong for lipid molecules in plasma and mitochondrial membranes [17,18]. In Figs. 3c and 3d, strong signals can be observed around the edge of the cell, corresponding to the plasma membrane. This shows that the VSFG microscope can provide molecular imaging even for cultured cells, and we believe that this is the first report of IR super-resolution imaging of a living single cell. Interestingly, the distribution in Fig. 3d is significantly different from the other wavelengths, and has become less-structured and unclear. A similar example of structure disruption in the cell was observed by a Raman microscope in a previous paper, and was related to the cell mortality [2]. That is, the unstructured distribution of Fig. 3d indicates the death of the cell and the membrane structures in the cell, such as nuclear membrane, mitochondrial membrane, endoplasmic reticulum and so on, were destructured ahead of other intracellular structures. We checked the cell vitality using trypan blue, which confirmed that it was alive after the measurement. Therefore, the cell in Fig. 3 was not completely dead, but had just began to lose its activity. In this measurement, we moderately focused both IR and visible light beams, and their spot sizes were ~100 μm. Therefore, the damage of the light never occurs on the living cell because the photon density of both IR and visible light is much lower. This result shows that the VSFG microscope has the ability to observe changes in the physiology and activity of living cells. Although we need to improve the S/N ratio and shorten the accumulation time of the measurements, to achieve detailed spectroscopy in each part of the cancer cell and provide richer biological information, this result highlights the great potential of the VSFG microscope for providing detailed molecular information for biological and medical purposes.

 

Fig. 3 IR wavelength dependence of the A549 cultured cell image. VSFG image obtained by simultaneous introduction of visible (610 nm) and IR beams whose wavelengths were λ _IR = 3175 nm (ν _IR = 3150 cm⁻¹) (a), λ _IR = 3273 nm (ν _IR = 3055 cm⁻¹) (b), λ _IR = 3419 nm (ν _IR = 2925 cm⁻¹) (c), λ _IR = 3568 nm (ν _IR = 2803 cm⁻¹) (d). The white scale bars in each figure indicate 5 μm.

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4. Conclusion

We successfully imaged an A549 cultured cell (from lung cancer) using a VSFG detected IR super-resolution microscope. In addition, we observed differences in the signal intensity at various IR wavelengths which appear to originate from the differing IR absorptions of specific vibrational modes, and reveal the distribution of molecular species for the first time. Finally, it is expected that alongside the Raman microscope, this kind of microscope will become a powerful technique for the vibrational spectroscopy of living cells.