Researchers have long sought to improve optical methods of extracting information from living samples by fluorescence, light scattering, or other noninvasive techniques. Raman scattering can be used to optically investigate the chemical properties of samples due to its ability to detect capability of detecting molecular vibration frequencies that characterize molecular species, structures, and environmental conditions. In combination with optical imaging, Raman scattering can be applied to the direct sensing of biological molecules without requiring preprocessing or fluorescence staining of samples.1, 2, 3, 4, 5, 6 Although Raman scattering is a powerful tool for analyzing biomolecules, it has rarely been attempted as a contrast mechanism for imaging living specimens due to the extremely low scattering efficiency. Since typical Raman scattering signals are weak (scattering cross-section ) compared to fluorescence yields (absorption cross-section ), the measurement of Raman spectra usually requires long exposure times, making observations of living specimens difficult.
Here, we extend Raman scattering spectroscopy to enable dynamic imaging of molecular distributions in living cells with high temporal and spatial resolution. We combined a slit-scanning detection technique and optimized the excitation wavelength to image molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained living HeLa cells. By investigating the wavelength dependencies of Raman signal yields and background signals, we found that -wavelength excitation can be used to generate Raman scattering signals strong enough for imaging and for suppressing the background signals that result from autofluorescence. Additionally, cytochrome c exhibits resonant Raman scattering by excitation, and we found that in situ imaging of cytochrome c in living cells can finally be performed using our technique. These results show that the activities of molecules, including exogenous molecules such as in pharmaceutical drugs, can be directly monitored in living cells by Raman scattering to identify cellular functions that conventional fluorescence techniques are incapable of revealing.
Raman Spectra of Living HeLa Cells
In order to select the excitation wavelength, we first compared Raman spectra of cultured HeLa cells obtained with different excitation wavelengths. We used the standard laser lines: from a semiconductor laser, from an Ar ion laser, from a frequency-doubled laser, and from a HeNe laser. Wavelength choice is a tradeoff; shorter wavelengths provide excellent Raman scattering efficiency and spatial resolution, both of which are inversely proportional to wavelength, while longer wavelengths produce significantly less background autofluorescence signals than shorter wavelengths. We used a 1.2 numerical aperture (NA) objective lens, both for illuminating the cells and collecting the Raman signal, using a spectrophotometer (320PI, Acton) and a cooled CCD camera (PIXIS 400BR, Princeton Instruments).
Figure 1 shows the effect of the excitation wavelength on Raman spectra obtained from cytosol regions in living HeLa cells. For each excitation wavelength, we obtained Raman spectra from 36 different positions in the cytosol of a single cell and averaged them to produce each spectrum in Fig. 1, separated by wavelength with no background removal applied. The laser intensity at the focus was , and the 36 spectra were obtained in parallel over an exposure time of . In Fig. 1, the Raman spectra obtained with the 488-, 514.5-, and excitation wavelengths exhibited much stronger scattering signals than the spectra obtained with the wavelength, which was expected due to the wavelength dependence of the scattering efficiency. The measured spectra contain peaks that are known to occur in biological samples, such as the ring breathing of phenylalanine ( ), deformation ( ) and stretching mode ( , ), stretching mode ( ), and Amide-I vibrational mode of peptide bonds ( ) (Refs. 7, 8).
In addition to these typical Raman shifts, strong Raman peaks appear at 753, 1127, 1314, and in the spectra obtained by the 514.5- and excitation wavelengths. These peaks can be assigned to vibration modes of cytochrome c.9 Since cytochrome c contains a heme protein that absorbs light at , strong resonant Raman scattering is observed when irradiated with this wavelength range. The Raman peak at , which shows the pyrrole breathing mode in cytochrome c, was previously measured in vitro by irradiation,9 and can be clearly observed in situ in this result. By observing the peaks at 753, 1127, 1314, and , this technique can be used to detect cytochrome c in living cells by resonant Raman scattering. We also measured Raman spectra from the nuclear regions of living HeLa cells (not shown). The Raman spectra from the nuclei were similar, and no substantial spectral dependence on the excitation wavelength was observed, which shows that resonant Raman signals were not a significant contribution to the total Raman emission from the nuclear regions.
We also investigated the contribution of autofluorescence to the background signal in the Raman spectra, which is of particular interest since spectroscopic sensitivity is dramatically reduced by any background contributions due to autofluorescence. The flavin coenzymes FAD and FMN are known to be sources of autofluorescence in the detected wavelength range.10, 11 Lipofucin is another possible source of autofluorescence; however, it absorbs light predominantly in the UV region and is not significantly excited by the wavelengths used in this experiment.12 We measured the average fluorescence intensity of FAD at the regions between 600 and for excitation wavelengths of 488, 514.5, and . We found that excitation produced an autofluorescence signal approximately 12 times lower than excitation, and 167 times lower than excitation. The autofluorescence background signal for excitation light was markedly decreased compared to excitation, and the Raman scattering signals were of comparable strength, which shows that excitation is superior for imaging living cell samples.
Slit-Scanning Confocal Raman Microscopy
We used a home-made Raman microscope with excitation and slit-scanning excitation and detection.13, 14 The slit-scanning technique allowed us to detect Raman spectra from different positions in parallel, and as a result, greatly improved the image acquisition rate. The sample was irradiated by a line-shaped focus, and Raman scattering signals from the illuminated line were imaged at the entrance slit of a spectrophotometer. Line illumination is also useful in reducing photodamage of the sample because the light intensity at the focal plane is much lower than that of single-focus scanning at the same exposure. Additionally, the slit of the spectrophotometer eliminates Raman scattering from out-of-focus planes, providing spatial resolution in three dimensions and improving of image contrast.15 To produce the line-shaped laser light, we used a cyrindrical lens and imaged the illumination line at the sample by a water immersion objective lens.
Raman Scattering Images of an Unstained Living Hela Cell
Using our slit-scanning Raman microscope, we obtained a hyperspectral image of living HeLa cells in the range of Raman shifts between and . The cell was observed in a HEPES-buffered Tyrode’s solution composed of (in mM) NaCl, 150; glucose, 10; HEPES, 10; KCl, 4.0; , 1.0; , 1.0; and NaOH, 4.0. Then the cell was irradiated with a light intensity of at the focal plane. Singular value decomposition (SVD) was used for noise reduction, and we chose seven loading vectors that significantly contribute to the image contrast for the image reconstruction.4 Following noise reduction, we subtracted the fluorescence background signal from the Raman spectra at each pixel in the image by a modified polyfit fluorescence removal technique.16
The distribution of cytochrome c is reconstructed in Fig. 2a from the intensity distribution of the Raman peak at . Since cytochrome c is used for electron transfer in oxidative phosphorylation in mitochondria, the image has a contrast similar to the distribution of mitochondria. Figure 2b shows the Raman signal distribution at given by the Amide-I vibration mode of peptide bonds in protein beta sheets.7 We chose this wave number because the C-C stretching vibration modes in hydrocarbon chains of lipid molecules overlap the shorter Raman shifts of the Amide-I band. Since beta sheets are commonly seen in proteins, Fig. 2b is strongly correlated with protein distribution in the cell, and consequently, there is a slightly higher protein concentration at the nucleus. Figure 2c shows the Raman signal distribution at where the signal due to the stretching vibration is strongly detected from the hydrocarbon chain of lipid molecules. Although proteins and other biological molecules also contain , the image contrast is provided mainly by the lipid vesicles that are rich in lipid molecules.17 By combining these images via the different color channels of a single image, we obtained the distributions of protein beta sheets, cytochrome c, and lipid vesicles shown in Fig. 2d.
Raman Observation of Dynamic Distributions of Biomolecules
Time-resolved, dynamic molecular distributions are shown in Video 1 , where Raman images of a living HeLa cell were taken during cytokinesis. The image acquisition time for each Raman image was , with an interval between images of . The images in Video 1 were obtained with 48 line exposures of each, for a total exposure time of . The difference between the image acquisition time and the exposure time is due to the data transfer time from the CCD camera to the data storage computer. For Video 1, we also applied noise reduction by the use of SVD, and the images were reconstructed using five loading vectors. The contrast due to Raman scattering in Video 1 indicates that proteins exist in relatively higher concentrations at the chromosomes than in other parts of in the cells, which allows us to trace the progress of cytokinesis by the temporal variation of protein distribution. We also observe that highly concentrated cytochrome c appeared near the cleavage furrow, presumably to provide sufficient energy to the contractile ring that divides the cell into two. In addition, the movement of lipid vesicles associated with cellular dynamics during mitosis can be discerned. During extended observation, photodamage of the cell is a possibility; however, our results show that any photodamage which may have occurred was not significant enough to stop cytokinesis from proceeding.10.1117/1.2952192.1
Using the Raman microscopy method described here, we demonstrated label-free observation of biological molecules in living cells using Raman scattering for a contrast mechanism. Label-free imaging provides us with opportunities to observe biological activities without the disturbances of labeling procedures and agents that usually degrade the viability of samples. It frees us from the photobleaching problems inherent in fluorescence staining techniques and allows us to obtain distributions of chemicals in samples that are impossible to stain or in locations where staining is undesirable. We also showed that the resonant Raman scattering of cytochrome c can be distinctly observed using a wavelength for excitation, allowing label-free observation of cytochrome c distributed in living cells. Cytochrome c is a protein well known for its important role in the production of ATP in mitochondria, and the distribution of cytochrome c is thought to change drastically during apoptosis.18, 19 The Raman imaging technique demonstrated in this paper will allow in situ studies of the role of cytochrome c in apoptosis as well as its function in other cellular activities.