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Spontaneous Raman microscopy is a potentially useful technique for imaging living cells, tissue and small animals without any probe or dye labeling. We have developed a spontaneous Raman imaging system in wide-field view, which we term ‘light sheet-excited direct Raman spectroscopy’ (LSDRS). This system, which we reported previously, consists of a background-free electrically tunable Ti:Sapphire laser (BF-ETL), a cylindrical lens, a CCD camera, and a narrow bandpass filter. Here, we have adapted the LSDRS system for microscopy systems, such as single-plane illumination microscopy (SPIM) for biomedical applications, and demonstrated spontaneous Raman imaging of a living fish. The results suggest that our Raman microscopy system enables investigation of the differentiation process and mechanism of iridocytes during development. This is the first report in which Raman imaging of a living animal was successfully demonstrated by spontaneous Raman scattering signals, but not nonlinear Raman effects such as CARS and SRS.
©2012 Optical Society of America
1. Introduction
Raman spectroscopy is potentially useful in real-time monitoring of biological samples without staining or induction of extrinsic probes. Recently, Raman spectroscopy has been explored in the context of biomedical applications because it can provide detailed information about the chemical composition of cells and tissues [1–5]. The Raman spectroscopy technique could be applied to molecular imaging in the visualization, characterization and measurement of biological processes at the molecular and cellular levels. Meanwhile, fluorescence imaging, a conventional molecular imaging technique, is used in fluorescence microscopy, which is an essential tool in biology and medicine. A number of fluorescent dyes and fluorescent proteins have been developed to visualize biomolecular dynamics in living cells and tissues. Wide-field fluorescence microscopy and confocal fluorescence microscopy are widely used for imaging analyses, both in vitro and in vivo. In particular, confocal fluorescence imaging enables generation of images with high spatial resolution and high signal-to-noise ratio, due to the combination of the laser scanner and the pinhole in front of the photomultiplier. However, photobleaching and phototoxicity are inevitable problems in confocal microscopy. Furthermore, image blurring occurs in deeper portions of tissue, because of scattering from outside of the focal plane. In order to overcome these problems in fluorescence microscopy, the light sheet microscope was developed. Light sheet microscopy is based on wide-field microscopy, but sheet-shaped illumination perpendicular to the angle detection is employed in place of epi-illumination. Single-plane-illumination microscopy (SPIM), which was reported by Engelbrecht et al., has been applied to live cell imaging in the field of developmental biology [6–10]. In light sheet microscopy, only a thin slice of the specimen is illuminated by the excitation laser and imaged on to a CCD detector through an objective and a tube lens. Because the light sheet is settled at the focal plane of the objective, no fluorescence emission and Raman scattering occur except the focal plane. This optical sectioning effect allows not only no out-focus blur but also to suppress a phototoxicity and bleaching of the fluorophores [11].
In Raman imaging techniques, nonlinear Raman effects, stimulated Raman scattering (SRS) and coherent anti-stokes Raman scattering (CARS) have been employed in order to image biological samples [12–16]. The nonlinear Raman signal is several orders of magnitude stronger than spontaneous Raman scattering. When a femto-second laser source is employed, the excitation source is amenable to inducing both the CARS and the multi-photon fluorescence. However, the experimental setup is complicated by the need for both pump beam and stocks beam for generation of an observable signal. In contrast, spontaneous Raman scattering can be observed using a single laser and a spectroscopic device. For biomedical applications, development of imaging techniques with spontaneous Raman scattering is still challenging, because the scattering signals from cells and tissues are usually extremely weak. Confocal mapping would enable generation of a clear image; however, as acquisition of a Raman image takes a long time, simultaneity of the image would be lost. In one biomedical application of spontaneous Raman imaging, slit-scanning confocal Raman microscopy was reported by Hamada et al. [17]. Those authors irradiated unstained living HeLa cells with a line-shaped excitation laser at 532 nm, imaged Raman scattering at a spectrometer slit equipped with a CCD detector, and reconstructed a hyperspectral image. The images were taken at a relatively high frame rate, 185 sec/image. Furthermore, wide-field Raman microscopic techniques have been developed in recent years. Single-plane illumination Raman imaging, incorporating SPIM and Raman imaging, has been reported by Barman et al. [18]. Those authors demonstrated that optically sectioned wide-field Raman images of polystyrene beads of 20 μm in diameter could be obtained using a single laser source and spectrometer. The authors also developed a wide-field Raman imaging system associated with the light sheet-based technique, for use in biomedical applications. We have previously described a light sheet-based Raman imaging technique [19], “light sheet-direct Raman spectroscopy (LSDRS)”, which incorporated a background-free electrically tunable Ti:Sapphire laser (BF-ETL) [20, 21] to allow multi-wavelength-excitation spontaneous Raman imaging of chemical components. Although several papers associated with the light sheet-based Raman imaging technique have been published, as mention above, no one has reported a biomedical application of light sheet-excited Raman microscopy.
Light sheet-based Raman imaging has great potential as a practical tool for studies in basic biology and medicine. We modified our original LSDRS system to obtain single-plane-illumination Raman image from a living teleost fish. A near-infrared laser was used for excitation, because it could suppress the photodamage and avoid generating autofluorescence in this sample. In our system, the excitation wavelength has to be tuned to an observation for extremely weak Raman signal. BF-ETL is much better at the accuracy, stability and rapidity than a typical tunable laser. That’s why we employed BF-ETL as the laser source for the Raman imaging. The laser was shaped into a sheet using a cylindrical lens, and the sample was placed in water. Raman scattering from the sample was collected using an objective lens, via a custom-made bandpass filter, and imaged using a tube lens projecting onto a highly sensitive CCD detector. This is the first report of light sheet excitation Raman imaging performed in a living organism. Teleost fish was used for this study because of their availability and versatility as experimental organisms. Medaka fish and zebrafish are the most commonly used experimental teleost fish in basic biology and experimental medicine. The light sheet excitation Raman microscopic technique is directly applicable to phenotyping analysis based on genome databases.
2. Materials and methods
2.1 Light sheet Raman microscope
A schematic diagram of the light sheet Raman microscopic system is shown in Fig. 1(a) . A background-free electrically tunable Ti:Sapphire laser (BF-ETL), pumped with Nd:YAG SHG nano-second pulse laser (532 nm, repetition rate 1 kHz, pulse width 60 ns; SXG-10LR Megaopto Co., Ltd.) was employed as the excitation light source. The detailed specifications of the BF-ETL are described elsewhere [20, 21]. Excitation wavelengths can be tuned from 720 to 830 nm by AOTF in this system. Output laser power was typically 60 mW. Laser beam diameter was adjusted using an afocal lens system, and the light sheet was formed using a cylindrical lens with a focal length of 40 mm. The sample was positioned at the beam waist position. Raman scattering was collected perpendicularly to the light sheet using an objective lens (Olympus LMPLN 5x IR) for lower-magnification observations (Fig. 1(b)). For higher-magnification observations, a water immersion objective lens (Olympus UMPLFLN 20xW) and a home-made chamber were used; its windows were made of fused silica glass (Fig. 1(c)). The sample was suspended from a motor stage unit above the chamber. A custom-made narrow bandpass filter (IRIDIAN CWL 830 nm FWHM 2.5 nm) was employed to detect the Raman peak; the filter also reduced the Rayleigh scattering signal and stray light. A tube lens of focal length 200 mm and a highly sensitive CCD detector (iKon-M DU-934N-BRD, 90% QE beyond 800 nm, active pixels 1024 x 1024, pixel size W x H 13 μm x 13 μm, image size 13.3 mm x 13.3 mm, thermoelectric cooling down to −60 degree ANDOR) were used for taking Raman images.
Fig. 1 The light sheet Raman microscopic system. (a) A schematic diagram of the experimental setup of the microscope with the light sheet illumination from the overview and the sideview. The sample was settled at the focal point crossing the illumination laser beam. (b) For lower magnification, the sample was put into a quartz cuvette and was observed with a 5X objective via air. (c) For higher magnification, the sample was settled into the home-made-chamber and was observed by a water immersion lens (20X).
2.2 Sample preparation
A see-through mutant strain of Oryzias latipes (Quintet), which has colorless chromatophores, was provided by NBRP Medaka (National BioResource Project Medaka). The young fishes were used within a week of hatching for acquisition of live Raman images and Raman spectra. Fish were embedded into 1% low melting temperature agarose gel (SeaPlaque GTG Agarose, LONZA) in a silica glass cuvette or cutting syringe prior to light sheet microscopy. For Raman microspectroscopy, the fish was embedded into the gel on 35 mm culture dish. In both cases, the sample gel was immersed in water to avoid drying during observation. Purified guanine (075-01093, Wako Pure Chemical Industries, Ltd.) was also embedded into the agarose gel for Raman imaging analysis.
2.3 Acquisition of Raman images
Samples were irradiated with sheet-shaped laser light during the acquisition of Raman images. Exposure time was 10 seconds for each of 6 acquisitions, i.e., a total of 60 seconds. Different Raman images were taken at different excitation wavelengths. The excitation laser power increased gradually as the wavelength longer (data not shown). A typical emission power was 60 mW.
2.4 Measurement of Raman spectra
Raman spectra of various anatomical regions of Quintet fish were measured using a laser Raman microscope (RENISHAW In via Raman microscope). The excitation laser (785 nm, 10 mW) was focused on the sample via a 60x water-immersion objective lens (Olympus LUMPLFLN 60xW, NA = 1.0). The exposure time was 60 seconds; the grating had 1200 lines/mm. The spectral resolution was less than 2 cm−1, and the diameter of the laser spot at the focal point was less than 1 μm.
3. Results and discussion
Figure 2(a) depicts a dark-field image of a young fish (Quintet line) taken using the light sheet microscopic system. The field of view (FOV) of the image is 2.349 x 2.349 (mm x mm). The properties of the light sheet were investigated by observing acetone. Figure 2(b) shows a Raman image of acetone excited at 779 nm. Raman scattering from acetone fluid that was generated by the light sheet illumination appeared along with the path of the laser. The signal disappeared when the excitation wavelength was switched to shorter or longer wavelengths. i.e., the signal was not Rayleigh scattering but Raman scattering, due to the dependency of the excitation wavelength. The center of the light sheet has the highest excitation intensity due to Gaussian intensity distribution. Once the sample is away from the center of the light sheet, the excitation effect will decrease along the sheet and the Raman signal may disappear. Judging by the distribution of the Raman signal intensity of acetone shown in Fig. 2(b), the effective width of the light sheet is approximately 300 μm. In this observation, the fish was enough small to observe at the light sheet width. The thickness of the light sheet, which determined the size of the optical section in this microscopic system, was estimated to be 62 µm, due to the performance of the cylindrical lens and the properties of the laser beam. In this condition, the excitation wavelength swept from 720 nm to 780 nm at 5 nm intervals, and images were taken at each excitation wavelength (see Media 1).
Fig. 2 (Media 1) Light sheet−excited spontaneous Raman images of a living fish. (a) A dark-field image of a young fish (Quintet line) taken by oblique illumination. (b) Raman image of acetone (fluid) in a cuvette that illuminated by 779 nm of the excitation laser sheet by which Raman signal of acetone can be observed. (c) Raman image of the Quintet taken by the excitation at 740 nm. (d) Merged image of (a) and (c). (e) Raman image of the Quintet taken by the excitation at 770 nm. (f) Merged image of (a) and (d).
At 740 nm, Raman scattering was detected concurrently with autofluorescence (Fig. 2(c) and 2(d)). A large bright spot in the center may represent the autofluorescence signal; several small signals (pointed with white arrows) may reflect Raman scattering (indicated in pseudocolor). Another excitation wavelength at 770 nm was chosen and imaged (Fig. 2(e) and 2(f)). At this wavelength, no Raman-like signal appeared, and only the autofluorescence signal was observed. Although the excitation laser intensity at 770 nm was higher than at 740 nm, the small signals that appear in Fig. 2(c) and 2(d) are absent in Fig. 2(e) and 2(f). A large signal in intestines was observed over the whole range of the excitation wavelength. In contrast, the Raman-like signal was observed in the eyes, and in a part of the head called the lateral patch (white arrows). To quantitatively verify these two different types of signals, regions of interest (ROIs) were defined on the image. Figure 3 shows ROIs encompassing the intestines (ROI 1), lateral patch (ROI 2) and eye part (ROI 3) and plots of mean signal intensity as a function of excitation wavelength for each ROI. This analysis suggests that the large and small signals have different origins: autofluorescence and Raman scattering, respectively. The origin of autofluorescence could be pigment in gall bladder. Medaka fish has metabolic intermediate of hemoglobin in gall bladder, spleen and liver. The distribution of the autofluorescence signal agrees with the location of these organs in Fig. 2(d) and 2(f). Furthermore, the Raman-like signal from the eye and lateral patch may be assigned to identical molecular components, because they exhibit the same dependency on excitation wavelength. According to the relationship between the excitation and observation wavelengths, the signal with a peak at 740 nm corresponds to the Raman shift around 1465 cm−1.
Fig. 3 Image intensity profile of the Raman and autofluorescence signals. The ROIs were defined for Fig. 2(c). Averaged intensities were plotted to the excitation wavelengths to be observed. Lateral axis were indicated with wavenumber unit for Raman shifts and the wavelengths for the excitations.
To identify the origin of the Raman signal observed in the light sheet microscope system, we also performed spectroscopic analysis of Quintet medaka. Figure 4 shows Raman spectra of Quintet medaka. At this developmental stage, pigmentation that forms iridocytes can be observed at the eyes and lateral patch. Raman spectra of eye, muscle and bone were measured. Figure 4(a) depicts iridocytes in the eye. The reflecting platelets consist mainly of purine, guanine, hypoxanthine and adenine [22, 23]. Figure 4(b) shows a magnified picture of the body. Raman spectra were obtained from the body trunk (muscle) and a bony region (spine). Figure 4 shows raw Raman spectra collected from each body part of Quintet medaka. In the Raman spectrum of iridocytes in the eye, which yielded the highest signal, several sharp bands (650, 930, 1267, 1360, 1465, and 1546 cm−1) may be assigned to guanine (Fig. 4, spectrum (a)) [24]. The Raman spectra of iridocytes in the eye and the lateral patch are very similar (data not shown). This result agrees with the Raman image in Fig. 2 with regard to the localization of iridocytes and the peak wavelength of the signal. The muscle also has the typical Raman spectrum of proteins (Fig. 4, spectrum (b)). The peaks at 1003, 1200−1350, 1450 and 1655 cm−1 can be assigned to the vibrational modes of phenylalanine: Amide III, CH2 bending, and Amide I, respectively [25, 26]. The Raman spectrum of the bony spine has no peak corresponding to calcium phosphate (~960 cm−1), but does have small peaks at 1003 and 1655 cm−1 that may originate from muscle surrounding the spine (Fig. 4, spectrum (c)). The reason why the calcium phosphate peak is absent from the spectrum is that the bone tissue may not be mature at this developmental stage. The agarose gel was measured in the same manner as the background component (Fig. 4, spectrum (d)). Thus, agarose did not affect measurement of Raman spectra or Raman images in this experiment. We tried to measure Raman spectrum of the intestine, but the autofluorescence background was too high to obtain a Raman signal.
Fig. 4 Raman spectra of Quintet medaka. (a) Iridocyte in the eye, (b) muscle in the body trunk, (c) spine, and (d) agarose gel in which the fish was embedded were measured by the commercially-available Raman microscope.
We also used higher-magnification equipment, in order to achieve Raman imaging with a higher signal-to-noise ratio, by using another objective lens (20x, numerical aperture 0.5). The optical setup for making the light sheet was not modified; the light sheet’s properties were the same as in the previous demonstration. Wavelength of the excitation laser was swept at intervals of 1 nm. Figure 5 depicts dark-field images and Raman images of the body and the eye, exposed to laser illumination at 740 nm and 745 nm, respectively. FOV of the image is 0.599 x 0.599 (mm x mm). Raman signals appeared in the body trunk as well as in the eye (Fig. 5(c) and 5(d)). Bright Raman images were obtained successfully after only 2 seconds of exposure in a single acquisition. Raman signal appeared along the dorsal stripe (spinal cord), as shown in Fig. 5(c). The plot curve of the images in the dorsal stripe was nearly identical to that of the images obtained from iridocytes in the eye. Hence, the origin of Raman signals both in the eye and the body trunk are probably the same. The image intensity curve is regards as an excitation Raman profile of iridocytes. Hence, the excitation Raman curve converted wavelength to Raman shift in x-axis is nearly equivalent to the usual Raman spectrum. Our original multiwavelength laser source, BF-ETL made it possible to obtain the hyperspectral Raman images not only with high speed acquisition, but also with high spectral resolution in this light sheet-excited microscopic system.
Fig. 5 Magnified images of the Quintet medaka taken with the 20X water immersion objective. (a) A dark-field image of the body trunk taken by oblique illumination and, (b) the Raman image taken by the excitation at 740 nm. (c) A dark-field image of the eye taken by oblique illumination and, (d) the Raman image taken by the excitation at 745 nm.
We also observed purified guanine powder in the system to verify the origin of the Raman signal from iridocytes. Figure 6 shows image intensity plots for the iridocytes and purified guanine. A peak around 770 nm, marked with an asterisk (*), could be attributed to stray light from the substrate in the optics or chamber window. No image contrast could be obtained at 770 nm (data not shown). Our observation used medaka of juvenile stage, when iridocytes are not observed in the dorsal stripe by bright field microscopy. The Raman signal was however detected in the both dorsal and ventral (left and right in Fig. 5(c)) sides, and the signal was stronger at the ventral than the dorsal. This pattern well agrees with the distribution of iridocytes in the adult medaka. Therefore, it is likely that the Raman signal shows guanine localization of chromatophore progenitor cells before differentiation. Any other methods cannot detect this kind of localization of guanine in the living fish.
Fig. 6 Image intensity profile of the Raman signals from iridocytes and guanine. Averaged intensities were plotted to the excitation wavelengths to be observed. Lateral axis were indicated with wavenumber unit for Raman shifts and the wavelengths for the excitations. A peak with an asterisk (*) could be stray light.
4. Conclusion
We developed light sheet Raman microscopy for biomedical applications. In this report, unstained molecular imaging of a living fish was demonstrated using a novel optical sectioning method based on the SPIM technique. This is the first report of light sheet-excited Raman imaging of a living specimen. In our results, bright Raman images were obtained after exposures of less than 1 minute. This new technique has a potential as a practical tool for phenotyping the chromatophores of medaka and zebrafish over the course of development. Since this is a Raman-based imaging technique, it can be combined with other laser applications, such as laser micro-dissection (LMD) or the infrared laser-evoked gene operator (IR-LEGO) system [27]. Furthermore, the light sheet technique is applicable to macro-imaging systems, such as cancer diagnosis using endoscopy or intra-operative margin assessment in medicine [28, 29].
Acknowledgments
The medaka were provided from NBRP Medaka (National BioResource Project Medaka). The authors are grateful to Ms. Yasuko Ozawa and Dr. Hiroyuki Takeda for allowing use of the Quintet medaka strain. The Raman spectral measurement was performed by using facilities of Instrument Center in Institute for Molecular Science (IMS). Funding for this work was supported by Grant-in-Aid for Scientific Research, JSPS (Japan Society for the Promotion of Science), SENTAN (Development of systems and technology for advanced measurement and analysis) and CREST (Core Research for Evolutional Science and Technology), JST (Japan Science and Technology) and the Hori Science and Arts Foundation.
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