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Abstract
A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Raman microscopy is a powerful method for chemically selective, non-invasive, label-free single-cell-analytical imaging for investigating living cells without cell disruption [1–9]. Thanks to the molecule specific vibrational contrast, various kinds of intracellular metabolites have been visualized in a label-free manner [10–20]. Since ordinary Raman scattering is a weak process, coherent Raman scattering such as coherent anti-Stokes Raman scattering (CARS) [10–12] and stimulated Raman scattering (SRS) [13–15,19,21] processes have been widely employed to boost the weak Raman signal, and obtain Raman images with high sensitivity and high speed. Recently, coherent Raman microscopy has been extended to a spectroscopic imaging technique [7,6]. In particular, ultra-broadband multiplex CARS spectroscopic imaging [22–25] enables us to obtain both CARS images and spectra with a spectral coverage of approximately 3000 cm−1, spanning the entire region of the fundamental vibrational modes [22,26–29]. CARS spectroscopic imaging has been applied to a study on liquid-liquid phase separation in vitro [30] and in-vivo visualization of algae [31,32] and bacteria [25,33], liver diagnosis [34], enzymatic reaction [35], and spatially resolved omics [36], revealing intracellular accumulation of a wide variety of metabolites. Moreover, new techniques have been reported using CARS spectroscopic imaging such as super-resolved imaging [37], surface-enhanced imaging [38], and Fourier-transformation CARS imaging [39].
Regarding the application of CARS microscopy in life sciences, the forward scattering properties of CARS can pose a practical problem. To detect the strong forward CARS signal enhanced with the phase matching condition [40], another objective lens is typically used on the opposite side of the illumination objective lens [3–5]. However, in the inverted microscope commonly used in the life science field, only one objective lens is used for both illumination and detection. This configuration makes it difficult to install a forward CARS detection system.
One solution to this problem is to use weak backward CARS detection. Several studies have reported the detection of backward CARS using an inverted microscope or a fiber endoscope with a pulsed laser source [2,41–43]. This backward CARS signal is primarily due to a diffused forward CARS signal from opaque samples such as tissue (see Fig. 1), which produces a relatively strong backscattered forward CARS signal. However, in thin, transparent samples such as cells, the diffused forward CARS signal is negligible and only a weak, phase-mismatched backward CARS signal appears [40,44,45]. To date, there have been no report on backward CARS spectra under a microscope, except for the results obtained from measuring the diffused forward CARS spectra [41]. Therefore, signal amplification techniques are needed to perform spectroscopic imaging of the backward CARS signal at a rate comparable to that of forward CARS.
Fig. 1. Illustration of the CARS signal generation. Concerning the backward configuration, the scattering of the forward CARS signal is dominant for opaque samples. On the other hand, for transparent or thin samples such as cells, the inherent and weak backward CARS signal is mainly detected.
Here we used an electron-multiplying charge coupled device (EM-CCD) camera to improve the detection efficiency and signal-to-noise ratio (SNR) of the backward CARS signal. Although EM-CCD cameras have been employed for spontaneous Raman microscopy [16,18,20,46,47], the application to coherent Raman microspectroscopy has still been limited [48]. This is because most studies on coherent Raman microspectroscopy, including ours [28,29], focus on the forward CARS detection, for which a conventional CCD camera is sufficient [33]. First, we describe the experimental setup with an EM-CCD camera. We then evaluated the SNR and impact of EM gain. Finally, we demonstrated CARS imaging of epithelial cells.
2. Methods
2.1 Backward multiplex CARS spectroscopic imaging system
The backward multiplex CARS signal was collected by a home-made nonlinear optical microscope equipped with a custom-made, dual-fiber output synchronized light source (OPERA HP, Leukos, Limoges, France) [33], which was modified for the backward signal collection as shown in Fig. 2. The first laser output had a wavelength of 1064 nm, a pulse width of 50 ps, and a repetition rate of 1 MHz, and was used as the pump beam (ω1) for the CARS process. The second laser output was passed through a photonic crystal fiber (PCF) to provide supercontinuum (SC) radiation ranging from visible to near infrared (NIR) with a broad spectral range of 1100-1800nm, which was used as the Stokes beam (ω2). The pump and Stokes laser beams were superimposed by an edge filter (LP02-1064RE-25, Semrock, Rochester, NY) and were guided into a modified inverted microscope (ECLIPSE Ti, Nikon Corporation, Tokyo, Japan). Two laser beams were tightly focused on the sample through an objective (CFI Plan Apo 60x NA 1.27, water-immersion, Nikon Corporation). The sample was placed on a piezoelectric stage (Nano-LP200, Mad City Labs, Madison, WI) for position selection. The full scanning range of the xyz-piezo stage was 200 µm3. The backscattered CARS signal (2ω1 - ω2) was spectrally separated by a dichroic mirror (Di02-R980-25 × 36, Semrock, NY), and was detected using a spectrometer (Kymera 193i, Andor Technology, Belfast, UK) equipped with an EM-CCD camera (Newton DU 970P, Andor Technology, Belfast, UK) to enhance the weak CARS signal. The groove and blaze wavelength of the grating in the spectrometer were 300 G/mm and 1200 nm, respectively. The spectral coverage and spectral resolution of the CARS signal were ∼3500 cm−1 and 14 cm−1, respectively. The forward CARS signal can also be collected using the second objective (Plan S Fluor 40× NA 0.6, Nikon Corporation), and detected using the same spectrometer and the EM-CCD camera just by switching the flip-mounted mirror indicated by A in Fig. 2.
Fig. 2. Experimental setup of a backward multiplex CARS spectroscopic imaging system with an EM-CCD camera. The conventional forward CARS signal can also be obtained using the same detector by flipping the mirror mount indicated by A.
2.2 Sample preparation
We utilized polystyrene beads (Polysciences Inc.) with a diameter of 10 µm to evaluate the performance of the present system. The original aqueous suspension of the beads was diluted more than 10 times, and approximately 5 µl of the suspension was sandwiched between a cover glass and a glass slide, then sealed with nail polish.
EpH4 cells derived from a mouse mammary epithelial cell were used as cultured cell samples. Cells were cultured on coverslips soaked in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and incubated in a humidified incubator containing 5% CO2 in air at 37 °C for 3 days. The coverslip was removed from the medium immediately before measurement. The cells on the cover glass were flipped over, sandwiched with a glass slide, and sealed with nail polish. An on-stage incubator was useful in particular for time-lapse CARS study [49], however in the present study an incubator was not necessary due to the short overall measurement time.
3. Results and discussion
3.1 Evaluation of EM-gain dependence of the backward CARS signal
The performance of the system with an EM-CCD camera was evaluated using the backward CARS signals of polystyrene beads with a diameter of 10 µm. Figure 3(a) shows CARS spectra with the EM-gain value of 200 and of 255 (maximum). For reference, the CARS signal with the conventional mode is also shown. We used photo-generated electrons as a vertical axis rather than the CCD counts to compare the EM mode with the conventional mode, because the conversion ratio of photo-generated electrons to CCD counts was different between the EM mode (4.3 count/electron) and the conventional CCD mode (1.3 count/electron). The CARS spectra with the EM gain 255 and 200 indicate that the signal intensity is not proportional to the value of the EM gain, and the signal is amplified enough to be visible in comparison with the conventional CCD. We also evaluated the issue of etaloning on the back-illuminated CCD, which gives fringing in the spectral profile in the NIR region. The 1064-nm pump generates the CARS signal in the range of 746-1010 nm. To suppress the effect of etaloning in this range, back-illuminated, deep-depletion CCD cameras are often used in measurements [28,29]. However, deep-depletion type EM-CCD cameras are not currently available. Since the EM-CCD camera used in this study consists of the normal back-illuminated CCD, the etaloning may be visible in the spectral range in the present study. Due to the relative intensity of the noise in the CARS spectrum (Fig. 3(a)), however, the impact of fringing is not prominent in the backscattered CARS signal. On the other hand, it is noticeable in the forward CARS signal in the fingerprint region shown in Supplement 1, Fig. S1.
Fig. 3. Backward CARS signal detected by an EM-CCD camera. (a) Spectral profiles of the raw CARS signal of a polystyrene bead with the EM-gain value of (i) 255, (ii) 200, and (iii) conventional mode. (b) EM-gain dependence of the photo-generated electrons corresponding to the CARS signal intensity (red). The CARS signal at 1003 cm−1 is evaluated. The vertical axis is indicated as enhancement factor, which is calculated by dividing the CARS signal intensity at each EM-gain value with that at the conventional mode. The exposure time was 50 ms on the software, corresponding to 18 fps. The solid lines represent the results fitted with an exponential function for the eye guide phenomenologically. It is proportional to exp(0.01x) for x < 100, while exp(0.03x) for x > 150, respectively. Here x represents the EM-gain value.
Next, we will focus on the nonlinear dependence of the CARS signal on the EM-gain value. Figure 3(b) depicts the EM-gain dependence of the backward CARS signal at 1003 cm−1. As shown in Fig. 3(a), the signal exhibits a sharp vibrational resonance attributed to the phenyl-ring breathing mode. The vertical axis in Fig. 3(b) is indicated as enhancement factor, which is calculated by dividing the CARS signal intensity at each EM-gain value with that at the conventional mode. The laser power for ω1 and ω2 was fixed to approximately 45 mW and 170 mW, respectively. The exposure time at each pixel was 50 ms, corresponding to 18 frame-per-second (fps) due to finite readout time. As pointed out in Fig. 3(a), the CARS signal intensity is amplified nonlinearly with respect to the EM gain. It initially increased roughly with exp(0.01x) against the EM-gain value x (x < 100), then increased with exp(0.03x) when x > 150. In both cases, the signal intensity was found to increase exponentially against the EM-gain value. Figure 4 summarizes the results of backward CARS imaging of polystyrene beads. Figure 4(a) indicates a bright field image. Figure 4(b) shows the comparison between the conventional mode and the EM-gain mode with the value of 255. The spectral profiles indicated by (i) and (ii) in Fig. 4(b) correspond to the raw CARS signals at the red crosses in Fig. 4(c) and (d), respectively. The aromatic CH stretching (3060 cm−1) and phenyl-ring breathing (1003 cm−1) vibrational modes were observed at the gray hatched regions for the EM mode (i), while these resonances were hardly observed for the conventional mode. Figure 4(c) and (d) show backward CARS images at 1003 cm−1 for the conventional and EM modes, respectively. The exposure time per pixel was 0.57 ms (on the software). Due to the finite readout time, the frame rate was 440 fps. The line profiles below the images shown in Fig. 4(e) and (f) correspond to the dotted horizontal line in Fig. 4(c) and (d). Comparing the two images, the CARS image in the EM mode gives a much clearer contrast than that in the conventional mode. The EM mode gives distinct signal intensity at the position of a bead. Therefore, it was demonstrated that the EM mode is better than the conventional mode.
Fig. 4. Backward CARS imaging of polystyrene beads with an EM-CCD camera. (a) Bright field image. (b) Spectral profiles of the raw backward CARS signal indicated by (i) and (ii), corresponding to the red crosses at (c) and (d). (c and d) Backward CARS images at 1003 cm−1 by the conventional mode (c) and EM-gain mode at the value of 255 (d). (e and f) Line profiles of the broken lines in (c) and (d). Inset in (d): binary image converted from (d). The white and black areas correspond to the signal and noise areas, respectively. The white areas were defined as noise and black areas as signal to calculate SNR. The image size (pixels and microns) and step size were 101 × 101, 50 × 50 µm2, and 0.5 µm, respectively. The scale bar is 10 µm. The exposure time was 0.57 ms on the software. Due to the finite readout time, the frame rate was 440 fps.
In order to evaluate the improvement quantitatively, we compared SNR in the EM mode with that in the conventional mode. To calculate SNR, white and black areas indicated in the inset of Fig. 4(d) were used as the noise and the signal regions. The SNR is calculated using the following equation,
whereHere $SN{R_{\textrm{RMS}}}$ is the SNR calculated by the RMS method. ${I_{\textrm{av}}}$ is average value of the CARS signal at the signal region indicated in Fig, 4(d), ${N_{\textrm{av}}}$ is average value of the noise at the noise region in Fig. 4(d), ${N_{\textrm{RMS}}}$ is the RMS, ${N_\textrm{i}}$, is the noise at i-th spatial pixel, n is the total number of the pixels at the noise region. The values of the SNR were calculated to be 0.73 and 1.98 for the conventional and the EM-gain mode at the value of 255, respectively. Based on these results, the SNR was improved by a factor of ca. 3 for the EM mode, indicating the usefulness of an EM-CCD camera in the backward configuration.
Finally, we will focus on the unique image formation using the backward CARS signal. The backward CARS image produces low contrast (dark) around the center of beads, while the image produces high contrast (bright) at the peripheral part of the beads, which can be understood by considering the phase-matching condition [40]. Around the center of a bead, the CARS radiation builds up in the forward direction through the focal volume, due to the phase matching condition. This is because the bead diameter (10 mm) is sufficiently larger than the focal volume (∼5 µm [33]). On the contrary, the CARS signal in the backward direction is efficiently suppressed by destructive interference, at the expense of constructive interference in the forward direction. Similar phenomena were observed in third harmonic generation (THG) imaging in the forward observation [50]. Intracellular lipid droplets (LDs) can be visualized by the THG signal. Small LDs give THG spots, while large LDs appear as rings.
Since the coherent buildup of the CARS radiation does not perfectly take place at the peripheral part of beads, the backward CARS signal is relatively more intense at the peripheral than at the center of beads. On the contrary, the situation is the opposite of the forward CARS image (Supplement 1, Fig. S1) which shows high contrast at the center of beads (inset in Supplement 1, Fig. S1).
3.2 Backward CARS imaging of EpH4 cells
To evaluate the applicability of this method to the life sciences, we demonstrated backward CARS imaging of EpH4 cells with the EM-gain mode. As for the cultured cell, the backward CARS signal is weak, and the forward CARS signal is hardly detected in the backward direction due to the thickness and transparency of the cells. Figure 5 represents the backward CARS image at 2850 cm−1 (CH2 stretching mode due to lipids) and 2930 cm−1(CH3 stretching mode due to protein and lipids) with the exposure time of 100 ms/pixel, accompanied with bright-field image obtained after the measurement. We used laser powers with approximately 250 and 170 mW for ω1 and ω2. Intracellular small organelles on the order of a few micrometers were visualized with an exposure time of only 100 ms/pixel. [44,45], small structures in the orders of incident laser wavelength are mainly observed, because backscattered forward CARS signal is negligible for EpH4 cells, and inherent backward CARS signal is dominant. Particles of approximately 2 µm are observed in CARS images both at 2850 cm−1 and 2930 cm−1 in Fig. 5(a). In addition, structures of approximately 5 µm can be seen in the CARS image at 2930 cm−1 within the region enclosed by a white dashed line. Figure 5(b) shows the spectral profile of the imaginary part of χ(3) (Im[χ(3)]) at the organelles indicated by the arrows (i) and (ii). The Im[χ(3)]) spectrum corresponds to vibrationally resonant spectrum, which is equivalent to the spontaneous Raman spectrum. The conversion from the CARS spectrum to the Im[χ(3)] is retrieved by using the maximum entropy method [51]. In Fig. 5(b), the spectral profile only in the CH region (2800 - 3000cm−1) is shown because the Raman bands in the fingerprint region (600 - 1800cm−1) were hardly observed due to fringing caused by etaloning of the EM-CCD camera. Although the fingerprint vibrational modes were not used in the present study, each spectrum is clearly different between (i) and (ii) even in the CH stretching region. The spectral profile of Im[χ(3)] indicated by (i) shows the vibrational signature of lipids at 2850 cm−1 due to CH2 stretching vibrational mode. On the other hand, the spectral profile indicated by (ii) shows the vibrational signature of proteins at 2930 cm−1 due to CH3 stretching vibrational mode [28], the small organelles indicated by (i) and (ii) are safely assigned as LDs and nucleoli, because the former is rich in lipids, while the latter is rich in protein.
Fig. 5. Backward CARS image of EpH4 cells with an EM-CCD camera. (a) Bright field and backward CARS images at 2850 cm−1 and 2930 cm−1 with exposure time of 100 ms. (b) Im[χ(3)] spectrum indicated by the white arrows (i) and (ii) in (a). (c) Bright field and backward CARS images at 2850 cm−1 and 2930 cm−1 with exposure time of 30 ms. (d) Im[χ(3)] spectrum indicated by the white arrow (iii) in (c). The image size (pixels and microns) and step size were 101 × 101, 50 × 50 µm2, and 0.5 µm. Scale bar is 10 µm. The broken lines in (c) indicate the positions of nuclei.
These trends have been effectively extracted using multivariate curve resolution-alternating least squares (MCR-ALS) method [52,53]. By specifically focusing on the CH stretching region, we have successfully decomposed the spectra and images associated with lipids, proteins, and water both for the forward and backward CARS data as shown in Fig. S2. However, in contrast to the successful decomposition in the CH stretching region, our attempts at spatio-spectral decomposition in the fingerprint region using MCR-ALS were hindered by a significant etaloning effect. Consequently, we were unable to achieve the desired decomposition results in this region.
Next, we tried to see if we could capture CARS images at a higher speed than 100 ms/pixel. The results obtained by 30 ms/pixel are shown in Fig. 5(c). Although the SNR is worse than that with 100 ms/pixel, we have successfully observed LDs (indicated by the white arrows (iii)) even with short exposure times such as our previous study [28,29]. The results above demonstrate that micrometer-sized organelles within cells can be spectroscopically identified in just 100 ms/pixel using our method. This confirms that the method developed in the present study is suitable for live-cell imaging.
4. Conclusion
In this study, we developed a backward multiplex CARS spectroscopic imaging system with an EM-CCD camera. As a standard sample, we measured the CARS spectrum of polystyrene beads in the backward configuration and were able to obtain the CARS spectrum in the spectral range of 3500 cm−1. Measuring the CARS signal using an EM-CCD camera improved SNR by approximately three times compared to that using a conventional CCD camera, validating the usefulness of an EM-CCD camera. The intra-cellular organelles such as LDs and nucleoli in cultured epithelial cells were visualized and spectroscopically identified in the backward CARS configuration even with the exposure time of 100 ms/pixel proving the powerfulness of the system as a spectroscopic imaging system. Therefore, we believe that these results justify further investigation into the combination of CARS and conventional inverted microscopes. This could potentially contribute to the development of more advanced imaging techniques including other multiphoton imaging methods. Our state-of-the-art imaging system will provide exceptional potential for real-time spectroscopic imaging of living cells, enabling revolutionary molecular diagnostics in both experimental and clinical applications.
Funding
Japan Society for the Promotion of Science (21H04961); JST-Mirai Program (JPMJMI22G5); Agence Nationale de la Recherche (ANR-10-LABX-0074 Sigma-LIM).
Acknowledgments
The authors would like to express their gratitude to J. Ukon of Ukon Craft Science, Ltd., for facilitating the collaboration between the Japanese and French laboratories. Additionally, the authors would like to extend their appreciation to Tokyo Instruments, Inc. for generously providing the EM-CCD camera used in this study. The authors would also like to thank Dr. Shinichi Miyazaki for his valuable assistance with the MCR-ALS analysis.
Disclosures
The authors declare that they have no conflict of interest.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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