Raman spectroscopy, Imaging, hollow optical fiber, fiber bundle

A Raman imaging system which combined a hollow fiber bundle and a direct imaging technique was constructed for high-speed endoscopic Raman imaging. The hollow fiber bundle is fabricated by depositing a silver thin film on the inner surface of pre-drawn glass capillary bundle. It performs as a fiber optic probe which transmits a Raman image with high signal-to-noise ratio because the propagating light is confined into the air core inducing little light scattering. The field of view on the sample is uniformly irradiated by the excitation laser light via the probe. The back-scattered image is collected by the probe and captured directly by an image sensor. A pair of thin film tunable filters is used to select target Raman band. This imaging system enables flexible and high-speed Raman imaging of biological tissues.

Raman imaging has received attention in the field of biomedical engineering because of its potential as a nondestructive and label-free method for a histologic diagnosis. The imaging equipment is constituted based on a laser microscope and useful for measurement of tissue sections but it is generally not appropriate for in vivo measurements. On the other hand, a few studies have reported on the use of an imaging fiber bundle for image transmission. However, it is difficult to detect a very weak Raman signal generated from biological tissues because of intense background noise induced in the silica core.

Recently, we have proposed the hollow fiber bundle for image transmission. The hollow fiber bundle has a honeycomb cross-section inner coated with a silver film, and laser induced background noise is negligible because light is confined into the air core. We have also demonstrated that the Raman image with full spectrum can be constructed by applying the line scan technique to proposed hollow fiber bundle.

In this report, high speed endoscopic Raman imaging system consisting of a combination of the hollow fiber bundle and the direct imaging method is proposed. Imaging of biological tissues is also demonstrated.

We constructed measurement systems based on the direct imaging method known as a high-speed imaging method.

Fig. 1 shows the schematic of a measurement system. The excitation laser beam with a wavelength of 785.2 nm is uniformly launched into the all cores of a hollow fiber bundle shown in Table 1.

Fig. 1. Schematic of the measurement system

Table 1. The characteristics of the hollow fiber bundle

[A design of fiber optic raman imaging system] Table 1

The output laser light is focused again on the sample by an objective lens of NA0.68 and irradiates the field of view on the sample. The Raman signal back scattered from a sample is collected by the bundle and transmitted to the spectrometer or the CCD camera. In this setup, Raman spectrum and Raman image are captured separately. In Raman spectrum measurement, the tunable bandpass filter is removed and the flipper mirror is installed. The incident light is dispersed by a spectrometer (F/4.9) and captured by the cooled CCD (128×1024 pixel). On the other hand, in the Raman image measurement, the flipper mirror is removed. The target Raman band is selected by the tunable bandpass filter and the 2D Raman image is directly captured by the cooled EMCCD (512×512 pixel).

In Raman image measurement, it is necessary to control the spectral resolution of the filter and ensure SN ratio as high as possible. The filter having high transmittance and tunable resolution is ideal for direct Raman imaging. We therefore studied the use of thin-film filter (TFTF, Versa Chrome, Semrock). The transmission band of the TFTF can be tuned by changing the incident angle. Fig. 2 shows TFTFs transmission spectra when the incident angle is changed from 0 to 60 degree.

Fig. 2. TFTF transmission spectrum tuning the incident angle

From this measurement, measurable spectral range was estimated at 0 to 1624 cm^-1 for 785 nm excitation, and it was confirmed that the TFTF performs as the band selector with sufficient spectral range to measure biological tissues. However, the band width of 222～291cm^-1 is too wide to select narrow Raman bands. Therefore, we use a pair of TFTFs to improve the spectral resolution.

Table 2 is the comparison with other tunable filters generally used for direct imaging equipment. Because the acousto-optic tunable filter(AOTF) and the liquid-crystal tunable filter (LCTF) generally have polarization-dependent characteristics, the transmittance for random polarization does not exceed 50%. On the other hand, TFTF pair has an overwhelmingly higher transmittance than other filters because of the polarization independent characteristics, while the minimum spectral resolution is inferior to that of LCTF. Furthermore, since the bandwidth can be tuned by the angle between TFTFs, the throughput can be maximized by adjusting the bandwidth to the targeted Raman band.

Table 2. Characteristics of the tunable bandpass filters

Figure 3 shows Raman spectra of TiO2 powder measured by using single TFTF, TFTF pair and the Raman spectrometer. In TFTF pair, the angle between filters is fixed at 2 degree, the spectral resolution was 33-210 cm^-1. The Raman bands at 471, 624 cm^-1 are assigned to TiOz. The result shows that these Raman bands which cannot be separated by single TFTF can be separated by TFTF pair. We confirmed that TFTF pair performs as band selector for direct Raman imaging.

Fig. 3 Raman spectrum using the single /double TFTF

In order to evaluate the constructed endoscopic Raman imaging system, we performed imaging of two different samples: polymer micro-beads and a biological tissue.

Figure.4 (a)shows the micrograph of the polymer beads. 10 um spherical beads of polystyrene (PS) and polymethyl methacrylate (PMMA) was mixed in the ratio of 1:1 and dispersed on an aluminum substrate. The magnification of the distal objective lens was set to 15× to obtain a magnified image of a minute sample. The Raman image is obtained in the following procedures. First, one measures Raman spectrum of wide area on the sample surface (Fig.4(b)A). Then, the targeted Raman band is selected by TFTF pair (Fig. 4(b)B, Fig.4(b)C). Finally, the optical path is switched to the camera side, and image is captured. Fig. 4(b)A is measured Raman spectrum of polymer particles. The Raman bands at 819, 1015 cm^-1 are assigned to the PMMA, PS, respectively.

Fig. 4 (a) Micrograph of polymer micro-beacds.(b)Raman spectrum of polymer micro-beads

Fig.5 is a composite of Raman images obtained by using these Raman bands. The intensity of excitation laser light was 48 mW, and measuring time was 8 second for each Raman image. While the basic microscope has difficulty in distinguishing transparent samples, the Raman image shown in Fig. 5 clearly indicates the difference of chemical structure between PS and PMMA. It should also be noted that since the system background is sufficiently small, high contrast Raman image is obtained without background subtraction.

Fig. 5 Raman image of(Red)PS and (Green) PMMA

A tissue of porcine muscle shown in Fig. 6(a) was used as a biological tissue sample. Fig. 6 (b)A shows measured Raman spectrum. The Raman bands at 1312, 1437, 1748, 1854 cm^-1 are assigned to the C=C stretching mode, C=C stretching mode, CH2 bending mode, CH2 twisting mode, respectively.

Fig. 6 (a) Micrograph of biological tissue.(b)Raman spectrum of biological tissue.

Fig.7 shows the Raman image measured by selecting 1437 cm band as shown in Fig.6 (b)B. The excitation laser light intensity was 48 mW at the output end of the fiber bundle, and the measuring time was 20 second. The Raman image of 1437 cm^-1 represents the distribution of lipid in the tissue. Therefore, we can clearly confirm the boundary between the lean and adipose from the image. This result suggests that proposed Raman imaging system may perform chemical imaging in vivo with a clinically acceptable measurement time.

Fig. 7 Raman image of biological tissue

The direct Raman imaging system using a hollow fiber bundle was constructed for high-speed endoscopic Raman imaging. A pair of thin film tunable filter with high transmission was applied as the band selector. To verify the performance of the constructed system, we performed imaging of polymer micro-beads and a porcine meet slice. From the captured image, we confirmed that the system produces high contrast Raman image within an acceptable duration of time.