Nonstaining Blood Flow Imaging Using Optical Interference Due to

Mar 21, 2018 - Aoi Electronics Co. Ltd., 455-1, Kohzai Minamimachi, Takamatsu , Kagawa 761-8014 , Japan. § Faculty of Engineering, Kagawa University,...
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Nonstaining Blood Flow Imaging Using Optical Interference Due to Doppler Shift and Near-Infrared Imaging of Molecular Distribution in Developing Fish Egg Embryos Mika Ishigaki,*,† Paralee Puangchit,† Yui Yasui,† Akane Ishida,‡ Hiroki Hayashi,‡ Yoshihiko Nakayama,‡ Hideya Taniguchi,‡ Ichiro Ishimaru,§ and Yukihiro Ozaki*,† †

School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan Aoi Electronics Co. Ltd., 455-1, Kohzai Minamimachi, Takamatsu, Kagawa 761-8014, Japan § Faculty of Engineering, Kagawa University, Hayashicho 2217-20, Takamatsu City, Kagawa Prefecture 761-0396, Japan ‡

ABSTRACT: In the present study, we successfully obtained nonstaining blood flow images of a developing fish egg embryo using optical interference caused by the Doppler shift. The spectral distribution of light reflected by moving objects such as the heart and red cells was found to be different from that of the incident light because of the Doppler effect. Interference between different frequency components was observed in an interferogram through heterodyne interaction using an imagingtype two-dimensional Fourier spectroscopic system, and information on the intensities of the spectral components was obtained by Fourier transformation. Beat signals with specific frequencies due to the heart beating and blood flow of the fish egg embryo were detected. When the signals were plotted in two dimensions, the heart part and vessel flows were clearly visualized without staining. In addition, nearinfrared (NIR) images were produced using absorbance spectra of the molecular vibrations of O−H and C−H groups included in water, hydrocarbons, and aliphatic compounds. Obtaining nonstaining blood flow images using heterodyne optical interference and images of molecular distribution using molecular vibrational information simultaneously manifests an exciting advance in NIR imaging.

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Many research groups have developed noninvasive real-time blood flow measuring devices using this technique for clinical and research purposes.3−8,11 Near-infrared (NIR) spectroscopy is a vibrational spectroscopy in the wavelength region of 800−2500 nm (4000−12 500 cm−1). The overtones and combination modes associated with molecular vibrations of stretching and bending modes can be observed in that wavelength region.12−17 In our previous works, we applied NIR spectroscopy and imaging to the investigation of embryogenesis of a medaka fish egg (Oryzias latipes).18,19 Information about the molecular composition and structure of water, proteins, and lipids in the embryo could be acquired noninvasively in vivo. NIR spectral signatures of lipids and proteins were detected, for example, at 4336 and 4864 cm−1, respectively, and the concentrations and distribution variations of these components in the course of egg development were visualized by plotting the second derivatives of the NIR absorbance spectral intensities.18 Furthermore, the results showed that the egg membrane spectrum was characterized by protein bands with α-helix and β-sheet structures, indicating that the membrane proteins formed such secondary structures.19

ichelson interferometers (MIs) are used in various methods of bioanalysis, and optical coherence tomography (OCT) can be cited as one of them.1,2 OCT is a technique to capture the internal structure of a specimen with high resolution and high speed using optical interference. The refractive index varies across the boundary between different organisms or lesions; the optical path length changes, and the spatial phase of light starts to differ.1,2 By knowing the depth of the sample surface layer contributing to reflection, it is possible to identify specific structures or lesions in an OCT image by investigating the position where the refractive index changes. As another example of the application of optical interference to bioanalysis, a laser blood flow system can measure blood flow noninvasively.3−8 Light scattered by moving objects such as red cells is frequency-shifted compared to the incident light, with the amount of shift proportional to the object velocity. Therefore, it is possible to obtain the speed of an object by measuring the frequency of the scattered light. This principle is adopted not only in blood flow measurements but also in laser Doppler speed meters that can measure the vibration speed of a structure or the speed of a vehicle on a road.9,10 Furthermore, when two waves with slightly different frequencies are superimposed, a beat signal at the difference frequency can be observed. This interference method is called optical heterodyne interferometry.4,11 The optical beat signal can be detected as a periodic change in the detected light intensity. © XXXX American Chemical Society

Received: December 30, 2017 Accepted: March 21, 2018 Published: March 21, 2018 A

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with a holder, and NIR measurements were performed in both the reflectance and transmission modes. In the reflectance mode, the measurement system was composed of a hyperspectral imaging unit (AOI ELECTRONICS CO., Ltd., NT00T011), an InGaAs camera (Hamamatsu Photonics K.K., C10633-13), a halogen light (USHIO LIGHTING, INC., JCR12 V100WBAU), and an objective lens at 8× magnification (MORITEX Corporation, MML8-80D-IR). In the transmission mode, it consisted of a hyperspectral imaging unit (AOI ELECTRONICS CO., Ltd., NT00-T012), the same halogen light, an NIR camera (Sumitomo Electric Industries, Ltd., CVN800), and an objective lens at 4× magnification (Nikon, f100). The spatial resolution with an objective lens at 8× magnification was about 8 μm. The measured wavelength region was 1000−2500 nm, and the wavelength resolution was 10 nm. In the spectroscopic part, a partial movable mirror moved by 250 μm in 30 s, resulting in the optical path difference of 250 √2 μm. During this time, 1800 imaging slides were obtained, which corresponded to 60 frames per second. The number of pixels in the two-dimensional image was approximately 81 000 points (319(X) × 255(Y)). The characteristic of the instrument is to adopt the wavefront split-type interferometer. The ITFS system traditionally used in astrophotography, on the other hand, takes in an amplitude division type. In that system, all light rays are similarly divided into two fluxes, and lights from out of the focal plane interfere in principle. However, in the present system, lights coming from a focal plane can interfere, and the others from out-focal plane can not interfere. These out-focal components are detected as a direct current (DC). The spectroscopic mechanism used in the system makes it possible to obtain confocal imaging data.

In the present study, we further expand the nonstaining NIR imaging of developing fish egg embryos. Not only NIR images are obtained from spectroscopic information about the molecular vibrations as in the previous work, but also nonstaining blood flow images are obtained by detecting interference signals caused by the Doppler effect with an imaging-type two-dimensional Fourier spectroscopy (ITFS) system.20,21 The system uses the so-called phase-shift interferometry between the detected optical signals and is able to obtain two-dimensional imaging data from the focal plane. Therefore, by changing the focal plane, three-dimensional spectral data can be acquired using this system.20,21 Absorption spectra associated with the molecular vibrations excited in the egg substance were observed in the NIR wavelength region, and the information about the molecular composition of the embryo could be acquired noninvasively in vivo. Furthermore, light reflected by a moving structure, which was associated with heart beat and blood flow, was observed to exhibit a slightly shifted frequency due to the Doppler shift. A beat signal was generated by heterodyne interference between the shifted and nonshifted frequency components. Hence, new peaks, characteristic of the motion such as heart beat and blood flow, appeared in addition to the ones due to molecular vibrations. The novelty and uniqueness of the present study are in that we obtain not only in vivo images of molecular distributions from developing eggs but also, simultaneously, images of the blood flow in a noninvasive manner without staining. The nonstaining imaging technique using optical interference has applications in biological research such as cardiogenesis and differentiation of induced pluripotent stem (iPS) cells into cardiomyocytes. In the very early stages of cardiogenesis and differentiation of iPS cells, optical interference can catch the slight beating between them. It can enable identification of the beat position and evaluation of the potential for proper differentiation of cells into cardiomyocytes.

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RESULTS AND DISCUSSION NIR Results. Figure 1 shows an enlarged optical image of a medaka fish egg on the fifth day after fertilization. The detailed

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MATERIALS AND METHODS Japanese Medaka (O. latipes) Fish Eggs. As a sample, we used Japanese medaka fish eggs. The fishes were bred in a tank kept at 25 °C. The size of the egg was ∼1.5 mm in diameter, and the eggs were almost transparent. Fertilized eggs stuck to the stomach of a female medaka after spawning. They were picked up and incubated in an incubator (CN-25C-1, Mitsubishi Electric Engineering Co. Ltd., Japan) at 25 °C. On the third day after fertilization, counting the spawning day as the first day, the fish body was beginning to form in the egg. They would hatch at ∼10 days after fertilization under the incubation at the specified temperature. Around the third day, the embryonic body could be visualized with a microscope. In the present study, the eggs on the fifth and seventh days were measured. On these days, the heart beat could be confirmed with a microscope. When the NIR measurement was performed, the egg was picked up from the water buffer and sandwiched between two glass slides with two pinch cocks to maintain the optical path length at 0.5 mm.18 We performed all experiments in accordance with the guidelines for proper conduct of animal experiments and related activities of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The study was approved by the ethics committee of Kwansei Gakuin University. Imaging-type Two-Dimensional Fourier Spectroscopy (ITFS). In the present study, an ITFS system was used. The prepared specimen was mounted in front of the light source

Figure 1. Enlarged optical image of an embryonic body of a medaka fish egg on the fifth day after fertilization.

structures of the eye, oil droplet, and embryonic body can be observed. Interferograms were obtained for each point of the measurement area. Figure 2a depicts the alternate current (AC) component of the interferogram obtained in the reflectance mode from the yolk part at point A in Figure 1. The x-axis exhibits the moving distance of the partial movable mirror. The maximum value of it is 250 μm, and the center of it at 125 μm expresses the position of fixed part of the mirror. The B

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Figure 2. (a) AC components of the interferogram obtained from the yolk part at point A in Figure 1. (b) Averaged NIR absorbance spectrum obtained in the vicinity of point A.

Figure 3. (a) Interferogram obtained from the heart part (B) in Figure 1. (b) Magnified view of the interferogram (a) corresponding to a phase shift of 50 μm.

interferogram showing the center burst is similar to the ones measurements with normal MI. The spectral intensity is obtained by Fourier transformation (FT), and the NIR absorbance spectrum can be calculated according to the definition of absorbance. Figure 2b depicts the averaged NIR absorbance spectrum calculated for 70 data points obtained from a yolk part in the 1300−2350 nm region. Two broad water peaks can be seen at ∼1500 and 2000 nm. These two bands come from the combination of symmetric and antisymmetric O−H stretching modes and of O−H bending and antisymmetric stretching modes of water, respectively.12,22 Molecular vibrations due to biomaterials can be confirmed nondestructively. During the monitoring of the egg, heart beat was observed at around point B in Figure 1. Of note is that the interferogram detected at the heart part (B) showed a noise-like background in addition to the center burst, as shown in Figure 3a. Figure 3b demonstrates a magnified view corresponding to the optical path difference of 50 μm in 6 s. The periodic wave shape was confirmed, and 19 wave peaks were observed, corresponding to a frequency of 3.2 Hz. However, the number of heart beats obtained from the visible monitoring was 47 in 30 s, corresponding to a frequency of 1.6 Hz. The frequency obtained from the interferogram was two times higher than that found through the visual inspection of the heart beat. A fish heart has one atrium and one ventricle.23,24 The atrium beats first, and then the ventricle.23 Therefore, the periodic wave is assumed to reflect the heart beating of the fish embryo, and the double count may come from the motion of the two main

components of the heart. The interferogram shown in Figure 3a was processed by FT, and corresponding spectroscopic information was obtained in the 1000−2500 and 2000− 15 000 nm regions (Figures 4a and b). The strongest peak can be seen at 3768 nm, and two pronounced peaks are also observed at 1884 and 1256 nm. To confirm the origin of the peaks, the periodic waveform due to the heart beat was converted such that the wavelength information after FT can be calculated. The number of oscillations in the periodic waveform due to the heart beat for an optical path difference of 250 √2 μm in 30 s was 3.2 (Hz) × 30. Therefore, the wavelength of the peak λ [μm] after FT can be calculated from the following equation: 3.2(Hz) =

250 2 (μm) 1 × 30(s) λ(μm)

(1)

The resulting wavelength is λ = 3.68 μm. The strongest peak in Figure 4b is due to the fundamental mode of the heart beat, and the two peaks (1.88 and 1.26 μm) in Figure 4a seem to correspond to the first and second overtones of the fundamental mode, respectively. Mechanism of ITFS and Heterodyne Interference. To understand the underlying mechanism of the heart beat signature in the interferogram and corresponding FT spectroscopic spectrum, we review the detailed structure of the ITFS system used in the study and the heterodyne optical C

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gram as with a usual interferometer by continuously changing the spatial phase difference. Interference due to the optical path difference is observed at all bright spots in the focal plane, and two-dimensional spectroscopic information can be obtained by FT of the interferogram observed at each point. The feature of the spectroscopic mechanism used in this system is that the phase-shift interference is generated by the signal coming from the object. This is different from the principle of MI, with phase-shift interference between the signal coming from the object and the reference signal, adopted for general FT-NIR spectroscopy. Because all optical rays are similarly divided into two paths and cause interference in the MI system, the interference of signals coming from outside the focal plane is also observed. However, in the current system, interference occurs only for rays that come from the same point in the system. Therefore, interference between signals coming from outside the focal plane is not observed in the AC component but detected as the DC part of the interferogram. Here, for simplicity, let us consider interference of two signals emitted from a monochromatic light source. As shown in Figure 5, optical rays reflected from a certain point O of the sample pass through the imaging lens and are reflected by the phase shifter, focused by the imaging lens, and detected by the CCD camera. The complex amplitude due to the two signals at the observation point r that were reflected at the point O can be expressed as E = A1e−iφ1(r)eiωt + A 2 e−iφ2(r)eiωt

(2)

Here, A1 and A2 indicate the amplitudes, and ω is the angular frequency. The variables φ1(r) and φ2(r), which are the phases of the signals at the observation point r, are expressed as φ1(r) = 2πli/λ, where li and λ are the optical path length and the wavelength, respectively. In the present case, φ1(r) and φ2(r) have different phases because of the phase shifter. Because the intensity of the observed light is proportional to the square of the amplitude, the signal at the observation point r is expressed as

Figure 4. Spectroscopic information obtained by Fourier transformation of the data in Figure 3a in the (a) 1000−2500 nm and (b) 2000−15 000 nm regions.

interference caused by superposition of waves with slightly different frequencies. As the spectroscopic mechanism of the device, a method is adopted that introduces an optical path difference to the light diffracted or scattered by the sample using a partial movable mirror as shown in Figure 5. Due to the introduced optical path difference, the state of imaging changes, and the distribution of imaging intensity varies. It is possible to acquire an interfero-

I = |E|2 = A12 + A 22 + 2A1A 2 cos ψ (r )

ψ (r ) ≡ φ1(r ) − φ2(r ) =

2π δ λ

(3) (4)

Here, δ is the optical path difference defined as l1 − l2. When the light intensity is detected by the CCD detector, the first and second terms in eq 3 are observed as the DC component, and the third term as the AC one. When the position of the movable mirror is on the other fixed one, the optical path lengths are the same for these two signals. Therefore, all intensities are enhanced in such a situation, and the phenomenon known as a center burst can be observed. In the present study, a fish egg was used as a sample. The surface of the egg is reflecting light, and its heart beat and blood flow are generated with the development of the sample. It is well-known that light reflected from objects moving at finite velocities will have a different frequency compared to the incident light due to the Doppler effect. Therefore, the light scattered by a moving red cell interferes with that reflected by static tissue. Laser Doppler flowmeters use these phenomena to measure the tissue blood flow noninvasively and in a noncontact manner.3−8 When signals with slightly different frequencies interfere with each other, an optical beat signal with a frequency equal to the difference of the original frequencies can be observed. The optical beat signal can be detected using

Figure 5. Schematic view of the imaging-type two-dimensional Fourier spectroscopic system (ITFS; AOI ELECTRONICS CO., Ltd., NT00T011). D

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Figure 6. Blood flow images of a medaka fish egg on the 5th day after fertilization obtained by plotting (a) detected light intensity from the sample, (b) absorbance, and (c) relative intensity calculated as the light intensity divided by the white light reference at (A) 1260 and (B) 1860 nm.

intensity (a), absorbance (b), and relative intensity (c) calculated as the detected light intensity divided by the white light reference. Here, the absorbance spectra with the heart beat signal show some dips corresponding to the wavelengths of the heart beat. The spectra can be interpreted as if the signals with these wavelengths were emitted from the sample. This enables us to clearly understand that, for the Doppler effect, components not included in the incident light are detected. In this way, the location where the flow of red blood cells linked with the motion of the heart beat occurs is successfully visualized, and blood flow images can be created without staining. Similarly, NIR measurements of the fish eggs in the transmission mode were also performed. Absorbance spectra can be calculated, showing a large contribution due to water, similar to that of the reflection NIR spectra shown in Figure 2b. Notably, two dips originated from the heart beat are observed at ∼1880 and 2260 nm. The heart rate seems to vary a little bit for different individuals or developmental stages, and the period derived from the spectral positions is different from that associated with the heart beat mentioned above for the reflectance measurement. Figure 7 depicts NIR images developed by plotting the light intensity at 2260 nm (a) and absorbance at 1880 (b), 1940 (c), and 2360 nm (d). Figure 7a highlights the exact part of the heart, and Figure 7b reveals the blood vessel stretched over the

interference caused by superposition of light waves. In the present sample, there is motion related to the heart beat and the flow of red blood cells through the vessels. Therefore, the frequency of the reflected light is partially shifted by the red cell motion. The reflected light includes the optical beat signal coming from the blood flow, which can be extracted through heterodyne optical interferometry. In this case, there are components with different phases caused by both the phase shifter and Doppler effect, as shown below: E = A1e−iφ1(r)eiωt + A 2 e−iφ2(r)eiω ′ t

(5)

Here, Ai, φi(r), and ω (ω′) are the amplitude, the phase at the observation point r, and the angular frequency of the signals, respectively. As in the previous case, the signal at the observation point r can be expressed as I = |E|2 = A12 + A 22 + 2A1A 2 cos ψ ′(r , t ) ψ ′(r , t ) ≡ φ1(r ) − φ2(r ) − (ω − ω′)t =

(6)

2π δ − δωt λ (7)

Here, δ is the optical path difference defined as l1 − l2, and δω indicates the angular frequency of the optical beat signal (ω − ω′). In this way, the Doppler frequency shift can be detected using the principle of optical interference. The principle is used in laser Doppler vibrometers, velocimeters, blood-flow sensors, etc., and the vibration and speed of objects can be measured in a noncontact way.3−8,25,26 Three peaks observed at 3768, 1884, and 1256 nm in the spectra shown in Figures 4a and b correspond to the fundamental, the first overtone, and the second overtone of the heart beating modes, respectively. The waveforms appearing in the interferogram due to heart beat are not simple cosine waves (Figure 3b) and should be expressed through superposition of higher-order overtones. The beat signal is generated by a mixture of light scattered by static and moving objects. Conversely, the measurement locations showing beat signals should contain moving objects. Therefore, it is possible to obtain the blood flow image by plotting the peak intensity of the beat signal. Figures 6A and B show the images produced by plotting the intensities in two dimensions at the wavelength of the second overtone (1256 nm) and first overtone (1884 nm), respectively, by using the detected light

Figure 7. NIR images obtained by plotting (a) light intensity at 2260 nm and absorbance intensity at (b) 1880, (c) 1940, and (d) 2360 nm. E

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ORCID

Mika Ishigaki: 0000-0002-6663-2911 Yukihiro Ozaki: 0000-0002-4479-4004 Author Contributions

M.I. designed the present study, conducted it as a leader, and played a major role in preparing the manuscript. The ITFS was designed and developed by I.I. and Aoi Electronics Co. Ltd. M.I., P.P., Y.Y., A.I., H.H., Y.N., and H.T. performed the experiments and data analyses. I.I. and Y.O. provided suggestions and constructive advice to all members for the detailed interpretation of the obtained data and contributed considerably to the preparation of the manuscript. All the authors participated in the discussion of the present results. Notes

The authors declare no competing financial interest.

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CONCLUSION The present study demonstrated nonstaining blood flow images obtained using the laser Doppler effect. With an imaging-type two-dimensional spectroscopic system, we detected twodimensional spectroscopic information from the focal plane by introducing spatial phase differences with a partial movable mirror. Of particular note is that the light reflected by the blood flow through the heart and blood vessels underwent a Doppler shift, and a beat signal linked to heart beat was detected through heterodyne interference with the nonshifted light. FT spectra appeared in the AC component of the interferogram showed not only molecular vibrational spectra due to water and lipids included in the fish egg but also pronounced peaks derived from the heart beat. By plotting the detected light intensities, we successfully obtained nonstaining blood flow images mapping the spectral information derived from the beat signal and showing the molecular distributions through the second derivative of the NIR absorbance spectra. In the NIR absorbance spectra, the band due to heart beating was observed in the negative direction, indicating that light with the frequency of the beat cycle emerged from the sample. Thus, the Doppler-shifted light reflected by a moving object has a particular frequency and a different ratio of its light intensity to that of the incident light. By detecting the new signal caused by the Doppler shift with optical interference, one can sensitively detect the location associated with the vibration or motion of interest. Therefore, it indicates possible application of the method to the study of cardiogenesis in the stage where the heart structure is yet not clear. Additionally, it may also be applicable for the detection of iPS cell differentiation into cardiomyocytes from both pulsation and molecular composition information.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. F

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