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Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope SRIVATHSAN VASUDEVAN,1,4,* GEORGE C. K. CHEN,2 ZHIPING LIN,3
AND
BENG KOON NG3
1
Discipline of Electrical Engineering, Indian Institute of Technology Indore, Indore, Madhya Pradesh 452017, India B. C. Photonics Tech. Co., Richmond, British Columbia V7E 1G9, Canada 3 Department of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798, Singapore 4 Centre for Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, Madhya Pradesh 452017, India *Corresponding author: [email protected] 2
Received 19 January 2015; revised 14 April 2015; accepted 14 April 2015; posted 14 April 2015 (Doc. ID 231595); published 7 May 2015
Photothermal microscopy (PTM), a noninvasive pump–probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications. © 2015 Optical Society of America OCIS codes: (180.6900) Three-dimensional microscopy; (090.1995) Digital holography; (350.5340) Photothermal effects. http://dx.doi.org/10.1364/AO.54.004478
1. INTRODUCTION Photothermal microscopy (PTM) is a noninvasive technique that has been applied to many biomedical studies. PTM utilizes two pulsed lasers as pump–probe lasers. The significant merit of this technique is that it is able to detect a sample smaller than the diffraction limit. The sample is irradiated with a pump pulse, which causes a temperature excursion. The absorbed thermal energy diffuses radially outward, forming a thermal field. When the thermal field expands beyond the diffraction limit of the microscope, a probe pulse is used to probe the sample plane [1,2]. The probe pulse would pick up the temperature-induced changes in the refractive index. The high-sensitivity high-resolution photothermal imaging can be realized by recording laser-induced temperaturedependent variations of the refractive index around the absorbing target. Suppose we have a triangular target, as shown in Fig. 1, which is smaller than the diffraction limit of the microscope. The laser excitation using a pump laser would excite the sample, thereby increasing the temperature of the sample. Here, each point of the triangular sample acts as a heat source, and, at a short time interval, the wavefront of the secondary wavelets (corresponding to the temperature profile) resembles that of the excited sample. At the same time, based upon the 1559-128X/15/144478-07$15/0$15.00 © 2015 Optical Society of America
characteristics of the medium, the refractive index of the medium changes due to temperature change. This creates a phase variation in the probe beam. To detect these phase changes, a phase contrast module is employed to convert the modulated phase into amplitude before detection by a CCD camera. Figure 2 illustrates the advantage of PTM by comparing a normal phase contrast image and the photothermal image of a lymphocyte cell obtained using the conventional photothermal imaging system developed by our group [3]. The PT image in Fig. 2(b) shows several bright and dark spots, which are due to spatial distribution of the refractive index change induced by the pump beam. The fact that the PT image depicts several components inside the cell, which cannot be seen in the conventional phase contrast microscope image [Fig. 2(a)], proves that a PT image can view cellular components better than a normal phase contrast microscope. Moreover, photothermal images could capture this information without adding any fluorescent tags; thus, PTM has become a popular technique for many biomedical imaging applications. Several studies [1,4,5] have been conducted using PTM. These images have been in tandem with fluorescent images [6]. It is clear from Fig. 2(b) that the photothermal content of the sample is stored as a change in the refractive index (due to temperature excursion)
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Fig. 1. Principles of photothermal imaging.
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Therefore, this paper attempts to solve the above-mentioned problems in the conventional PTM by acquiring quantitative photothermal images. This is implemented by integrating digital holographic microscope with a photothermal microscopy technique. Using a digital holographic microscope is a powerful well-known technique for obtaining quantitative phase information, and it has been widely used for many biomedical studies. Particularly, a digital holographic microscope has been used for real-time continuous monitoring of many biomedical cellular assays. Hence, the proposed method of integrating PTM with a digital holographic microscope would yield separate amplitude and phase images. In addition, the extracted phase values from these images can be used for further processing. The proposed method would provide first-hand information of the phase values created by the temperature excursion of the pump beam of PTM. This can provide important information, which can act as a cell signature. In addition, the proposed integration would also pave the way for continuous nonfluorescent real-time monitoring of assays, which can provide quantitative information. 2. EXPERIMENTAL DEVELOPMENT FOR OFF-AXIS DIGITAL HOLOGRAPHY
Fig. 2. (a) Phase contrast image of a lymphocyte cell. (b) Photothermal image of the same lymphocyte cell (60×, N.A., 0.65).
or, in other words, phase information. In order to obtain this information, a conventional phase contrast microscope is utilized for this purpose. While several studies have reported the applicability and advantage that the photothermal technique has over fluorescent techniques, the next step is to take PTM forward to obtain useful information from the photothermal images. The information can be quantitative information that can act as a signature of the sample. For example, information such as temperature distribution of the sample, particularly in the case of biological cells, could provide information on the absorption contrast. Absorption contrast of cells could be used as a potential tool to identify normal cells from malignant cells. Therefore, providing useful information from photothermal techniques can make a photothermal microscope an efficient diagnostic tool for many diseases including but not limited to cancer. A phase contrast microscope, used in conventional phase contrast microscopes, would not help to retrieve quantitative information. The reason is that current photothermal microscopes use a Zernike filter to convert the phase changes into amplitude for CCD camera recording. The images obtained using a Zernike filter are nonlinear [7–9]; hence, these images could not be used to extract phase information. Moreover, existence of halos in the image due to the phase contrast module [10,11] of the PTM also deters the quality of the photothermal images. However, quantitative phase values at each and every point of the PT image can yield important information such as temperature distribution, refractive index gradient with temperature, thermal diffusivity, etc.
As a first step of integration, the digital holography setup is constructed using the lasers available for a photothermal imaging setup. Figure 3 shows the experimental setup of the digital holographic photothermal microscope. The basic architecture for a digital holographic microscope is that of a Mach–Zender interferometer. Traditional digital holographic microscopes utilize a continuous highly stabilized laser as the laser source [12]. Stabilized lasers would have a long coherent length that could help the two beams (reference and the object) to interfere at the CCD plane. However, for photothermal imaging, pulsed lasers are used for pumping and probing the samples. Pulsed heating of the sample ensures that the biological sample is not heated up (approximately 10 ns). Also, since the photothermal field formed can be imaged only by a short duration laser, a pulsed nanosecond laser is used as a probe laser. The digital holographic experiments were performed using the developed experimental setup, as schematically depicted in Fig. 3. The probe laser, also called the imaging laser, is a frequency-doubled Nd:YAG pulsed laser with a wavelength of 532 nm with energy of 0.12 μJ. The laser output, which is spatially filtered and collimated (using a beam expander, not shown in Fig. 3), is split into a reference beam (R) and an
Fig. 3. Schematic of a digital holographic system.
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object beam (O) using a nonpolarized beam splitter. The setup is a transmissive interferometer setup based on the Mach– Zehnder configuration. The benefit of the experimental setup is that the path length in the reference and object arms is equal due to the symmetry of the setup. Hence, this setup can be used to calculate the amount of phase modulation caused by a sample that is placed in one of the arms. The sample or biological specimen is placed on the X –Y translational microscope sample stage. A pair of similar microscope objectives is used in the two optical arms of the experimental setup to match the curvatures of the two wavefronts. A slight angle is introduced between the object and the reference beams for off-axis holography. The microscope objectives and the beam splitter are adjusted such that the interference fringes are straight, thereby avoiding the need to perform any digital correction due to spherical aberration introduced by the microscope objectives. The object and the reference beams were allowed to interfere at the hologram plane. The hologram with intensity [13,14] I H x; y jRj2 jOj2 R O RO
(1)
is recorded using a standard black and white CCD camera (1280 × 980 pixels, Imaging Source, 41BF02). For recording the holograms, the CCD camera is placed at the hologram plane, as shown in Fig. 3. The reconstruction distance would be the distance between the image plane and the hologram plane. A simple program is written in MATLAB to transfer the image captured by the camera to the computer. In order to reconstruct the digital hologram recorded, a replica of the reference wave is needed. However, in this case, a digitally computed reference wave (R D ) is used to reconstruct the digital transmitted wavefront (ψ), as given by [12,15,16]: ψ R D jRj2 R D jOj2 R D R O R D RO :
(2)
The first two terms of Eq. (2) correspond to the zero-order diffraction, the third term to the twin image and the fourth term to the real image [17]. Hence, the reference arm is adjusted in the experimental setup so that the hologram is recorded in the off-axis geometry [14]. The incidence angle of the reference arm is adjusted such that there is a separation between the real image, twin image, and the zero-order diffraction in the observation plane. For the present experimental setup, it is necessary to verify the acquisition of the hologram and also the reconstruction of the amplitude and phase images. This step will ensure that the resolution of the microscope is preserved before performing PT imaging. A USAF target was placed at the sample plane and the hologram was acquired. The acquired hologram is shown in Fig. 4. By implementing a simple Fourier transform algorithm, the twin image, real image, and the zero-order diffraction terms are visible, as shown in Fig. 5(a). This clearly helps the experimentalist to verify the off-axis holograms. For the reconstruction process, the digitally constructed reference wave would be multiplied with the hologram amplitude transmission. The reconstructed wavefront ψ for the acquired hologram is represented by [14,18]
Fig. 4. Hologram of USAF target acquired using the developed experimental setup.
iπ 2 2 ψξ; η A exp ξ η λd ZZ iπ 2 x y 2 × I H x; y exp λd i2π xξ yη dxdy; × exp λd
(3)
where I H x; y is the recorded hologram, λ is the wavelength, d is the reconstruction distance, and x; y and ξ; η are the coordinates of the hologram image and the reconstructed image, respectively. A is a complex constant given by exp i2πd ∕λ∕iλd . For phase contrast imaging, a digital reference wave (RD ), which is a replica of the original reference wave, is multiplied with the hologram. Assuming a perfect plane wave for the reference wave, RD can be calculated as R D AR expi2π∕λkx x ky y;
(4)
where AR is the amplitude, and kx and ky are the components of the wave vector. By adjusting these two components, the
Fig. 5. (a) Twin image, real image, and the diffraction terms of the USAF hologram acquired in Fig. 3. (b) Intensity image of USAF target (reconstruction distance is 116.8 mm).
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reference wave could be matched with that of the original reference wave. The intensity of the reconstructed wavefront is calculated as Iξ; η jψξ; ηj2 ;
(5)
and the phase is given by φξ; η arctan
Imψξ; η : Reψξ; η
(6)
Reconstructing the USAF hologram shown in Fig. 4 by the above-mentioned equations, a portion of the reconstructed intensity image is shown in Fig. 5(b).
3. DIGITAL HOLOGRAPHY EXPERIMENTS It is evident from these images that the resolution of the microscope is achievable in digital holography, and the ability to obtain separate intensity and phase images is shown. In addition to the resolution, it is also essential to verify whether the phase values can be obtained from the digital holography experimental setup. The phase images obtained from the holographic reconstruction would be wrapped. Hence, phase unwrapping needs to be performed on the extracted phase image to acquire accurate results. Several phase unwrapping techniques are already available [19–21]. Suitable phase unwrapping methods are applied to the phase image to obtain continuous phase, and then the phase values can be extracted. Figure 6 shows the hologram of a polystyrene particle placed in the sample holder of the developed experimental setup. The size of the polystyrene particle is 45 μm. A phase unwrapping algorithm [17,22] is implemented on the phase image to obtain the unwrapped phase of the polystyrene particle. The hologram plane was 65 mm distance. The Fourier analysis shows the real, twin, and the diffraction term in Fig. 7(a). After reconstruction of the image from the hologram, the intensity image is shown in Fig. 7(b). After applying the phase unwrapping algorithm, the corresponding phase image is shown in Fig. 8(a). A line is drawn across the center of the phase image, and the corresponding phase values are extracted. The phase values are shown in Fig. 8(b). These phase values would help to decipher
Fig. 6. Hologram of a polystyrene particle (reconstruction distance, 65 mm).
Fig. 7. (a) Twin image and the diffraction terms of the polystyrene particle captured using the hologram in Fig. 6. (b) Intensity image of the polystyrene particle after reconstruction.
information on photothermal fields later on during photothermal imaging. From Fig. 8(b), the maximum phase corresponds to 37.36 rad, while the minimum phase across the edges is 2.6 rad (since the phase values across the two edges are not the same, the average of these two values, namely, 0.9761 and 4.242, is taken). Hence, the phase change created due to the particle is 34.76 (37.36–2.6) rad. The size of the particle is calculated by ϕ 2π∕λnp − nm d p , where np is the refractive index of the particle, nm is the refractive index of the medium, d p is the diameter of the particle, and ϕ is the phase in radians. Since the refractive index of the microscope immersion oil is 1.518, the size of the particle comes to dp
34.76 × 532 × 10−9 43.86 μm: 6.28 × 1.59 − 1.518
(7)
The real size of the particle, according to the specifications, is 45 μm (S:D: 0.3), thus proving that the calculation obtained from the hologram is very close to the true result. In order to check the consistency of the results, the same experiment has even been repeated with various polystyrene particles of different sizes. The summary of the size calculations is provided in Table 1. The results are quite consistent with the actual sizes of these particles. Therefore, the calculation substantiates the fact that the phase values obtained are accurate.
Fig. 8. (a) Three-dimensional visualization of the phase image obtained from the unwrapping algorithm. (b) Phase values of the polystyrene particle over a line drawn across the center of the particle.
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Table 1. Experimental Size Calculations of Various Samples Using the Developed Experimental Setup Sample Red polystyrene bead Transparent polystyrene bead Black polystyrene bead Transparent polystyrene bead
Actual Size (μm)
Experimentally Calculated Size (μm)
5.1 15
4.96 0.4 14.86 0.3
10 45
9.6 0.4 46.2 0.5
Having confirmed the results obtained from the digital holographic technique, a preliminary experiment was also conducted on red blood cells (RBCs) to ascertain the phase image and the viability of this technique for biological samples. Figure 9(a) shows the phase image obtained through digital holography and the corresponding phase values, obtained by drawing a line profile on the phase image, as shown in Fig. 9(b). The disc shape of the RBC is clearly evident from Fig. 9(a), which shows the advantage of the digital holographic technique. 4. QUANTITATIVE PHOTOTHERMAL MICROSCOPY OF RED BLOOD CELLS For conducting the photothermal experiments, the pump beam is needed for pumping the samples to induce a temperature excursion. Hence, the digital holographic microscopy setup is appended with a pump laser. The pump laser used is a Nd:YAG laser pumped optical parametric oscillator working
Fig. 9. (a) Phase image of the RBC through numerical reconstruction (color bar in radians). (b) Phase profile obtained from the phase image depicting the phase values.
Fig. 10. Modified experimental schematic incorporating photothermal imaging and digital holographic microscopy in a single setup.
at 540 nm wavelength. The pump laser is irradiated through BS2, which is focused by the microscope objective. The focused pump laser excites the sample, thus creating a temperature excursion required for photothermal studies. The revised experimental setup is shown in Fig. 10. After passing through the sample, a pump filter was used to block the pump beam from reaching the CCD camera, which is placed at the hologram plane. Three holograms were captured during this process. The first hologram is a no-pump image captured before pumping the sample. The second hologram is the PT image captured at a specific time delay (∼ ns) after pumping, while the third is taken without pumping the sample. The third hologram was captured at a time frame much longer (∼ minutes) after pumping. This image would ensure that the sample has returned back to normalcy after heating. Figure 11 shows the phase values of a polystyrene particle, which has been imaged three times: (1) no-pump image; (2) photothermal image (PT); and (3) no-pump image taken after a very long time from pump (∼ minutes). These phase profiles prove that, after the photothermal excitation, the particle returns back to its normalcy, which is clear from the phase values of the No-pump2
Fig. 11. Photothermal phase profile of a polystyrene particle taken (a) before pumping (No-pump1), (b) 50 ns after pumping (PT), and (c) long period after pumping (No-pump2).
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Table 2. Phase Change at the Center of the Particle Due to PTI
Sample Black polystyrene bead Red polystyrene bead
Phase Change Due to PTI at 50 ns (rad)
Phase Change Due to PTI at 400 ns (rad)
0.6 0.2
0.1 0.1
0.4 0.1
0 0.1
image when compared with the No-pump1 image. It is also visible that the phase change is due to temperature increase in the photothermal image. Since the d n∕d T is negative for polystyrene particles, the phase actually decreases due to increase in temperature. The same experiment is also repeated on black and red polystyrene particles for the repeatability, and the results are shown in Table 2. After proving that the proposed technique of adopting digital holography with photothermal imaging can extract accurate phase values, the PT imaging technique was applied to RBCs. Fresh human blood was obtained by venipuncture from healthy donors into 0.5% BSA solution in a phosphate buffer saline (PBS) solution pH 7.4. Erythrocytes were then collected through centrifugation at 4°C, 1000 G for 15 min. Experiments were performed with 0.3% hematocrit at 37°C in Ringer solution (that contains 125 mM NaCl, 5 mM KCl, 1 mM MgSO4 , 32 mM HEPES, 5 mM glucose, 1 mM CaCl2 , pH 7.4). Figures 12(a) and 12(b) show the phase images of the RBCs taken before pumping and 350 ns after pumping the sample (Nd:YAG laser, 540 nm, 0.4 μJ). Out of the three cells shown in the figure, the pump beam is focused on the bottom cell. A line was drawn across the RBCs, as shown in the figures, and the corresponding phase profiles were extracted. The phase
Fig. 12. Phase images taken (a) before pumping and (b) 350 ns after pumping of RBCs (pump energy, 0.4 μJ; probe energy, 0.12 μJ; scale bar, 5 μm; beam spot size, 15 μm; color bar in radians).
Fig. 13. Phase profiles of RBCs before pumping and 350 ns after pumping (X axis, distance in micrometers, Y axis, phase in radians).
profile of the RBCs before and after pumping is shown in Fig. 13. This proves that quantitative phase can be extracted from photothermal images through the integration of the digital holographic microscopy technique. 5. DISCUSSION AND CONCLUSION From the phase values shown in Fig. 13, it is clearly evident that the photothermal field expands compared to a “no-pump” image. Moreover, it can also be seen that the phase values at the center of the RBC are significantly reduced due to temperature excursion. For example, at the lowest portion of the RBC, the phase has decreased from 1 to 0.4 rad. It can be observed from Fig. 13 that the two peaks on the two sides of the cell become narrower, while the phase at the center of the cell shows a decrease compared to a no-pump image. This could be caused due to the effect of the thermal wave propagation at 350 ns. At 350 ns, the thermal wave has affected only the center region of the cell but not the entire cell yet. As the center portion of the cell is heated up, this portion is slightly expanded in size and its refractive index is lowered. The expanded center would momentarily compress the cell because the outer region is still at room temperature. Since the cell is compressed, its thickness is reduced leading to the narrowing of the two peaks. Also, because of the cell compression, its thickness is slightly higher and gives rise to an increase in phase at the two peaks. The above inference can be corroborated by the following: The thermal 2 diffusivity of the RBC is 0.0015 pffiffiffiffiffiffiffifficm ∕s [5]. The thermal diffusion length is given by 2 αt, where α is the thermal diffusivity and t is the time taken. Based on this equation, the thermal diffusion length comes to 0.46 μm. Using this analogy, since the focus spot is 1 μm, the thermal field expansion at 350 ns comes out to be 2 μm. Contemplating the experimental result shown in Fig. 13, using average size of the RBC as 6.5 μm [23], the separation between the two peaks is estimated about 2 μm. As the thermal wavefront at 350 ns is only 2 μm, this means only the center portion of the RBC is heated up, but the outer region of the cell is still at room temperature.
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There could be two reasons that change the phase of the photothermally excited sample. One is the change in refractive index, and the other is the tissue expansion effect. At the center of the RBC, where the pump beam is focused, the temperature is high, and the effect of negative d n∕d T is stronger than the effect of thermal expansion giving rise to an overall phase reduction within the diameter of the 1 μm region (the decrease in phase value is attributed to refractive index change (Δn), which is given by d n∕d T ΔT , where d n∕d T is the refractive index gradient with respect to temperature [5]). At a time delay of 350 ns, the effective thermal penetration has reached 2 μm; however, the outer region of the cell is still at room temperature, thus creating a thermal gradient. The radial thermal expansion around the center would then momentarily compress the cell, producing thicker cell material in the longitudinal direction (i.e., along the probe beam direction). This can be the cause behind the two peaks narrowing on the edges of the cell. At the same time, the lowering phase due to negative d n∕d T effect competes with increasing phase due to the thicker cell material along the longitudinal direction, giving an overall net effect of slight phase increment around the two peaks. In summary, a digital holographic photothermal technique is proposed for acquiring quantitative phase images. This technique is applied to RBCs, and the quantitative phase values were extracted. These phase values can be correlated to the thermal field. Besides obtaining high-quality images, the digital holographic photothermal technique can yield phase and amplitude images separately. This allows detailed study of the changes that have occurred due to photothermal excitation. Moreover, the phase values can be utilized to obtain more information such as the temperature distribution during photothermal studies, absorption of different organelles inside a cell etc. REFERENCES 1. D. O. Lapotko, T. R. Romanovskaya, A. Shnip, and V. P. Zharov, “Photothermal time-resolved imaging of living cells,” Lasers Surg. Med. 31, 53–63 (2002). 2. V. P. Zharov, “Far-field photothermal microscopy beyond the diffraction limit,” Opt. Lett. 11, 733–751 (2005). 3. C. K. Teu, G. Chen, M. Andika, S. Vasudevan, P. Chen, H. S. Yoon, Y. Zhao, and B. H. Choi, “Apoptosis monitoring of lymphocytes using photothermal imaging and response techniques,” in Optics within Life Sciences (OWLS) (ICO, 2008). 4. V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nano particles and cells,” IEEE J. Sel. Top. Quantum Electron. 28, 1314–1316 (2003).
Research Article 5. M. Andika, G. C. K. Chen, and S. Vasudevan, “Excitation temporal pulse shape and probe beam size effect on pulsed photothermal lens of single particle,” J. Opt. Soc. Am. B 27, 796–805 (2010). 6. D. Lasne, G. A. Blab, F. De Giorgi, F. Ichas, B. Lounis, and L. Cognet, “Label-free optical imaging of mitochondria in live cells,” Opt. Express 15, 14184–14193 (2007). 7. U. Schnars, T. M. Kreis, and W. P. O. Jüptner, “Digital recording and numerical reconstruction of holograms: reduction of the spatial frequency spectrum,” Opt. Eng. 35, 977–982 (1996). 8. C. J. Mann, L. Yu, and M. K. Kim, “Movies of cellular and sub-cellular motion by digital holographic microscopy,” Biomed. Eng. Online 5, 21–30 (2006). 9. C. J. Mann, “Quantitative biological microscopy by digital holography,” Ph.D dissertation (University of South Florida, 2006). 10. E. Hecht and A. Zajac, Optics (Addison-Wesley, 2002), Chap. 9. 11. C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19, 71–75 (1942). 12. E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett. 24, 291–293 (1999). 13. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, and C. Depeursinge, “Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy,” Opt. Lett. 30, 468–470 (2005). 14. E. Cuche, P. Marquet, and C. Depeursinge, “Spatial filtering for zeroorder and twin-image elimination in digital off-axis holography,” Appl. Opt. 39, 4070–4075 (2000). 15. D. Gabor, “A new microscopic principle,” Nature 161, 777–778 (1948). 16. P. Langehanenberg, B. Kemper, D. Dirksen, and G. Von Bally, “Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging,” Appl. Opt. 47, D176– D182 (2008). 17. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. J. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13, 9361–9373 (2005). 18. E. Cuche, P. Marquet, and C. Depeursinge, “Simultaneous amplitudecontrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms,” Appl. Opt. 38, 6994– 7001 (1999). 19. T. R. Judge and P. Bryanston-Cross, “Cellular-automata method for phase unwrapping,” Opt. Lasers Eng. 21, 199–239 (1994). 20. D. C. Ghiglia, G. A. Mastin, and L. A. Romero, “Cellular-automata method for phase unwrapping,” J. Opt. Soc. Am. A 4, 267–280 (1987). 21. M. A. Schofield and Y. Zhu, “Fast phase unwrapping algorithm for interferometric applications,” Opt. Lett. 28, 1194–1196 (2003). 22. D. C. Ghiglia and L. A. Romero, “Robust two-dimensional weighted and unweighted phase unwrapping that uses fast transforms and iterative methods,” J. Opt. Soc. Am. A 11, 107–117 (1994). 23. S. Vasudevan, G. C. K. Chen, M. Andika, S. Agarwal, P. Chen, and M. Olivo, “Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion,” J. Biomed. Opt. 15, 57001 (2010).
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Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope SRIVATHSAN VASUDEVAN,1,4,* GEORGE C. K. CHEN,2 ZHIPING LIN,3
AND
BENG KOON NG3
1
Discipline of Electrical Engineering, Indian Institute of Technology Indore, Indore, Madhya Pradesh 452017, India B. C. Photonics Tech. Co., Richmond, British Columbia V7E 1G9, Canada 3 Department of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798, Singapore 4 Centre for Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, Madhya Pradesh 452017, India *Corresponding author: [email protected] 2
Received 19 January 2015; revised 14 April 2015; accepted 14 April 2015; posted 14 April 2015 (Doc. ID 231595); published 7 May 2015
Photothermal microscopy (PTM), a noninvasive pump–probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications. © 2015 Optical Society of America OCIS codes: (180.6900) Three-dimensional microscopy; (090.1995) Digital holography; (350.5340) Photothermal effects. http://dx.doi.org/10.1364/AO.54.004478
1. INTRODUCTION Photothermal microscopy (PTM) is a noninvasive technique that has been applied to many biomedical studies. PTM utilizes two pulsed lasers as pump–probe lasers. The significant merit of this technique is that it is able to detect a sample smaller than the diffraction limit. The sample is irradiated with a pump pulse, which causes a temperature excursion. The absorbed thermal energy diffuses radially outward, forming a thermal field. When the thermal field expands beyond the diffraction limit of the microscope, a probe pulse is used to probe the sample plane [1,2]. The probe pulse would pick up the temperature-induced changes in the refractive index. The high-sensitivity high-resolution photothermal imaging can be realized by recording laser-induced temperaturedependent variations of the refractive index around the absorbing target. Suppose we have a triangular target, as shown in Fig. 1, which is smaller than the diffraction limit of the microscope. The laser excitation using a pump laser would excite the sample, thereby increasing the temperature of the sample. Here, each point of the triangular sample acts as a heat source, and, at a short time interval, the wavefront of the secondary wavelets (corresponding to the temperature profile) resembles that of the excited sample. At the same time, based upon the 1559-128X/15/144478-07$15/0$15.00 © 2015 Optical Society of America
characteristics of the medium, the refractive index of the medium changes due to temperature change. This creates a phase variation in the probe beam. To detect these phase changes, a phase contrast module is employed to convert the modulated phase into amplitude before detection by a CCD camera. Figure 2 illustrates the advantage of PTM by comparing a normal phase contrast image and the photothermal image of a lymphocyte cell obtained using the conventional photothermal imaging system developed by our group [3]. The PT image in Fig. 2(b) shows several bright and dark spots, which are due to spatial distribution of the refractive index change induced by the pump beam. The fact that the PT image depicts several components inside the cell, which cannot be seen in the conventional phase contrast microscope image [Fig. 2(a)], proves that a PT image can view cellular components better than a normal phase contrast microscope. Moreover, photothermal images could capture this information without adding any fluorescent tags; thus, PTM has become a popular technique for many biomedical imaging applications. Several studies [1,4,5] have been conducted using PTM. These images have been in tandem with fluorescent images [6]. It is clear from Fig. 2(b) that the photothermal content of the sample is stored as a change in the refractive index (due to temperature excursion)
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Fig. 1. Principles of photothermal imaging.
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Therefore, this paper attempts to solve the above-mentioned problems in the conventional PTM by acquiring quantitative photothermal images. This is implemented by integrating digital holographic microscope with a photothermal microscopy technique. Using a digital holographic microscope is a powerful well-known technique for obtaining quantitative phase information, and it has been widely used for many biomedical studies. Particularly, a digital holographic microscope has been used for real-time continuous monitoring of many biomedical cellular assays. Hence, the proposed method of integrating PTM with a digital holographic microscope would yield separate amplitude and phase images. In addition, the extracted phase values from these images can be used for further processing. The proposed method would provide first-hand information of the phase values created by the temperature excursion of the pump beam of PTM. This can provide important information, which can act as a cell signature. In addition, the proposed integration would also pave the way for continuous nonfluorescent real-time monitoring of assays, which can provide quantitative information. 2. EXPERIMENTAL DEVELOPMENT FOR OFF-AXIS DIGITAL HOLOGRAPHY
Fig. 2. (a) Phase contrast image of a lymphocyte cell. (b) Photothermal image of the same lymphocyte cell (60×, N.A., 0.65).
or, in other words, phase information. In order to obtain this information, a conventional phase contrast microscope is utilized for this purpose. While several studies have reported the applicability and advantage that the photothermal technique has over fluorescent techniques, the next step is to take PTM forward to obtain useful information from the photothermal images. The information can be quantitative information that can act as a signature of the sample. For example, information such as temperature distribution of the sample, particularly in the case of biological cells, could provide information on the absorption contrast. Absorption contrast of cells could be used as a potential tool to identify normal cells from malignant cells. Therefore, providing useful information from photothermal techniques can make a photothermal microscope an efficient diagnostic tool for many diseases including but not limited to cancer. A phase contrast microscope, used in conventional phase contrast microscopes, would not help to retrieve quantitative information. The reason is that current photothermal microscopes use a Zernike filter to convert the phase changes into amplitude for CCD camera recording. The images obtained using a Zernike filter are nonlinear [7–9]; hence, these images could not be used to extract phase information. Moreover, existence of halos in the image due to the phase contrast module [10,11] of the PTM also deters the quality of the photothermal images. However, quantitative phase values at each and every point of the PT image can yield important information such as temperature distribution, refractive index gradient with temperature, thermal diffusivity, etc.
As a first step of integration, the digital holography setup is constructed using the lasers available for a photothermal imaging setup. Figure 3 shows the experimental setup of the digital holographic photothermal microscope. The basic architecture for a digital holographic microscope is that of a Mach–Zender interferometer. Traditional digital holographic microscopes utilize a continuous highly stabilized laser as the laser source [12]. Stabilized lasers would have a long coherent length that could help the two beams (reference and the object) to interfere at the CCD plane. However, for photothermal imaging, pulsed lasers are used for pumping and probing the samples. Pulsed heating of the sample ensures that the biological sample is not heated up (approximately 10 ns). Also, since the photothermal field formed can be imaged only by a short duration laser, a pulsed nanosecond laser is used as a probe laser. The digital holographic experiments were performed using the developed experimental setup, as schematically depicted in Fig. 3. The probe laser, also called the imaging laser, is a frequency-doubled Nd:YAG pulsed laser with a wavelength of 532 nm with energy of 0.12 μJ. The laser output, which is spatially filtered and collimated (using a beam expander, not shown in Fig. 3), is split into a reference beam (R) and an
Fig. 3. Schematic of a digital holographic system.
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object beam (O) using a nonpolarized beam splitter. The setup is a transmissive interferometer setup based on the Mach– Zehnder configuration. The benefit of the experimental setup is that the path length in the reference and object arms is equal due to the symmetry of the setup. Hence, this setup can be used to calculate the amount of phase modulation caused by a sample that is placed in one of the arms. The sample or biological specimen is placed on the X –Y translational microscope sample stage. A pair of similar microscope objectives is used in the two optical arms of the experimental setup to match the curvatures of the two wavefronts. A slight angle is introduced between the object and the reference beams for off-axis holography. The microscope objectives and the beam splitter are adjusted such that the interference fringes are straight, thereby avoiding the need to perform any digital correction due to spherical aberration introduced by the microscope objectives. The object and the reference beams were allowed to interfere at the hologram plane. The hologram with intensity [13,14] I H x; y jRj2 jOj2 R O RO
(1)
is recorded using a standard black and white CCD camera (1280 × 980 pixels, Imaging Source, 41BF02). For recording the holograms, the CCD camera is placed at the hologram plane, as shown in Fig. 3. The reconstruction distance would be the distance between the image plane and the hologram plane. A simple program is written in MATLAB to transfer the image captured by the camera to the computer. In order to reconstruct the digital hologram recorded, a replica of the reference wave is needed. However, in this case, a digitally computed reference wave (R D ) is used to reconstruct the digital transmitted wavefront (ψ), as given by [12,15,16]: ψ R D jRj2 R D jOj2 R D R O R D RO :
(2)
The first two terms of Eq. (2) correspond to the zero-order diffraction, the third term to the twin image and the fourth term to the real image [17]. Hence, the reference arm is adjusted in the experimental setup so that the hologram is recorded in the off-axis geometry [14]. The incidence angle of the reference arm is adjusted such that there is a separation between the real image, twin image, and the zero-order diffraction in the observation plane. For the present experimental setup, it is necessary to verify the acquisition of the hologram and also the reconstruction of the amplitude and phase images. This step will ensure that the resolution of the microscope is preserved before performing PT imaging. A USAF target was placed at the sample plane and the hologram was acquired. The acquired hologram is shown in Fig. 4. By implementing a simple Fourier transform algorithm, the twin image, real image, and the zero-order diffraction terms are visible, as shown in Fig. 5(a). This clearly helps the experimentalist to verify the off-axis holograms. For the reconstruction process, the digitally constructed reference wave would be multiplied with the hologram amplitude transmission. The reconstructed wavefront ψ for the acquired hologram is represented by [14,18]
Fig. 4. Hologram of USAF target acquired using the developed experimental setup.
iπ 2 2 ψξ; η A exp ξ η λd ZZ iπ 2 x y 2 × I H x; y exp λd i2π xξ yη dxdy; × exp λd
(3)
where I H x; y is the recorded hologram, λ is the wavelength, d is the reconstruction distance, and x; y and ξ; η are the coordinates of the hologram image and the reconstructed image, respectively. A is a complex constant given by exp i2πd ∕λ∕iλd . For phase contrast imaging, a digital reference wave (RD ), which is a replica of the original reference wave, is multiplied with the hologram. Assuming a perfect plane wave for the reference wave, RD can be calculated as R D AR expi2π∕λkx x ky y;
(4)
where AR is the amplitude, and kx and ky are the components of the wave vector. By adjusting these two components, the
Fig. 5. (a) Twin image, real image, and the diffraction terms of the USAF hologram acquired in Fig. 3. (b) Intensity image of USAF target (reconstruction distance is 116.8 mm).
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reference wave could be matched with that of the original reference wave. The intensity of the reconstructed wavefront is calculated as Iξ; η jψξ; ηj2 ;
(5)
and the phase is given by φξ; η arctan
Imψξ; η : Reψξ; η
(6)
Reconstructing the USAF hologram shown in Fig. 4 by the above-mentioned equations, a portion of the reconstructed intensity image is shown in Fig. 5(b).
3. DIGITAL HOLOGRAPHY EXPERIMENTS It is evident from these images that the resolution of the microscope is achievable in digital holography, and the ability to obtain separate intensity and phase images is shown. In addition to the resolution, it is also essential to verify whether the phase values can be obtained from the digital holography experimental setup. The phase images obtained from the holographic reconstruction would be wrapped. Hence, phase unwrapping needs to be performed on the extracted phase image to acquire accurate results. Several phase unwrapping techniques are already available [19–21]. Suitable phase unwrapping methods are applied to the phase image to obtain continuous phase, and then the phase values can be extracted. Figure 6 shows the hologram of a polystyrene particle placed in the sample holder of the developed experimental setup. The size of the polystyrene particle is 45 μm. A phase unwrapping algorithm [17,22] is implemented on the phase image to obtain the unwrapped phase of the polystyrene particle. The hologram plane was 65 mm distance. The Fourier analysis shows the real, twin, and the diffraction term in Fig. 7(a). After reconstruction of the image from the hologram, the intensity image is shown in Fig. 7(b). After applying the phase unwrapping algorithm, the corresponding phase image is shown in Fig. 8(a). A line is drawn across the center of the phase image, and the corresponding phase values are extracted. The phase values are shown in Fig. 8(b). These phase values would help to decipher
Fig. 6. Hologram of a polystyrene particle (reconstruction distance, 65 mm).
Fig. 7. (a) Twin image and the diffraction terms of the polystyrene particle captured using the hologram in Fig. 6. (b) Intensity image of the polystyrene particle after reconstruction.
information on photothermal fields later on during photothermal imaging. From Fig. 8(b), the maximum phase corresponds to 37.36 rad, while the minimum phase across the edges is 2.6 rad (since the phase values across the two edges are not the same, the average of these two values, namely, 0.9761 and 4.242, is taken). Hence, the phase change created due to the particle is 34.76 (37.36–2.6) rad. The size of the particle is calculated by ϕ 2π∕λnp − nm d p , where np is the refractive index of the particle, nm is the refractive index of the medium, d p is the diameter of the particle, and ϕ is the phase in radians. Since the refractive index of the microscope immersion oil is 1.518, the size of the particle comes to dp
34.76 × 532 × 10−9 43.86 μm: 6.28 × 1.59 − 1.518
(7)
The real size of the particle, according to the specifications, is 45 μm (S:D: 0.3), thus proving that the calculation obtained from the hologram is very close to the true result. In order to check the consistency of the results, the same experiment has even been repeated with various polystyrene particles of different sizes. The summary of the size calculations is provided in Table 1. The results are quite consistent with the actual sizes of these particles. Therefore, the calculation substantiates the fact that the phase values obtained are accurate.
Fig. 8. (a) Three-dimensional visualization of the phase image obtained from the unwrapping algorithm. (b) Phase values of the polystyrene particle over a line drawn across the center of the particle.
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Table 1. Experimental Size Calculations of Various Samples Using the Developed Experimental Setup Sample Red polystyrene bead Transparent polystyrene bead Black polystyrene bead Transparent polystyrene bead
Actual Size (μm)
Experimentally Calculated Size (μm)
5.1 15
4.96 0.4 14.86 0.3
10 45
9.6 0.4 46.2 0.5
Having confirmed the results obtained from the digital holographic technique, a preliminary experiment was also conducted on red blood cells (RBCs) to ascertain the phase image and the viability of this technique for biological samples. Figure 9(a) shows the phase image obtained through digital holography and the corresponding phase values, obtained by drawing a line profile on the phase image, as shown in Fig. 9(b). The disc shape of the RBC is clearly evident from Fig. 9(a), which shows the advantage of the digital holographic technique. 4. QUANTITATIVE PHOTOTHERMAL MICROSCOPY OF RED BLOOD CELLS For conducting the photothermal experiments, the pump beam is needed for pumping the samples to induce a temperature excursion. Hence, the digital holographic microscopy setup is appended with a pump laser. The pump laser used is a Nd:YAG laser pumped optical parametric oscillator working
Fig. 9. (a) Phase image of the RBC through numerical reconstruction (color bar in radians). (b) Phase profile obtained from the phase image depicting the phase values.
Fig. 10. Modified experimental schematic incorporating photothermal imaging and digital holographic microscopy in a single setup.
at 540 nm wavelength. The pump laser is irradiated through BS2, which is focused by the microscope objective. The focused pump laser excites the sample, thus creating a temperature excursion required for photothermal studies. The revised experimental setup is shown in Fig. 10. After passing through the sample, a pump filter was used to block the pump beam from reaching the CCD camera, which is placed at the hologram plane. Three holograms were captured during this process. The first hologram is a no-pump image captured before pumping the sample. The second hologram is the PT image captured at a specific time delay (∼ ns) after pumping, while the third is taken without pumping the sample. The third hologram was captured at a time frame much longer (∼ minutes) after pumping. This image would ensure that the sample has returned back to normalcy after heating. Figure 11 shows the phase values of a polystyrene particle, which has been imaged three times: (1) no-pump image; (2) photothermal image (PT); and (3) no-pump image taken after a very long time from pump (∼ minutes). These phase profiles prove that, after the photothermal excitation, the particle returns back to its normalcy, which is clear from the phase values of the No-pump2
Fig. 11. Photothermal phase profile of a polystyrene particle taken (a) before pumping (No-pump1), (b) 50 ns after pumping (PT), and (c) long period after pumping (No-pump2).
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Table 2. Phase Change at the Center of the Particle Due to PTI
Sample Black polystyrene bead Red polystyrene bead
Phase Change Due to PTI at 50 ns (rad)
Phase Change Due to PTI at 400 ns (rad)
0.6 0.2
0.1 0.1
0.4 0.1
0 0.1
image when compared with the No-pump1 image. It is also visible that the phase change is due to temperature increase in the photothermal image. Since the d n∕d T is negative for polystyrene particles, the phase actually decreases due to increase in temperature. The same experiment is also repeated on black and red polystyrene particles for the repeatability, and the results are shown in Table 2. After proving that the proposed technique of adopting digital holography with photothermal imaging can extract accurate phase values, the PT imaging technique was applied to RBCs. Fresh human blood was obtained by venipuncture from healthy donors into 0.5% BSA solution in a phosphate buffer saline (PBS) solution pH 7.4. Erythrocytes were then collected through centrifugation at 4°C, 1000 G for 15 min. Experiments were performed with 0.3% hematocrit at 37°C in Ringer solution (that contains 125 mM NaCl, 5 mM KCl, 1 mM MgSO4 , 32 mM HEPES, 5 mM glucose, 1 mM CaCl2 , pH 7.4). Figures 12(a) and 12(b) show the phase images of the RBCs taken before pumping and 350 ns after pumping the sample (Nd:YAG laser, 540 nm, 0.4 μJ). Out of the three cells shown in the figure, the pump beam is focused on the bottom cell. A line was drawn across the RBCs, as shown in the figures, and the corresponding phase profiles were extracted. The phase
Fig. 12. Phase images taken (a) before pumping and (b) 350 ns after pumping of RBCs (pump energy, 0.4 μJ; probe energy, 0.12 μJ; scale bar, 5 μm; beam spot size, 15 μm; color bar in radians).
Fig. 13. Phase profiles of RBCs before pumping and 350 ns after pumping (X axis, distance in micrometers, Y axis, phase in radians).
profile of the RBCs before and after pumping is shown in Fig. 13. This proves that quantitative phase can be extracted from photothermal images through the integration of the digital holographic microscopy technique. 5. DISCUSSION AND CONCLUSION From the phase values shown in Fig. 13, it is clearly evident that the photothermal field expands compared to a “no-pump” image. Moreover, it can also be seen that the phase values at the center of the RBC are significantly reduced due to temperature excursion. For example, at the lowest portion of the RBC, the phase has decreased from 1 to 0.4 rad. It can be observed from Fig. 13 that the two peaks on the two sides of the cell become narrower, while the phase at the center of the cell shows a decrease compared to a no-pump image. This could be caused due to the effect of the thermal wave propagation at 350 ns. At 350 ns, the thermal wave has affected only the center region of the cell but not the entire cell yet. As the center portion of the cell is heated up, this portion is slightly expanded in size and its refractive index is lowered. The expanded center would momentarily compress the cell because the outer region is still at room temperature. Since the cell is compressed, its thickness is reduced leading to the narrowing of the two peaks. Also, because of the cell compression, its thickness is slightly higher and gives rise to an increase in phase at the two peaks. The above inference can be corroborated by the following: The thermal 2 diffusivity of the RBC is 0.0015 pffiffiffiffiffiffiffifficm ∕s [5]. The thermal diffusion length is given by 2 αt, where α is the thermal diffusivity and t is the time taken. Based on this equation, the thermal diffusion length comes to 0.46 μm. Using this analogy, since the focus spot is 1 μm, the thermal field expansion at 350 ns comes out to be 2 μm. Contemplating the experimental result shown in Fig. 13, using average size of the RBC as 6.5 μm [23], the separation between the two peaks is estimated about 2 μm. As the thermal wavefront at 350 ns is only 2 μm, this means only the center portion of the RBC is heated up, but the outer region of the cell is still at room temperature.
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There could be two reasons that change the phase of the photothermally excited sample. One is the change in refractive index, and the other is the tissue expansion effect. At the center of the RBC, where the pump beam is focused, the temperature is high, and the effect of negative d n∕d T is stronger than the effect of thermal expansion giving rise to an overall phase reduction within the diameter of the 1 μm region (the decrease in phase value is attributed to refractive index change (Δn), which is given by d n∕d T ΔT , where d n∕d T is the refractive index gradient with respect to temperature [5]). At a time delay of 350 ns, the effective thermal penetration has reached 2 μm; however, the outer region of the cell is still at room temperature, thus creating a thermal gradient. The radial thermal expansion around the center would then momentarily compress the cell, producing thicker cell material in the longitudinal direction (i.e., along the probe beam direction). This can be the cause behind the two peaks narrowing on the edges of the cell. At the same time, the lowering phase due to negative d n∕d T effect competes with increasing phase due to the thicker cell material along the longitudinal direction, giving an overall net effect of slight phase increment around the two peaks. In summary, a digital holographic photothermal technique is proposed for acquiring quantitative phase images. This technique is applied to RBCs, and the quantitative phase values were extracted. These phase values can be correlated to the thermal field. Besides obtaining high-quality images, the digital holographic photothermal technique can yield phase and amplitude images separately. This allows detailed study of the changes that have occurred due to photothermal excitation. Moreover, the phase values can be utilized to obtain more information such as the temperature distribution during photothermal studies, absorption of different organelles inside a cell etc. REFERENCES 1. D. O. Lapotko, T. R. Romanovskaya, A. Shnip, and V. P. Zharov, “Photothermal time-resolved imaging of living cells,” Lasers Surg. Med. 31, 53–63 (2002). 2. V. P. Zharov, “Far-field photothermal microscopy beyond the diffraction limit,” Opt. Lett. 11, 733–751 (2005). 3. C. K. Teu, G. Chen, M. Andika, S. Vasudevan, P. Chen, H. S. Yoon, Y. Zhao, and B. H. Choi, “Apoptosis monitoring of lymphocytes using photothermal imaging and response techniques,” in Optics within Life Sciences (OWLS) (ICO, 2008). 4. V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nano particles and cells,” IEEE J. Sel. Top. Quantum Electron. 28, 1314–1316 (2003).
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