We propose a novel digital holographic microscopy (DHM) by integrating surface plasmon holographic microscopy (SPHM) with reflection DHM based on the angular and polarization multiplexing techniques. Taking advantages of the high sensitivity of surface plasmon resonance (SPR) and the high reflectivity of gold film, the tiny variations of specimen’s refractive index (RI) can be measured by using SPHM, and meanwhile, the thickness changes of the specimen can be determined by means of reflection DHM. We experimentally monitor the volatilization process of an alcohol-water mixture droplet to verify the validity of the integrated DHM. The proposed microscopy is very promising in the objective-coupling SPR microscopy for multi-information measurements of diverse specimens with low-contrast RI distributions (biomolecules, nanofluids, etc.) in a dynamic and nondestructive way.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Surface plasmon resonance (SPR) is widely applied in the areas of physical property characterization and biochemical monitoring with the advantage of sensitive response to tiny variations of specimens in the near field of metal surface [1–4]. By measuring the intensity of the light wave reflected from the metal surface during SPR, numerous applications are experimentally achieved including nanoparticle concentration measurements , graphene layer characterizations , biomolecule dot and living cell imaging [7,8], etc. Compared with the intensity measurement, phase interrogation of the reflected light wave shows much higher sensitivity to the specimen’s refractive index (RI) change in the near field of metal surface . Thus, it can be applied to measuring mixture RI [10,11], sensing protein interactions , monitoring DNA absorptions , and so on.
As an advanced imaging technique, digital holographic microscopy (DHM) can realize dynamic, wide-field, and nondestructive measurements for the complex amplitude of the light wave [14–17]. Taking these advantages, surface plasmon holographic microscopy (SPHM) is proposed to simultaneously acquire the amplitude- and phase-contrast SPR images of different specimens with high sensitivity [18,19]. Compared with total internal reflection fluorescence (TIRF) microscopy, SPHM demonstrates the superiority of non-intrusive reflection imaging with high imaging contrast. Recently, we have proposed and improved a common-path SPHM to monitor the tiny variations of the specimen RI [20,21]. We have also developed a compact objective-coupling SPHM to measure the thickness distribution of a thin film . However, the current SPHM is not capable to measure the RI and thickness of a specimen simultaneously. To solve this problem, an effective approach is proposed to combine the total internal reflection DHM and transmission DHM together for determining the RI and thickness variations of the ultraviolet curing adhesive in real time . Another method is demonstrated to monitor the dynamic evaporation process of a deionized water droplet from the transmission and reflection phase images, calculating the RI and thickness changes of the specimen simultaneously . Regrettably, these methods above lack the high sensitivity to measure the tiny variations of the specimen RI in a dynamic process, which limits their potentials to characterize specimens with low-contrast RI distributions in the near-field detection. Consequently, the simultaneous measurements of specimen’s RI and thickness with high sensitivity require more research interests especially in the biological monitoring area.
In this paper, we firstly integrate SPHM with reflection DHM to simultaneously monitor the tiny variations of RI and thickness changes for dielectric specimens by employing the angular and polarization multiplexing techniques. Particularly, the gold film is coated on the prism in the experimental setup to simultaneously excite SPR and realize the reflection DHM.-Therefore, the tiny variations of the specimen RI can be determined with high sensitivity via the reflection phase shift of the reconstructed object waves using SPHM. While the thickness changes of specimens can be obtained from the reflection phase of the reconstructed object waves by reflection DHM. As a verification, the volatilization process of an alcohol-water droplet is monitored by simultaneously characterizing the tiny variations of RI and thickness changes of the droplet. With the advantages of dynamic, non-contact and wide-field measurements, the integrated DHM is applicable to monitoring specimens with low-contrast RI distributions especially in the biological and chemical areas.
2.1 Digital holographic microscopy
In DHM, the hologram is recorded in the image plane of the object, and the complex amplitude of the object wave is generally reconstructed using the angular spectrum method. In order to remove the background noise and the influence of the reference wave distortion, the double-exposure holographic interferometry is employed to obtain the relative phase distribution of the reconstructed object waves [20–24]. The relative phase distribution can be written as Δϕ(x, y) = arg[O(x, y)/O'(x, y)] = ϕO(x, y)–ϕO'(x, y), where O(x, y) and O'(x, y) denote the complex amplitudes of the reconstructed object waves with and without specimens, respectively, ϕO(x, y) and ϕO'(x, y) are the corresponding phase distributions.
2.2 RI measurement by SPHM
Figure 1 shows the three-layer Kretschmann configuration which consists of a prism, a gold layer, and a dielectric layer. In order to excite SPR, a p-polarized light beam passes through the prism at a certain incident angle (i.e. SPR angle) with a dielectric specimen adhered on the gold surface. Under the wavevector-matching condition, the light wave will resonantly couple with the surface plasmon wave (SPW) which is the electron density wave in the gold layer . The penetration of SPW into the dielectric layer leads to the high sensitivity of SPR to the tiny changes of specimens in the near field of the gold surface.
According to the Fresnel formulas for multi-layer reflection, the complex reflection coefficient r of the light wave reflected from the gold layer is determined by multiple parameters and can be described asFigure 2(a) depicts the theoretical curve between Δφ and n3, in which the quite steep slope in the blue rectangle can be used to measure the tiny variations of n3 by Δφ with high sensitivity. Because the change of reflection phase shift corresponds to the wavefront phase change of the reflected light wave, Δφ is equal to Δϕ1 which is measured by the double-exposure holographic interferometry in SPHM.
Since the Fresnel formulas are complex  and it is difficult to deduce a function between n3 and Δφ, we characterize their mathematical relationship by using fifth polynominal fitting tool in the Matlab software to calculate n3 from Δφ, as shown in Fig. 2(b) . The fitting function can be expressed as
2.3 Thickness determination by reflection DHM
Owing to the high reflectivity of gold layer, a light beam which vertically hits on the gold layer is reflected back by the gold surface and goes through the specimen adhered on the gold surface twice. The relative phase distribution Δϕ2(x, y) of the reflected light wave can be determined by the double-exposure holographic interferometry in reflection DHM and can be expressed asEq. (2), the thickness of the specimen can be determined by
Combining the advantages of the high sensitivity of SPR and the high reflectivity of gold film together, an integrated DHM can be constructed based on the prism-coupling SPR setup to measure the specimen’s RI and thickness simultaneously according to Eqs. (2) and (4).
3. Experimental setup
Figure 3 depicts the experimental setup of the integrated DHM. Here, we combine two off-axis Mach-Zehnder interferometers together employing the angular and polarization multiplexing techniques. In particular, a light beam from a He-Ne laser (λ = 632.8 nm) is divided into two beams by the fiber coupler FC. The two beams are expanded, collimated and polarized to 45° via the beam expanders BE1, 2, lenses L1, 2 and half-wave plates HP1, 2, respectively. One beam is further divided into p- and s-polarized ones via polarized beam splitter PBS1. The p-polarized one acts as the object beam Op in SPHM, which passes through the rectangular prism at an incident angle of 72.82° to excite SPR with a specimen adhered on the gold film. The s-polarized one acts as the object beam Os in reflection DHM, which vertically hits on the gold layer and is reflected back by the gold surface, going through the specimen twice. The two object beams further pass through the long working distance microscope objectives LWDMO1, 2 (Mitutoyo M Plan Apo 5 × ), respectively, and then are combined together through BS2 and imaged on the CCD target (Basler acA2040-90μm, 2048H × 2048V, pixel size 5.5μm) by L3. The other beam from the FC is also divided into p- and s-polarized ones via PBS2. The two divided beams act as the reference beams Rp and Rs in SPHM and reflection DHM, respectively. Finally, all the beams are combined by BS4 to form a composite hologram on the CCD target. Here, the two object beams are projected on the CCD target coaxially, while the incident angles of reference beams on the CCD target are adjusted to generate two sets of interference fringes with different orientations. The inset shows the routes of the two object beams.
4. Experiment results
4.1 Preliminary measurements
To demonstrate the feasibility of the integrated DHM, we firstly measure the RI and thickness distributions of an alcohol-water mixture droplet with the volume ratio of 2:3 at the initial time. When the droplet is adhered on the gold surface, a hologram is immediately recorded. Then, a background hologram is captured after cleaning out the mixture. As shown in Fig. 4(a), there are two sets of interference fringes with nearly orthogonal directions in the composite background hologram, which effectively eliminates their crosstalk. Figure 4(b) depicts the spatial spectra of the composite hologram, where the spatial spectra in the red and yellow rectangles correspond to the object waves in SPHM and reflection DHM, respectively.
Employing the reconstruction algorithm, the relative reflection phase distribution Δϕ1(x, y) is obtained, which is equal to the reflection phase shift difference distribution Δφ(x, y). As shown in Fig. 5(a), the phase image of the specimen is compressed in one direction due to the oblique incidence of the light beam on the prism. To recover the phase image of the droplet, the reconstruction algorithm based on the angular spectrum method in digital holography is adopted to compensate for the tilted angle caused by the inclined SPR plane on the prism with respect to the optical axis . Figure 5(b) shows the recovered phase image, in which all the area of the specimen image is focused for calibration. Figure 5(c) depicts the RI distribution along the black line in Fig. 5(b), according to Eq. (2). Since alcohol is completely miscible with water, the alcohol-water mixture is reasonably considered as a homogenous liquid. Thus, the RI value of the alcohol-water mixture is averaged as n3 = 1.3397 RIU. Figure 5(d) plots the reconstructed relative reflection phase distribution Δϕ2(x, y) in reflection DHM. According to Eq. (4), the three-dimensional (3D) thickness distribution of the mixture droplet is depicted in Fig. 5(e). Figure 5(f) depicts the thickness profile of the mixture droplet along the black line in Fig. 5(d).
4.2 Measurements of a dynamic process
Subsequently, we monitor the volatilization process of an alcohol-water mixture droplet with the volume ratio of 1:2 to verify the dynamic measurement capability of the proposed setup. Firstly, 43 holograms with the specimen are captured at the rate of 1 fps. Then, the background hologram is recorded after removing the droplet. With numerical reconstruction, we calculate the value of relative reflection phase Δϕ1 (i.e. Δφ) by averaging the relative reflection phase distribution in the mixture area. As shown in Fig. 6(a), Δϕ1 displays a very distinct change during the volatilization process. According to Eq. (2), the corresponding RI variation of the mixture droplet is quantitatively determined. Figure 6(b) shows that the specimen RI decreases from 1.3374 RIU to 1.3318 RIU when the mixture volatilizes to pure water. So the RI variation Δn3 is 0.0056 RIU. The RI of pure water is measured as 1.3318 RIU, which is in good accordance with the measured value of 1.3317~1.3319 RIU (@20°C~25°C) by commercial devices at 632.8nm. In reflection DHM, the maximum value of relative reflection phase distribution Δϕ2 decreases continuously with the monitoring time, as depicted in Fig. 6(c). According to Eq. (4), the maximum value of the droplet thickness distribution hmax also descends gradually, as shown in Fig. 6(d). The small fluctuations of the measured values in Figs. 6(a)-6(d) result from the unstable laser source and the environmental disturbances.
The dynamic changes of the specimen 3D thickness distributions during the volatilization process can be seen in Visualization 1. In particular, the 3D thickness distributions of the droplet at the time of t = 0s, 9s, and 42s are shown in Figs. 7(a)–7(c), respectively. Figure 7(d) depicts the corresponding thickness profiles along the horizontal line which goes through the center of the droplet. It can be seen that the mixture droplet shrinks quite fast in the early stage, revealing the quick volatilization of alcohol. Thus, both the RI and thickness of the mixture droplet reduce successively during the monitoring time.
The specimen RI measurement based on SPHM shows very high sensitivity, which can be given by |dΔφ(x, y)/dn3(x, y)| . So the RI measurement sensitivity is as high as 1.71 × 104 deg/RIU in monitoring the volatilization process of an alcohol-water mixture droplet, which is comparable to the sensitivity of 1.29 × 104 deg/RIU in the existing phase-sensitive SPR system . Since the total internal reflection (TIR)-DHM can also measure the RI changes of specimens , we further compare the theoretical sensitivities of the specimen RI measurement based on TIR and SPR. As shown in Fig. 8, the sensitivity of the specimen RI measurement based on SPR is two orders of magnitude higher than that based on TIR, demonstrating the great advantages of monitoring both the tiny variations of specimen RI and various processes which are related to the small changes of specimen RI by using SPHM.
In conclusion, we have demonstrated an integrated DHM which can be used to simultaneously monitor the tiny variations of RI and thickness changes for dielectric specimens by combining SPHM with reflection DHM through the angular and polarization multiplexing techniques. In SPHM, the tiny variations of the specimen RI can be determined with high sensitivity based on the three-layer SPR model. The specimen thickness can be calculated from the relative reflection phase distribution in reflection DHM. The gold film coated on the prism in the experimental setup is used to simultaneously excite SPR and realize the reflection DHM. The experiment of monitoring the volatilization process of an alcohol-water mixture droplet has been successfully performed to demonstrate the effectiveness of the integrated microscopy. The sensitivity of the RI measurement is as high as 1.71 × 104 deg/RIU in the experiment. With such high sensitivity of RI measurement, our system will pave a new route to monitor the biological specimen interactions based on the objective-coupling SPR configuration in a noninvasive and dynamic way.
Key R&D Program of Shaanxi Province (2017KW-012); National Natural Science Foundation of China (NSFC) (11634010); Joint Fund of the National Natural Science Foundations of China and China Academy of Engineering Physics (NSAF) (U1730137).
We would like to thank Cheng Li at the School of Science in Northwestern Polytechnical University for the fabrication of gold film.
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