1. Introduction

Coherence-gated optical imaging techniques that enable wide-field time- and depth-resolved imaging through turbid media like atmosphere, plastic or biological tissue have attracted much attention in recent years [1–9]. Optical coherence tomography (OCT) [1, 2, 6] with impressive performance e.g. in ophthalmology [10] is the dominating imaging technique in this field. A less mature coherence-gated imaging technique is the adaptive [3, 4] holographic method of optical coherence imaging (OCI) [3–5, 7, 11]. Equipped with our recently discovered all-optical phase coherent photorefractive (PCP) II-VI quantum wells (QWs) [7, 11–14], OCI and a modified novel technique which we named contrast enhanced holographic imaging (CEHI) have the potential to become powerful methods for high-speed three-dimensional (3D) imaging through turbid media or for highly sensitive particle tracking in solution.

In the last two decades homovalent III-V QWs have been extensively used as high-speed photorefractive films. These structures reveal strong Franz-Keldysh [15–20] and quantum confined Stark effects [21–24] when transverse or longitudinal external fields are applied normal or parallel to the QW growth direction. Because of the high diffraction efficiencies and fast response time of these films, two dimensional optical imaging devices [25], femtosecond pulse shaping [26] and laser based ultrasound detection [27] have been demonstrated. Furthermore 3D optical coherence imaging (OCI) at high frame rates [5, 28, 29] and with arbitrary low coherence light sources [5, 29] has been performed. These investigations include imaging through turbid media [5, 8, 9] and on biological systems [3, 4]. However, photorefractive III-V QWs easily degrade due to Joule heating at the contacts.

Heterovalent II-VI/III-V quantum wells (ZnSe quantum wells grown on GaAs substrate) exhibit a strong all-optical photorefractive (PR) effect without requiring external electric fields [12–14]. The all-optical PR effect is caused by the different conduction and valence band offsets between the II-VI and III-V semiconductors which leads to the injection of optically excited GaAs substrate electrons into the ZnSe quantum well. A long living electron density grating is formed in the ZnSe QW by the repulsive interaction of captured electrons with the exciton density grating. The generated electron density grating in the QW is stabilized by strongly localized holes at the barrier/GaAs interface. The spatially modulated electron density grating in the QW and the hole grating at the interface create space-charge fields which are responsible for the occurrence of the PR effect [12, 13].

The phase coherent photorefractive effect (PCP) has a fast response time of the electron grating (in the order of few microseconds) even at low excitation intensities [12–14] and in addition a high diffraction efficiency of about ~1 x 10⁻³ [11]. Together with the time-gating capabilities of PCP these properties enable imaging of stationary and moving objects [7, 11]. In this paper we demonstrate imaging of floating glass beads of ~30 µm and ~150 µm size and of living Paramecium and Euglena cells using OCI in the contrast enhanced holographic imaging (CEHI) mode. CEHI is also capable of imaging a ~100 µm diameter metal wire concealed by a chicken skin using a two color configuration.

2. Experimental details

The PCP QW structure was pseudomorphically grown on a (001) oriented GaAs substrate by molecular beam epitaxy [30]. It consists of an 8 nm thick ZnSe single QW sandwiched between two 30 nm Zn_0.92Mg_0.08Se barriers, with a 20 nm thick ZnSe buffer layer between the barrier and the substrate. A frequency-doubled mode locked Ti-sapphire laser providing 100 fs pulses at a repetition rate of 80 MHz served as light source. The laser energy was resonantly tuned to the ZnSe QW exciton transition at ~2.805 eV. For the holographic imaging a four-wave-mixing setup in a back scattering configuration was used [7]. The linearly polarized pulses with wave vectors k₁ and k₂ can be mutually delayed with a micrometer positioning stage by a time τ. The angle θ between the writing beams had been set to ~3.5° which corresponds to a grating spacing of ~7 µm. The PCP QW structure, with an active area of ~7 x 6 mm², was mounted in a Helium-flow cryostat maintained at liquid nitrogen temperature of ~77 K. At this temperature, the diffraction efficiency of the ZnSe QW structure had been measured to be ~1 x 10⁻³ [11].

A sketch of the essential part of the CEHI set-up is shown in Fig. 1(a). The front surface of a glass cuvette (1 x 1 cm² inner basis area, 3 cm height and 1.25 mm glass thickness) is set parallel to the PCP QW surface in the cryostat. In earlier experiments on large diameter glass beads [11] the weak reflected laser beam from the rear cuvette’s glass/air interface was used as the erasing beam. Few 10 micrometer size glass beads and small Paramecium and Euglena cells were not clearly visible in this case. In order to increase the sensitivity of the CEHI set-up and the visibility of small particles it was crucial to increase the ratio between the erasing beam and object beam intensity but at the same time keeping the incident beam intensity low to avoid overexposure of the cuvette’s holographic image or harming the living organisms. We therefore coated the rear cuvette surface with silver or used an uncoated cuvette with a mirror placed at the back glass surface resulting in a significant contrast enhancement of the imaged objects. In order to minimize reflection losses of both the object and the erasing beam a polarization dependent beam splitter (PBS) was introduced into the set-up which allows reducing the incident beam power without losing the improved image contrast. The incident beam k₂ that first passes a λ/2 waveplate is almost perfectly reflected at the PBS towards the cuvette. The double pass through the λ/4 waveplate rotates the reflected light from front and rear surfaces of the glass cuvette by π/2, which leads to an almost complete transmission at the PBS. A real image of the front glass surface of the cuvette is generated on the QW with an f - 2f - f lens configuration. The delay between the object pulse k₂ and reference pulse k₁ is set to zero at the front surface of the glass cuvette which creates a hologram of the cuvette’s surface by generating an electron density grating in the PCP QW. This hologram is simultaneously read out by reference beam k₁ in direction 2k₁ – k₂.

 

Fig. 1 (a) Schematic diagram of the contrast enhanced holographic imaging (CEHI) set-up for imaging moving glass beads and living unicellular organisms in transparent solution. PBS: polarization dependent beam splitter. (b) For imaging through turbid media a microscope slide has been covered with chicken skin on the back side. A 95 µm thick metal wire was subsequently attached at the rear side. A He-Ne laser beam is directed from the left, normal to the microscope slide surface.

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As imaged objects we used silver coated glass beads of 160 ± 20 µm size which were immersed in a 3:1 bromobenzene-bromoform solution in the glass cuvette. The solution has been chosen to match the density of the heavy glass beads in order to achieve a floating motion of the beads. As mentioned above, the improved CEHI set-up also allowed to investigate lighter silver-coated glass beads of 30 ± 15 µm size as well as living Paramecium and Euglena cells which were immersed in a water solution in the glass cuvettes.

The ability to enhance the imaging sensitivity by increasing the ratio between the erasing beam and object beam further led to the development of the two-color CEHI setup shown in Fig. 1(b). The set-up was used to demonstrate imaging through turbid media. We covered a microscope slide with chicken skin and attached a 95 ± 5 µm thick metal wire behind the skin, see Fig. 1(b). A continuous wave (cw) He-Ne laser erasing beam with variable intensity was directed from the back side normal to the microscope slide surface, penetrated the skin and injected electrons in the PCP QW. CEHI movies or images were recorded by a standard CCD video camera at a frame rate of 25 Hz.

3. Experimental results

3.1 CEHI of glass beads and living unicellular organisms

For CEHI of 30 µm glass beads and living unicellular organisms the front and silver coated back glass surfaces of the cuvette are oriented normal to the incident beam direction. The average intensities of the reference and object beams at the PCP QW were measured to be ~60 and ~40 µW/cm², respectively, with a laser focus dimension (width x height) of ~3 x 1.5 mm². The reflected light from the cuvette’s front surface (with wavevector k₂) was set to zero delay with respect to reference pulse k₁ producing a hologram of the cuvette’s surface. The temporal coincidence of pulses k₁ and k₂ was further indicated by a thin dark vertical line at the centre of the holographic image due to the PCP signal dip at temporal pulse overlap [12, 13]. The coherence time of ~1 ps of the excited excitons in the ZnSe QW corresponds to a coherence length of ~300 µm. This long coherence length enables a wide-field imaging that covers the entire laser beam width despite beam walk-off [9]. Reflected light from the silver coated back surface significantly reduces the hologram brightness due to the injection of excess substrate electrons into the PCP QW and hence acts as the erasing beam.

Moving objects in the cuvette prevent light pulses from reaching the cuvette’s back surface and the PCP QW which leads to a local brightness enhancement of the front surface hologram resulting in bright images of the beam-blocking objects. Figure 2 shows a sequence of background subtracted frames of imaged glass beads of ~30 μm diameter floating in a water solution. The recording time increases with ascending frame number, the time period between the frames is 1/25 s and the image area is ~0.4 x 0.4 mm². Bead images obtained with an optical microscope are shown on the bottom of Fig. 2 for comparison. Different holographic bead images possess a less or more pronounced Fresnel diffraction pattern appearing as nearly full circles or showing a signal minimum in its center (called Poisson or Arago spot), respectively. As discussed in section 3.2 these differences in the diffraction patterns are correlated to the beads’ sizes and their distances with respect to the front surface of the cuvette and can thus be used to determine the local depth of the beads inside the solution.

 

Fig. 2 Sequence of background subtracted frames of a CEHI movie of silver coated glass beads of 30 µm diameter floating in water solution. The image area is 0.4 x 0.4 mm². The recording time increases with ascending frame number, the time period between the frames is 1/25 s. A microscope image of glass beads is given on the bottom for comparison.

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After optimizing the signal contrast with the 30 µm glass beads an identical cuvette filled with Paramecium or Euglena cells was positioned onto the cuvette holder. Figure 3 demonstrates background subtracted contrast enhanced holographic images of living Paramecium cells. The image area is ~0.45 x 0.5 mm². The recording time increases with ascending frame number, the time period between the frames is 1/25 s. (A short movie clip is shown in ”Media 1”). The images show a fast moving mature Paramecium cell of ~150 μm size and slower moving ~50 μm size Paramecium cells. Their Fresnel diffraction patterns differ significantly depending on their distance to the cuvette’s front surface. One of the smaller cells has been encircled for better visibility. A microscope image of Paramecium cells is given on the bottom of Fig. 3 for comparison.

 

Fig. 3 Sequence of background subtracted frames of a CEHI movie of living Paramecium cells. The image area is ~0.45 x 0.5 mm². The recording time increases with ascending frame number, the time period between the frames is 1/25 s (see also Media 1). A microscope image of Paramecium cells is given on the bottom for comparison.

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Figure 4 shows background subtracted CEHI frames of Euglena cells. The recording time increases with ascending frame number with a period of 2/25 s. The imaged area is ~0.4 x 0.4 mm². A microscope image of Euglena cells is given on the bottom of Fig. 4. Due to the significantly smaller size of Euglena the images of cells which are more than 1 mm away from the front surface reveal significant Fresnel diffraction (see section 3.2), whereas Euglena cells which are swimming in close vicinity of the cuvette’s front surface appear as 50 µm long and ~10 µm wide cells as in the microscope image. One of the Euglena cells has been circled for better visibility. (A short movie clip is shown in “Media 2”).

 

Fig. 4 Sequence of background subtracted frames of a CEHI movie of living Euglena cells. The image area is ~0.4 x 0.4 mm². The recording time increases with ascending frame number, the time period between the frames is 2/25 s (see also Media 2). A microscope image of Euglena cells is given on the bottom for comparison.

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3.2 Depth resolution in contrast enhanced holographic imaging

Imaging in the contrast-enhanced holographic imaging mode enables very sensitive real-time detection of both reflecting and absorbing particles with a large depth of field but does not directly provide depth information as in single-shot 3D OCI [7, 11]. However, depth information can be obtained when the incident laser beam is slightly tilted with respect to the normal of the cuvette surface which has been briefly discussed in [11]. A detailed description is illustrated in Figs. 5(a) and 5(b) which show two different ways in which a double image of an object can be formed, namely either by rotating the cuvette’s normal by an angle α ≈1̊ with respect to the incident laser or by rotating a mirror located at a distance L’ from the rear cuvette surface by an angle α, respectively. Due to the rotation the glass beads block the laser beam twice, on its path toward the cuvette’s rear surface and after its reflection from the rear surface, thus producing a double image with lateral separation Δ. The dashed red lines in Fig. 5(a) and 5(b) represent the “shadow” paths of the bead. The brighter and slightly smaller one of each double image represents the bead with the shorter shadow path. This is also indicated by the less pronounced Fresnel diffraction. Depth information of the bead can be obtained geometrically according to

 

Fig. 5 Two possible alignments of a glass cuvette and the object beam enabling depth resolved CEHI. (a) The glass cuvette is rotated by an angle α with respect to the incident laser light; (b) the glass bead is at distance L from the rear inner glass surface and Lʹ is the distance from the rear cuvette surface to the remote mirror surface which is rotated by an angle α.

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The refractive index of the bromobenzene-bromoform solution n_sol has been determined using a simple Cauchy dispersion equation given by n(λ) = A + B / λ². For bromobenzene the parameters A = 1.5167 and B = 0.0138 [31] have been used. For bromoform the parameters A = 1.5758 and B = 0.007 were derived from published data [32, 33] resulting in a refractive index of n_sol ≈ 1.596 for the 3:1 solution at λ = 442 nm. The refractive index of glass is n_glass = 1.49. Using Snell’s law for small angles and a refractive index of 1 for the ambient air leads to γ/α = 1/n_glass and β/α = 1/n_sol, respectively. The distance of the bead with respect to the inner rear surface thus becomes L ≈ n_sol (Δ – 2Cα/n_glass)/(2α). The minuscule angle of rotation α was determined by the separation Δ of the hologram images obtained from a wire of known thickness (95 ± 5 µm, measured with a microscope) which was located at the inner front surface of the cuvette (at L = 10 mm) as shown in the middle Fig. 6 (which has been composed from three different image frames). The thinner left wire image which is minimally affected by Fresnel diffraction (as discussed in the next paragraph) also served as a size calibration of the beads. The separation Δ₁ and Δ₂ of the left and right split bead images is ~130 and ~220 µm, respectively. With a rotation angle of α ≈0.95 ± 0.05° the left and right bead are L = 4.92 ± 0.31 mm and 9.25 ± 0.53 mm away from the rear inner glass surface, respectively. The error in the distance L is predominately caused by the uncertainty of the rotation angle α. A high precision rotational stage with an angular resolution of 10 arc seconds would result in an uncertainty of L that is smaller than the object’s size.

 

Fig. 6 CEHI of floating glass beads inside the cuvette and of a 95 µm thick wire at the inner front surface of the cuvette possessing double images using a configuration shown in Fig. 5(a). Different distances Δ₁ and Δ₂ of double images of beads correspond to different depth levels in the solution.

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Depth resolution can also be achieved if a transparent cuvette with a high quality remote mirror as shown in Fig. 5(b) is used. The extra distance L’ from the rear surface of the cuvette to the mirror increases the separation Δ of the double image of the bead and thus enhances the measurement precision of this quantity. The distance L of the bead with respect to the rear outer cuvette glass surface is now given by

Figure 7 shows the background subtracted double image of a 160 µm diameter silver coated glass bead recorded in this cuvette-remote mirror configuration for a mirror distance L’ ≈15 ± 0.5 mm. The distance L and the rotation angle α were measured by the double image splitting of a 95 µm thin wire which was located first at the front and then at the rear inner glass surface of the cuvette. The distance of the bead from the inner rear surface was subsequently calculated to be L = 6.8 ± 0.5 mm and the rotation angle resulted to α ≈0.72 ± 0.02°.

 

Fig. 7 (a) Double image of a floating ~160 µm diameter glass bead with using a CEHI configuration shown in Fig. 5(b).

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As mentioned in section 3.1 an object’s depth information can also be obtained by modeling its Fresnel diffraction pattern [34] which depends on the distance of the object with respect to the cuvette’s front surface. The double image of the beads in Fig. 7 clearly demonstrates this effect. The brighter left image is caused by blocking the reflected laser beam from the remote mirror having the shorter “shadow” path as illustrated as red dashed line in Fig. 5(b). The bead image that possesses a central black dot (Arago spot) is caused by blocking the incoming laser beam. The long distance of the “shadow” path being reflected at the remote mirror surface leads to a distinct Fresnel diffraction pattern of the bead at the cuvette’s front surface.

For a quantitative analysis we computed the diffraction pattern of the “double” bead image shown in Fig. 7 according to a mathematical diffraction theory for opaque spheres [34]. Assuming an excitation with a plane wave the field amplitudes U within and outside the “shadowed” area are given by

(3)$$U\left(\gamma ,\delta \right)\propto \{\begin{array}{cc}{U}_0(\gamma ,\delta )exp\left[ikz\right]exp\left[\frac{i\gamma }{2}\right]{\displaystyle \sum _{n=0}^{\infty }{\left(\frac{\delta }{i\gamma }\right)}^n{J}_n\left(\delta \right)}& \delta <\gamma \\ U_0(\gamma ,\delta )exp\left[ikz\right]\left[exp\left[\frac{-i{\delta }^2}{2\gamma }\right]-exp\left[\frac{i\gamma }{2}\right]{\displaystyle \sum _{n=0}^{\infty }{\left(\frac{\gamma }{i\delta }\right)}^{n+1}{J}_{n+1}\left(\delta \right)}\right]& \delta \ge \gamma \end{array}$$

In Eq. (3) we used γ = (ka²)/z and δ = (kaR)/z, where k = (2π)/λ is the wavevector of the incident laser light with λ = 441 nm, a is the radius of the spherical obstacle, z is the on-axis propagation distance and R is the radial distance from the center of the obstacle. U₀ (γ, δ) = exp(ikR²/2z) and J_n(δ) are Bessel functions. In order to account for the different refractive indices for the individual light paths we used the on-axis propagation distance z = 3C/n_glass + (D + L)/n_sol + 2L’ for the right image (in beam direction) and z = C/n_glass + (D – L)/n_sol for the left image. C = 1.25 mm is the thickness of the cuvette glass and D = 10 mm is the distance between inner front and rear surface of the cuvette. As defined earlier, L represents the distance of the bead with respect to the inner rear cuvette glass surface and L’ = 15 mm is the distance of the remote mirror from the outer rear surface of the cuvette, see also Fig. 5(b). The small path elongation of distance z due to the rotation angle α has been neglected.

Figure 8 shows the experimentally observed and calculated, inverted intensity profile of the Fresnel pattern as a function of radial distance R from the center of the left (a) and right (b) bead image. The calculated profile of the left bead image with the shorter “shadow path” shows narrowly spaced diffraction rings. Experimentally, only the first ring is resolved. The diffraction rings of the bead image with the long “shadow path” are experimentally resolved. The best reproduction of the experimentally observed radii and thicknesses of the diffraction pattern as well as of the Arago spot for the right bead image is reached for a distance of L = 6.8 ± 0.7 mm. This value for L is in very good agreement with the value determined from the double image distance Δ described earlier. The uncertainty of ± 0.7 mm in the L value is predominantly attributed to the weak image contrast which could be improved using a high bit CCD camera with better light intensity resolution. In Fig. 9 we further demonstrate the calculated two-dimensional inverted Fresnel diffraction pattern (bright areas and rings appear as dark areas and rings) for comparison for the left (a) and the right (b) bead image shown in Fig. 7. The very small Arago spot in the left bead image is missing due to overexposure.

 

Fig. 8 Intensity profile (a) of the left bead and (b) of the right bead image shown in Fig. 7 indicated as blue full line. R denotes the radial distance from the center of the bead. Calculated inverted intensity profiles using Eq. (3) as described in the text are shown as red lines.

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Fig. 9 Calculated inverted two-dimensional intensity profile (a) of the left bead image and (b) of the right bead image shown in Fig. 7 using Eq. (3) as described in the text.

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Using a rectangular shaped obstacle the Fresnel diffraction from cylindrically-shaped Euglena cells can be calculated similarly to circular obstacles (not shown here). More complicated shaped objects like Paramecium cells could approximately be modeled by combining the diffraction from rectangular and circular obstacles.

3.3 Contrast enhanced holographic imaging through turbid media

Biological tissue and cells have a transparency maximum in the red and near infrared spectral range. Using this transparency advantage we performed CEHI experiments on an approximately ~150 µm thick chicken skin (from food) which was pressed against the rear side of a microscope objective slide. Then a 95 μm diameter thick metal wire was attached to the back side of the skin. The metal wire was not visible with the naked eye when looking at the front side of the microscope slide. Figure 10(a) and 10(b), respectively, show both front and rear-side views of the chicken skin with the attached metal wire.

 

Fig. 10 Photograph of a ~150 µm thick chicken skin on a glass plate with a metal wire of ~100 µm in diameter attached to the rear side of the chicken skin. (a) View from the front, (b) view from the back side, (c) view from the front side with incident blue laser light and (d) view from the back side including red laser light illumination from rear and blue laser light illumination from the front.

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For the CEHI experiments the cuvette in Fig. 1(a) was replaced with the prepared microscope slide. The clean front surface was oriented parallel to the QW film in the cryostat, see Fig. 1(b). The delay between the reflected pulse k₂ and the reference pulse k₁ was adjusted to zero producing a strong holographic image of the slide surface. Next, a continuous wave (cw) He-Ne laser beam (incident power of ~10 mW) was aimed onto the back side of the slide at normal incidence to the slide surface. The red laser beam (wavelength 633 nm) partially penetrates the chicken skin and weakens the electron density hologram in the QW due to the injection of substrate electrons. The power of the red He-Ne laser light after passing through the chicken skin was measured to be ~1.75 μW in front of the ZnSe PCP QW. Figure 10(c) and 10(d) show photographs with the incident ~100 fs pulsed blue object laser beam k₂ on the front glass surface and the cw He-Ne laser light entering from the back side, respectively. The holographic image of the wire, which partially blocks the penetrating He-Ne laser light, is shown in Fig. 11. The image area is ~0.8 x 0.7 mm². In the frame sequence the spatial cw He-Ne laser position on the chicken skin has been slightly changed. The areas next to the bright image of the wire are dark, because the transmitted red laser erases the hologram of the microscope slide. Thus the concealed wire on the back side of turbid media can be visualized.

 

Fig. 11 CEHI of a ~100 µm diameter wire behind the 150 µm thick layer of chicken skin. The image area is 0.8 x 0.7 mm². In the frame sequence the spatial cw He-Ne laser position on the chicken skin has been changed.

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4. Summary

Real-time CEHI using all-optical PCP ZnSe QWs can provide wide-field real-size holographic images with a large depth-of-field. The high refresh rate of the PCP QW devices enables direct-to-video monitoring of moving silver coated glass beads of ~160 µm and ~30 µm diameter in solution. Living unicellular Paramecium cells and Euglena in water were also imaged.

CEHI does not directly provide the depth information as single-shot 3D OCI [7, 11]. However, depth information can be retained when the incident laser beam is slightly tilted with respect to the normal of the cuvette surface. This creates a double image of the investigated objects. From the distance between the images the object’s depth relative to the cuvette’s inner front surface can be derived. The depth resolution is in the order of the object’s size. In addition, the diffraction pattern of the objects can be used to evaluate their depth with respect to the cuvette’s inner front surface. Model calculations of the diffraction patterns of a 160 µm diameter glass bead agree well with the experimentally observed diffraction patterns.

CEHI can also image through turbid media. In order to demonstrate this, we made a ~95 µm thick wire behind a ~150 µm thick chicken skin visible which was attached to a microscope objective slide. The local enhancement of the slide’s hologram due to the concealed wire was facilitated by shining a He-Ne laser beam of 633 nm wavelength from the back side through the skin.

Our investigations demonstrate that PCP QWs are powerful tools for real-time depth-resolved CEHI of stationary and non-stationary micrometer sized biological objects in transparent media and of obscured objects in turbid media. The combined recording of objects in both 3D-OCI [7, 11] and CEHI mode bears potential for sensitive particle or bacteria detection at very low concentration. The new methods also allow to track unicellular organisms in real-time to study e.g. the influence of drugs and toxic materials as well as of electric and magnetic fields on organisms in an unconfined 3D environment.

Acknowledgments

The experimental support of M. Kaveh and Dr. X. Wang is acknowledged. Dr. T. Johnston (Nanoparticulate Surface Adhesion Ltd.) is kindly acknowledged for providing the 30 μm diameter silver-coated glass beads. This research work has been supported by the University Research Council of the University of Cincinnati.

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