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The long-term and real-time monitoring the cell division and changes of osteoblasts under simulated zero gravity condition were succeed by combing a digital holographic microscopy (DHM) with a superconducting magnet (SM). The SM could generate different magnetic force fields in a cylindrical cavity, where the gravitational force of biological samples could be canceled at a special gravity position by a high magnetic force. Therefore the specimens were levitated and in a simulated zero gravity environment. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet’s bore. The results presented the first-phase images of living cells undergoing dynamic divisions and changes under simulated zero gravity environment for a period of 10 hours. The experiments demonstrated that the SM-compatible DHM setup could provide a highly efficient and versatile method for research on the effects of microgravity on biological samples.
©2012 Optical Society of America
1. Introduction
The microgravity exhibited in the extraterrestrial space environment has attracted the attention of scientists, since gravity plays an important role in evolution of life on the Earth. The research fields of space biology and space medicine have bloomed because of the growing number of human space flights in recent years. The weightlessness environment alters the normal physiologic course of living things. Bone loss caused by microgravity is one of the most common and serious health problems faced by the astronauts [1, 2]. Therefore, it is urgent to investigate the molecular mechanism of bone loss induced by microgravity.
The low gravity environment during space flights is the ideal environment for studying cellular processes under microgravity. However, the limited space flights make it impossible to carry out extensive experiments for research on the effects of microgravity on biological specimens. Therefore, several ground-based platforms, including clinostat, rotary cell culture system, and random positioning machine, have been widely employed to simulate microgravity condition. Moreover, microgravity can also be achieved in drop tubes/tower, balloons and parabolic flights. Following the development of superconducting materials, a superconducting magnet (SM) with large gradient high magnetic field can achieve magnetic levitation of diamagnetic materials, such as biological macromolecules, cells, and tissues [3, 4]. In addition, magnetic levitation has the capability of sustaining a stable levitation for many hours and has been employed as a ground-based method to vary net gravitational forces experienced by biological systems [5, 6]. Hammer et al. have reported that MC3T3-E1 osteoblastic cells could be grown in magnetically simulated hypo-g and hyper-g environments and studied the gene expression of cells under these conditions [7]. Our previous studies have demonstrated that magnetic levitation can be used to simulate the gravitational environment for studying the microgravity effects on osteoblast and osteocyte’s morphology, cytoskeleton architecture, and function [8, 9].
Real-time observation of cell changes can be obtained with digital holographic microscopy (DHM). The technique provides phase measurements of transparent biological specimens with interferometric resolutions, in particular living cells in culture [10~13]. Previous studies [14, 15] demonstrated that the DHM was an ideal microscopic tool for real-time monitoring of living cells on the ground-based microgravity simulation platforms, such as the random positioning machine and the parabolic flights. Preliminary results from these studies presented sensible changes of cytoarchitecture under states of microgravity. However, because of the experimental limitation, the allowed exposure time of microgravity was not easily more than 1.5 hour on the random positioning machine [14] and about 20 seconds during the parabolic flights [15].
In this paper, we report a methodology to real-time monitoring the division and changes of osteoblasts under simulated zero gravity condition for a period of 10 hours by combining DHM and SM. Here, the SM was used as a ground-based microgravity simulation platform, which could produce a stable large gradient high magnetic field to levitate the specimen. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet’s bore. The experiments were performed to monitor sensible changes and division of osteoblasts under simulated zero gravity environment.
2. Materials and methods
2.1 Cell culture
The murine osteocytic cell line MLO-Y4 was kindly provided by Dr. Lynda Bonewald of University of Texas Health Science Center. The cells were seeded in 35mm tissue culture dish (Corning, Inc., Corning,NY) and grown in α-MEM culture medium (Gibco, Invitrogen Corporation, Carlsbad, CA) supplemented with 2mM L-glutamine, 2.2g/L sodium bicarbonate, 0.1mM nonessential amino acids, 1mM sodium pyruvate, and 5% fetal bovine serum (FBS) and calf serum (CS) (Hyclone, Logan, UT) at 37°C, 5% CO2 in a humidified atmosphere. For culturing of the cells in the bore of SM, α-MEM was changed to CO2 independent medium (Gibco) with 5% FBS and CS and the tissue culture dish were sealed with parafilm. The temperature during the experiment was controlled by a self–made water bath system, and the temperature range was 37 ± 0.5°C.
2.2 Microgravity platforms
The SM used in experiment was depicted in Fig. 1(D, E) , which was described in details in the reference [6]. An overview of the actual experimental configurations was shown in Fig. 2 . The SM (JMTA-16T50MF) was made by Japan Superconductor Technology (JASTEC). The height of the SM was 195cm and a cylindrical cavity of Φ51mm × 450mm in the SM could be used for the experiment, as show in Fig. 1(D). The magnetic induction (B) in the cavity varied along with the vertical direction (z). In the cavity, three different magnetic force fields [B(dB/dz)] of −1360, 0, and 1312 T2/m could be generated and the magnetic field intensity was 12, 16, and 12 T at three special positions, as shown in Fig. 1(E). The magnetic forces could be used to simulate variable gravity environments and the corresponding apparent gravity was 0, 1, and 2g. In addition, the magnitude and direction of magnetic force acting on the specimen varied in different locations, so increased and decreased apparent gravity environments could be achieved by changing the location of the biological samples in the cavity. Therefore, the SM could simulate gravitational environment from hypo-gravity to hyper-gravity. The maximum magnetic force field was −1513T2/m, and the magnetic force field, which could make biological specimens be levitated, was higher than −1360T2/m along a 30mm length in the bore. The measured field stability was about 0.27ppm/h. At this stability, the levitation capability could be maintained for more than four years. Furthermore, in order to develop a long time and stable simulated microgravity environment for carrying out the biological experiment, some equipments matched with the SM was designed, including temperature control system, object stage, and gas control system. For the sake of brevity, these devices were not concretely shown in Fig. 1.
Fig. 1 Experiment configuration of DHM-SM prototype. The diagrams of (A), (B), (C) were depicted the schematic of the DHM, PBS, polarizing beam splitter; BE, beam expander with spatial filter; λ/2, half-wave plate; O, object wave; R, reference wave; Obj, specimen; MO, microscope objective; TL, tube lens; BS, beam splitter; Inset: detail showing the off-axis geometry. The diagrams of (D), (E) were depicted the properties of the SM. (D) illustrated the distance from the bottom of SM to three apparent gravity positions, arrow represents magnetic field direction. (E) showed the gradient distribution of SM and the corresponding magnetic force field.
Fig. 2 Picture of the actual experimental configurations, (a) configuration of DHM in the cavity, (b) jig designed to hold cell containers and integrate optical device.
2.3 DHM setup
The DHM used in the experiment was a transmission setup based on a modified Mach-Zender interferometer configuration, as depicted in Fig. 1(A, B, C) and Fig. 2. The light from a frequency-doubled Nd:YAG laser (the wavelength was 532nm, the output power was 50mW) was divided into object arm and reference arm by using a polarized beam splitter (PBS). A half-wave plate (λ/2) was used in conjunction with the PBS to adjust the intensity ratio between the two arms. In the reference arm, another half-wave plate (λ/2) was introduced to obtain parallel polarization at the exit of the interferometer. Both the beams were coupled into polarization-maintaining single-mode optical fibers (PMF1 and PMF2) by laser-to-fiber couplers (FC1 and FC2), respectively. The reference beam (R) was guided to a beam expander (BE2) and collimated to a plane wave. The beam of object arm was guided into a cylindrical cavity in the SM, as shown in Fig. 1(D). In order to fitting the cavity, a vertically-configured jig was designed to hold cell container and integrate optical device, as shown in Fig. 2(b). It is important to note that the cell container position in the jig was designed to ensure the sample was exactly located at the zero gravity position. Here, the beam of PMF1 was collimated by a beam expander (BE1) and illuminated the specimen (Obj), as shown in Fig. 1(B) and Fig. 2(a). The object wave (O) was produced by collecting transmitted wave with an infinity-corrected microscope objective (MO, the magnification was 20 × , the numerical aperture was 0.4, the working distance was 8.5mm) combined with a tube lens (TL, the focal length was 167.7mm, the diameter was 30mm). Considering the high magnetic field environment, the brass and acrylic materials were chosen for the MO and jig. For the off-axis geometry, the R was reflected by a beam splitter (BS) and reached a camera with a small incidence angle θ with respect to the propagation direction of the O (see inset in Fig. 1). The holograms were recorded by a standard black and white CMOS camera (the region of interest was 1024 × 1024 pixels, a pixel size was 6.7μm × 6.7μm) and transferred to a computer via a USB 2.0 interface, as shown in Fig. 1(C). The power of incidence on the camera was about 0.1mW, and the exposure time of camera was 12μs. Although the DHM was fiber-based with both object and reference beams injected in different fibers, the fringe contrast of hologram was kept stable during the experiments by using polarization-maintaining single-mode optical fibers. Considering the effects of the high magnetic field on the laser and computer, the lengths of optical fibers (PMF1 and PMF2) and the data wire of camera were chosen as 8m and 10m, respectively. The magnetic intensity around the devices was less than 5G. The camera still operated stably even it was exposed to high magnetic field (about 0.2T) during the experiments.
Once the digital hologram has been acquired, a few steps were necessary before retrieving the digital complex object wave front. Firstly, the so-called hologram apodization was performed to avoid unwanted diffractions on the wave front due to the non-infinite nature of the camera chip, by multiplying the hologram with a two dimensional window function [16]. Secondly, the hologram spatial filtering was applied to filter out the high-intensity zero order and the twin object image in the off-axis configurations, by computing the Fourier transform of the hologram [17]. Thirdly, the compensation of tilt aberration due to the off-axis geometry was carried out in the hologram plane to correct tilted propagation direction with respect optical axis, by using the method of Numerical Parametric Lens (NPL) with the mathematical model of a plane wave [18]. Finally, the convolution propagation algorithm was employed to retrieve the object complex amplitude with the reconstruction distance of 25.6cm. For obtaining aberration-free phase contrast image, the re-application of the NPL defined with high order polynomials compensated the phase aberrations induced by the setup, in particular the curvature produced by the MO. By recording and reconstructing a blank hologram without any specimens in the setup, the standard deviation of phase distribution was determined as 8.5° in central region of 512 × 512 pixels.
3. Results and discussions
The DHM-SM system was used to observe the cell division and change of living osteocyte under simulated zero gravity. For investigation of the influence of zero gravity on osteocyte, the experiments were also performed out of SM, namely under normal gravity condition. The quantitative phase contrast images (coded to 256 gray levels) were shown in Fig. 3 , with images recorded every other 30s under simulated zero gravity condition. The Media 1 presented a fast-movie of the phase contrast images for entire experimental period of 10 hours.
Fig. 3 Frame extracted from DHM phase image movie (Media 1) of living osteoblasts under simulated zero gravity condition for the whole experimental period.
3.1 Observation of cell division cycle
Firstly, we followed the cell divisions during the growth of osteoblasts in simulated zero and normal gravity conditions. In the experiments, the cells were adhered on the substrate. The phase images were determined by the cell shape, the protein concentration and the projection of the cell thickness.
Figure 4 showed the contrast images under simulated zero condition with the gray level coded pseudo three dimensions. The mother cell was designated as A and the corresponding daughter cells were designated as a1, a2. Similarly, Fig. 5 showed the contrast images under normal gravity condition. The mother cell was designated as B and the corresponding daughter cells were designated as b1, b2. In two figures, the images on the top row were acquired prior to the cell division, and the images on the middle and the bottom were obtained after cell division.
Fig. 4 Three dimensions rendering of phase images presented cell division of osteoblast under simulated zero gravity condition.
Fig. 5 Three dimensions rendering of phase images presented cell division of osteoblast under normal gravity condition.
To clearly illustrate the correlation with the main stages of the cell division cycle, the maximum phase Δφmax of the phase images under simulated zero and normal gravity conditions were depicted in Fig. 6(a) and 6(b), respectively. For evaluating the Δφmax, the two dimensional cell tracking was employed in the field of view given in Fig. 4 and 5. During the evaluation procedure, the trajectories of cells, including the mother and daughter cells, were manually selected after the cell division process.
Fig. 6 Real-time monitoring of the maximum phase obtained from the phase images obtained under simulated zero and normal gravity conditions, respectively.
As seen in Fig. 6, the Δφmax remained constant prior to cell division. After this phase, the Δφmax increased significantly and reached the peak preceding cytokinesis (division of cytoplasm by formation of a septum in the center of the cell). This peak was probably related to the recruitment of proteins required for the mitosis machinery, for the formation of the septum required for cytokinesis and for the S phase (DNA replication, histone synthesis and Nucleus division). After the cell division, the Δφmax of the sister cells decreased significantly and reached a steady state.
By comparing the results obtained under these two gravity conditions, some striking differences could be observed. As shown in Fig. 6(a), the Δφmax of the cell under simulated zero gravity condition increased significantly and reached the peak in 35 minutes, and the cytokinesis began immediately when the peak was reached. After the cell division, the Δφmax of the sister cells decreased to a steady states in 35 minutes. In Fig. 6(b), it could be seen that the Δφmax of the cell under normal gravity condition changed from the constant to peak in 35 minutes. Afterwards, the Δφmax was keeping at peak level during 30 minutes preceding cytokinesis. After the cell division, it took 90 minutes for the Δφmax of the sister cells to reach a steady states.
3.2 Observation of cell changes
Secondly, the experiments were carried out to dynamically observe changes in cell optical thickness. The phase images represented the cell changes of osteoblasts under simulated zero and normal gravity conditions in Fig. 7 and Fig. 8 , respectively.
Fig. 7 Phase images showed the cell changes of osteoblast under the simulated zero gravity condition.
To evaluate the alteration process of cell optical thickness, pixel intensity profiles were extracted from the phase images. Two profiles were taken at different orientations, depicted by the dotted line in Fig. 9 (a) and 9(b). The phase measurements at observation time 0 h, 1h30, 3 h, and 4 h30 were presented in Fig. 9 (c) and 9(d) for different gravity conditions, respectively. As shown in Fig. 9(c), the phase measurements increased generally with exposure time of simulated zero gravity. In Fig. 9(d), it could be seen that the phase measurements under normal gravity condition increased generally with time.
Fig. 9 Phase measurements along the two cross sections (indicated in Fig.(a) and (b)) through the phase contrast images at observation time 0 h, 1h30, 3 h, and 4 h30.
Comparing the results shown in Fig. 6 and Fig. 9, there were significant differences of cell division process and changes in cell optical thickness under different gravity conditions. It hinted that the cytoskeleton, cytosol and associated organellar structure also change distribution in response to reduced gravitational force.
The current DHM-SM platform did not calculate either the intracellular refractive index, which linked to cellular content, or absolute cytomorphological topography from the measured phase data. Therefore, distinguishing a morphological alteration and an intracellular content density change remained difficult to achieve by using phase distribution changes. The further retrieve of both refractive index and topographical value from the phase measurement can be achieved by DHM based decoupling procedures for instance, sequential culture medium exchange [19], fixed chamber channel height squeezing the cells [20], or recently true real-time decoupling by dual-wavelength using dispersion with a dye-enhanced surrounding medium [21]. Moreover, considering that the experimental results were only preliminary observations without statistical confirmation, future work involving detailed analysis and interpretation of the phase response collected from a large number of cells with statistical significance under different microgravity environment is the next phase of the project.
4. Conclusions
In this paper, the experiments demonstrated the applicability of SM-compatible DHM setup for long-term and real-time monitoring the cell division and change of living osteoblasts in simulated zero gravity environment. The SM with large gradient high magnetic field was used as a ground-based microgravity simulation platform, where the specimens could be levitated in the magnet’s bore by a high magnetic force, namely simulating zero gravity environment. The proposed DHM system was granted with sufficiently flexible and reliable to observe cell changes in a cylindrical cavity, by using single mode optical fibers and a designed vertically-configured jig. The preliminary results provided observations on the effect of gravitational unloading on living osteoblasts. To our knowledge, the presented results are the first-phase images of living cells undergoing dynamic divisions and modifications in simulated zero gravity environment for a period of 10 hours. Future work will be undertaken in the next phases, involving detailed analysis and interpretation of the phase response collected from a large number of osteoblasts with statistical significance, and relating the quantitative phase measurement to true physical data in term of absolute cytomorphological topography and intracellular refractive index by using decoupling procedures. Such an advance will further reinforce the potential of DHM for the understanding of the mechanism of bone loss induced by microgravity.
Acknowledgments
This work was supported by the National Science Foundation of China (No.31000387 and 30970689).
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