Marys Medicine

Xic.postech.ac.kr

Imaging Cells with Radiology visible and—as we demonstrate here—microradiology of vertebrates in real time, which is not possible with optical live cells is feasible. Our experimental results show indeed microscopy due to opacity.
that fine details can be imaged with high lateral and timeresolution in a variety of biology materials. These includedleaf skin cells, human tumor cells, mouse neurons, and rabbitbone cells.
MATERIALS AND METHODS Fig. 1 shows a nice example of our results: the image of a Xenopus oocyte. Note that one can clearly identify theanimal pole (left hemisphere with even gray level), the The tests were performed with a microradiography system whose details aredescribed in Baik et al. (2004). Unmonochromatized x-ray beams from the vegetal pole (right hemisphere with granular appearance), 7B2 beamline of the Pohang Light Source and the PCI beamline of the and the nucleus within the animal pole (see the magnified Singapore Synchrotron Light Source were used to illuminate the sample.
and filtered inset). The edge enhancement is extremely clear The transmitted x rays were captured by a cleaved CdWO4 single-crystal for the outer boundary of the oocyte and also visible for the scintillator and converted to a visible image. This image was then magnified boundary between the nucleus and the surrounding region by an optical lens before being captured and stored by a charge-coupleddevice camera.
The samples were mounted on a translation/rotation stage that allowed The different appearance of the left and right hemisphere precise positioning. For biological samples with intrinsically limited x-ray can be attributed to an inhomogeneous distribution of cyto- absorption, a single image (typically 1280 3 1024 pixel, horizontal field of skeletons creating an asymmetry between the two hemi- view (FOV) 500 mm) could be taken within 100 ms. The corresponding spheres. The animal pole has much more densely connected radiation dose did not produce any detectable damage.
keratin than the vegetal pole (Gard et al., 1997). The meshsize is 10 mm in the vegetal pole, 10 times larger than those Sample preparation in the animal pole. The much smaller mesh size of the The leaf cell samples were obtained from Hippeastrum epidermis. Tissue cytoskeleton network and the presence of pigment granules specimens were obtained by peeling off the bottom skin of the leaf and then (;1 mm in diameter) blur the edges of the network and only sandwiched between two moisturized Kapton foils to preserve water. The an evenly distributed gray level is observed in the animal thickness of the leaf skin is ;30–50 mm and the Kapton foil is 50 mm. Such a procedure was not required to observe cells; it was possible to see These arguments are consistent with the electron micros- individual cells even in the leaf without peeling. The peeling andsandwiching procedure, however, eliminated a problem: the superposition copy results (Gard et al., 1997). In addition, our technique of many cell layers results in complicated images. An alternate solution for leads to nondestructive and three-dimensional (3D) obser- this problem is tomography. We demonstrated the tomography reconstruc- vation of the oocyte interior. Also note that the observation tion of plant cell by using thick samples (;5 mm) taken from the skin part of of the nucleus within the animal pole is important because an aloe leaf.
it could lead to the observation of the embryogenesis of Oocytes were selected for our tests to assess the penetration and the spatial resolution of our approach. Oocytes at stage 6 were surgically re-moved from the ovary of a Xenopus and incubated at 18°C in the standardND96 medium (in mM) (NaCl, 96; KCl, 2.0; CaCl2, 1.8; MgCl2, 1.0; andHEPES, 5, pH 7.6).
Cerebral neurons from rat brains were prepared from an 18-day-old rat embryo (Lin et al., 2002). They were placed on a coverslip glass locallycoated with poly-D-lysine so that neurons accumulated over the poly-D-lysine patches. Cell cultures were incubated at 37°C with 95% humidity and5% CO2. The samples for the microradiology tests were prepared from theculture after 9–11 days of incubation. A medium with collagen was usedto seal the neurons on the coverslip and a gel-like layer was formed after;20 min in ambient air at room temperature. The average thickness of thesamples was ;200 mm.
Fibroblast cells were used to test the feasibility of detecting changes in the cell morphology. The cell line (HIG-82) was obtained from the FoodScience Research Institute (Industrial Technology Research Institute,Taiwan). It was prepared from the fibroblast of rabbit joints. The cellswere cultured in 35-mm culture dishes without specific coating at the bottomand incubated at 37°C with 95% humidity and 5% CO2. The medium wasHam's F-12 medium (2 mM L-glutamine and Earle's buffered salt solutionadjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential aminoacids, and 1.0 mM sodium pyruvate 90%; fetal calf serum, 10%). Dishes Phase-contrast micrograph showing the interiors of a Xenopus with ;80% cell confluence were prepared for microradiology tests after oocyte. A sharp-tipped glass capillary (visible in the middle of the image) was used to fix the sample to the holder (the bottom dark region). The sample All animal cell samples were fixed with 4% paraformaldehyde overnight diameter was 1.5 mm. The inset shows a magnified version of the marked before being mounted on the sample holder. The oocyte was placed in air.
left-hand portion of the image. This magnified picture was processed to The coverslips with the neurons were placed in a sealed plastic bag to emphasize the boundary of the nucleus.
preserve sufficient moisture. The HIG-82 fibroblast cells were observed with Biophysical Journal 87(6) 4180–4187

the culture dish water-tight sealed and vertically placed in the path of thex-ray beam.
For preparation of mouse aorta, adult C57/BL6 mice were anesthetized with ether inhalation and were perfusion fixed via direct intracardiacinjection, initially with heparinized PBS (10 units/ml) followed byphosphate-buffered 2% paraformaldehyde (pH 7.4) for 10 min (Yeh et al.,2003). The thoracic aortas were dissected and cut into transverse rings.
Thereafter the aortas were embedded in resin using standard preparationprocedures for thin-section electron microscopy (Blackburn et al., 1995).
The above-described preparation procedures are much less complicated than the standard methods for optical microscopy and other microscopytechniques—and resulted in specimens very close to their natural state.
Specifically, no staining or other contrast-enhancing procedure was used.
The cultured cells were fixed to preserve their morphology because the x-raybeam propagates horizontally and therefore the specimens had to be placedvertically. The specimens were much thicker than for optical microscopy Microradiograph showing the entire leaf depth.
and an opaque medium could be used.
the gaps among the spongy parenchyma cells under thepalisade cells. Note that x ray can penetrate into the leaf and RESULTS AND DISCUSSION reveal the different morphologies of these cells and their The specific objectives of our tests were the following. First, packing. Fig. 3 stresses the difference with respect to optical we wanted to demonstrate sufficient (submicron) lateral res- microscopy: not only cells in the outermost layer are visible olution and contrast with hard x rays (wavelength ,0.1 nm) but also deeper cells.
to detect cellular details. Second, we wanted to show such The arrows in Fig. 2 identify stomata in the epidermis as a performance with thick samples and without complicated they open up. Such cells enabled us to demonstrate real-time sample processing. Third, we sought specific evidence of imaging of live specimens. Fig. 4 shows in fact individual cellular-level imaging of live specimens. The fourth images captured from a movie taken with a video camera.
objective was detection in real time on a video (
33 ms) The images clearly show the opening of a live stoma cell.
A careful examination of the sequence of images in Fig. 4 All such objectives were reached with tests on the already- reveals features beyond the mere opening. These include mentioned variety of specimens. Figs. 2 and 3 show micro- subcellular features and the induced changes (such as com- radiographs of a Hippeastrum leaf taken with broadband pression and displacement) of the surrounding cells. We (unmonochromatized) x rays. The (horizontal) FOV was suspect, for example, that the darker lines on the guard cells are those from the cellulose microfibrils. The live detection These images constitute, to the best of our knowledge, the of such minute movements is quite remarkable if one con- very first example of cell radiographs obtained with hard siders the complete absence of staining.
x rays without staining. Fig. 2 shows the leaf skin (thickness We also tested a procedure to enhance the moving cell ;30–50 mm) whereas Fig. 3 shows the entire leaf (thickness with respect to the nonmoving ones by subtraction. This is ;300 mm). The little triangle-shaped features in Fig. 3 are quite desirable because the nonmoving cells inside the leaf(particularly the parenchyma cells) have much strongercontrast than the epidermal cells and outer cuticle and there-fore complicate the image when one tries to observe themovement in a natural, unpeeled leaf. The first tests areencouraging and may provide a simple and feasible alternateprocedure with respect to tomography.
In parallel, we performed successful tests of tomographic reconstruction of microradiographs of plants cells. We useda standard filtered back projection algorithm from 1000projections. We found that the boundaries of individual cellcan be effectively reconstructed. Fig. 5 shows an example ofreconstruction performed for the skin of an aloe leaf ;5-mmthick. Note that only the area near the surface (shown in Fig.
5) is reconstructed due to the limited field of view whereas Microradiograph of a Hippeastrum leaf taken with broadband the entire thick sample was kept during the image acquisition (unmonochromatized) x rays. The image size was 1.5 3 1 mm. The figure for the required tomography reconstruction. The quality of shows cells from the leaf skin membrane. The arrows identify three of the the reconstruction was not optimized due to the slight move- stoma cells. To the best of our knowledge, these results constitute the firstexample of cell radiographs without staining.
ment of the sample during the measurements. Moreover, Biophysical Journal 87(6) 4180–4187

Imaging Cells with Radiology Tomographic reconstruction of an aloe leaf sample. The re- construction is based on 1000 projections taken within 2 min. Scale bar is50 mm.
show that tomographic reconstruction is feasible for thicksamples.
To better demonstrate this high ‘‘selectivity'' resulting from the enhanced contrast due to phase effect and from thehigh-penetration nature of x rays, we show in Fig. 6 a similarlocal tomography reconstruction of a toothpick. The di-ameter of the toothpick is ;3 mm and the reconstructed areais 240 3 240 mm. The quality of the reconstructed image isimproved with respect to the previous case by the eliminationof the artifacts due to dehydration-induced sample move-ment as seen for the aloe leaf. In the inset, it is clear that smallfeatures can still be easily detected despite the rather largesample examined. Fig. 6 b shows the 3D volume-generatedsurface contour image constructed from the tomography re-constructed data as exampled in Fig. 6 a. Note that not onlyeach channel is clear imaged, but also the texture of the cellwalls along the direction parallel to the rotation axis.
The added phase effect, however, complicated the tomog- raphy reconstruction based on the filtered back projectionalgorithm. One can use a more rigorous approach to extractthe embedded phase information (Bronnikov, 2002; Barty Images captured from a movie taken with a video camera et al., 2000; Cloetens et al., 2002) and obtain the real-space showing the opening movement of a live stoma cell.
image before initiating the normal tomography reconstruc-tion. Another simpler approach is to consider the phase although the image sequence was taken within 2 min, effects, in this case the dark and bright fringes on the edge, notable dehydration was observed. The reconstruction was simply as a region of light adsorption. By adjusting the also blurred by the ‘‘local tomography'' effects (Faridani sample to detector distance, such phase effect can be opti- et al., 1997; Anastasio et al., 2003) due to the large dimen- mized for best visual clarity without adding unwanted effect sion of the sample, which is larger than the field of view of to the tomography reconstruction as demonstrated in the each individual projection. In this specific case, the di- previous examples.
mension of the sample was 5 3 5 3 5 mm but only ;400 3 Note that in conventional tomography the reconstruction 400 3 300 mm was reconstructed. The corresponding arti- based on absorption works because absorption follows a facts are most noticeable in the area away from the center of linear dependence along the line of projection. This is in rotation. Such problems notwithstanding, the tests clearly fact a consequence of the small magnitude of the imaginary Biophysical Journal 87(6) 4180–4187

Radiograph of cultured and fixed neuron cells from mouse brain. The field of view (horizontal) was 500 mm.
Note that the neuron specimens were not even optimized for x-ray imaging. In fact, the collagen used for sealing wasobtained from three rat tails and dissolved in 0.1% acetate atthe concentration of 2 mg/ml, which is not specially designedfor microradiology and therefore leads to weak contrast. Thefixation collagen are also quite thick, ;300 mm, much morethan normally required for optical microscopy. The slightblurring in the microradiographs is likely due to collagen-induced diffuse scattering and can be improved by furtheroptimization of the collagen materials.
Fig. 8 shows cultured fibroblast cells from rabbit joints.
The cells can be fixed with paraformaldehyde beforemounting the specimen in a vertical position. The fixed cellswere then tightly packed and all with a fibril or spindle shape.
In contrast, Fig. 8 shows the same type of cells imagedwithout being fixed: the cells are not close packed and (a) Tomographic reconstruction of a toothpick, used to assess morphological modifications can be seen. Nevertheless, the the resolution performances of our apparatus and procedure. The diameter of image quality is still quite reasonable, indicating that our the sample is ;3 mm but only a central area of diameter ;240 mm isreconstructed. Features of mm size can be observed. (b) The volume- approach can be used for mammalian cells in their natural rendered 3D structure of the same toothpick sample.
With suitable fixation procedures, we could even obtain high-resolution tomography and 3D reconstructed images of part of the refractive index. Similarly, the phase shift due to mammalian cells. Fig. 9 a shows an example. The sample is the real part of the refractive index is approximately linear,because the real part is very close to unity and the imaginaryexponential in the wave function leads to a constant-phasefactor multiplied by a linearly changing phase factor.
Plant cells are quite easy to image with our approach. Figs.
7 and 8, however, show that animal cells can be seen as well.
Fig. 7 shows cultured and fixed neuron cells from mousebrain. Not only are the neuron bodies clearly visible but alsothe interconnected axons.
Specifically, it is possible to observe the region under the ganglion neurons so that the attachment of the neuronswith the extracellular matrix and with one another can berevealed. The x-ray penetration to the region under theganglion surface could be exploited to analyze neurons-glia Radiograph of cultured fibroblast cells extracted from a rabbit interactions and even the interaction between cells and bone. The cell samples were again vertically mounted but not fixed. The field of view (horizontal) was 500 mm.
Biophysical Journal 87(6) 4180–4187 Imaging Cells with Radiology a piece of aorta fixed in resin; the overall sample dimension,including the resin, is ;5 3 7 3 10 mm. Fig. 9 b showsone of the tomography reconstructed slices. In this cross-sectional view of mouse aorta, ;240- 3 240-mm field ofview, the detailed structure of the vessel wall is clearlyobserved after tomography reconstruction despite the factthat the sample is larger than the imaged area. The aorta wallis layered by the waving elastic laminae, the innermost ofwhich, the internal elastic lamina (marked by black arrows),is isolated from the lumen (L) by a monolayer of endothelialcells (white arrows). Fig. 10 is the volume-rendered 3Dstructure of this sample. The white arrows point to bordersbetween neighboring cells, whereas the black arrow pointsto a cell nucleus that extrudes from the surface and whoseoutline is well preserved in the 3D reconstruction analysis. Inthis case, the imaging capability goes beyond the mereimaging of the outline and shape of the individual cell andprovides subcell information.
Our results, thus, show that refractive index imaging based on coherent x rays can produce clear images at the cell andsometimes subcell level without staining. It is quite im-portant to compare this approach to other techniques andassess its advantages, limitations, and possible future im-provements. The resolution obtained so far is similar tooptical microscopy and can be substantially improved. Softx-ray microscopy has better resolution but has not beenimplemented on samples thicker than a single layer of cells.
Transmission electron microscopy and scanning electronmicroscopy require specific sample preparation proceduresand are limited in the sample thickness. Overall the biggestadvantage of our approach is offered by the high penetrationof hard x rays, easily the whole body of an animal, and theconsequent potential to examine systems close to naturalliving conditions. No microscopy in fact matches refractiveindex microradiology in examining thick and opaque spec-imens with high resolution (W. Yun, private communica-tion).
This approach is certainly not without limitations. The possible radiation damage is certainly less severe than inabsorption-contrast radiology but it cannot be neglected. Inmost of the two-dimensional measurements, where exposureto synchrotron x rays can be limited, radiation damage ordehydration is not observed. Although not evident in ourtests, this issue must nevertheless be carefully explored toassess its impact. The longer data-taking time required forthe tomography (sometimes .1000 snapshots at differentangles is necessary to preserve the resolution) could ag-gravate the problem. On the other hand, the current time perframe (1 ms) (Hwu et al., 2004) and the corresponding dose (a) X-ray micrographs of a mouse aorta sample fixed in resin; than the imaged area. The aorta wall is layered by the waving elastic the overall sample dimension, including the resin, is ;5 3 7 3 10 mm.
laminae, the innermost of which, internal elastic lamina (marked by black Scale bar is 400 mm. (b) One of the tomography reconstructed slices. The arrows), is isolated from the lumen (L) by a monolayer of endothelial cells detailed structure of the vessel wall is clearly seen even if the sample is larger (white arrows). Scale bar is 15 mm.
Biophysical Journal 87(6) 4180–4187 tests, should be systematically explored. Although ourtechnique can already clearly detect different cell morphol-ogies and cell details, even finer internal structure detailsshould become visible at higher magnification. Furthermore,future x-ray lasers will provide ultraintense and ultrashortpulses to perform one-shot imaging before damage. Evenwithout waiting for these future developments, our testsclearly show that coherent microradiology is a very helpfulinstrument in cell biology, largely complementary to opticalmicroscopy.
We are grateful to W. Y. Chow and Y. C. Chang (Department of LifeScience, National Tsing Hua University, Taiwan) for providing oocytes andneurons for our experiments.
This work was supported by the National Science Council (Taiwan), theAcademia Sinica (Taiwan), the BK21 Project, the Korea Institute of Scienceand Technology Evaluation and Planning through the National ResearchLaboratory and SKORE-A projects, the Fonds National Suisse de laRecherche Scientifique, the Ecole Polytechnique Fe´de´rale de Lausanne, and The volume-rendered 3D structure of the same aorta sample the National University of Singapore.
of Fig. 9. The white arrows point to borders between neighboring cells,whereas black arrow points a cell nucleus that extrudes from the surface andwhose outline is well preserved in the 3D reconstruction. Scale bar is 50 mm.
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Biophysical Journal 87(6) 4180–4187

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