1 Department of Medicine, Division of Nephrology and Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis 46202-5116; and 2 Department of Computer and Information Science, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana 46202-5132
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ABSTRACT |
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Confocal and two-photon fluorescence microscopy have advanced the exploration of complex, three-dimensional biological structures at submicron resolution. We have developed a voxel-based three-dimensional (3-D) imaging program (Voxx) capable of near real-time rendering that runs on inexpensive personal computers. This low-cost interactive 3-D imaging system provides a powerful tool for analyzing complex structures in cells and tissues and encourages a more thorough exploration of complex biological image data.
confocal; three-dimensional; two-photon; voxels
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INTRODUCTION |
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ADVANCES IN BIOCHEMISTRY and molecular biology have provided unprecedented insights into molecular interactions, but they also have increased our appreciation of the architectural organization of cells. With the development of specific probes, optical microscopy has allowed biochemical experiments to be conducted within individual cells, essentially using the cell as a test tube. The development of methods to express fluorescent chimeras of endogenous proteins has permitted the distribution and dynamics of specific proteins to be analyzed in living cells and in intact animals. Optical microscopy has become increasingly essential to an integrated approach to modern biomedical research.
Most microscopic studies produce two-dimensional (2-D) images that provide limited information about the three-dimensional (3-D) organization of cells and tissues. Although the development of confocal and multiphoton microscopy has made it possible to collect high-resolution 3-D images, true 3-D microscopy is still in its infancy. This is primarily due to the difficulty of analyzing 3-D images. In the past, 3-D visualization and image analysis systems were expensive, so they have not been widely available to biomedical researchers (6). Consequently, researchers have collected large quantities of images that sometimes have been incompletely explored and analyzed.
Fortunately, computer graphics hardware has developed to the point where high-speed 3-D graphics capabilities are common even on inexpensive personal computers (PCs). We have developed volume visualization software (Voxx) that takes advantage of such low-cost 3-D graphics hardware. Voxx promotes the exploration of complex microscopy data by providing interactive inspection and manipulation of 3-D images on PCs, tasks that until recently required much more expensive workstations or special-purpose voxel processors (2). As explained later in this article, we are making Voxx freely available to interested researchers.
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METHODS |
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Image acquisition. Image volumes may be generated from vertical series of 2-D images collected with the use of a microscope system that provides optical sectioning, such as a confocal or multiphoton microscope. Examples of images from such microscopy systems are shown in RESULTS. In each case, samples were mounted in aqueous medium, and images were collected with the use of a Bio-Rad MRC1024 confocal/two-photon system (Hercules, CA) fitted to a Nikon Eclipse inverted microscope (Melville, NY) with a ×60 water-immersion, NA 1.2 objective. Illumination for the multiphoton fluorescence excitation was provided by a Spectra-Physics (Mountain View, CA) Tsunami Lite Titanium-Sapphire laser. By matching the refractive index and the immersion medium with that of the mounting medium, this system avoids spherical aberration and has enabled us to collect high-contrast image volumes up to 200 µm in depth.
The 3-D visualization process should be considered during image acquisition. In addition to acquiring images with the required contrast and resolution, one should attempt to collect image volumes with a vertical spacing chosen so that consecutive optical sections overlap, but without oversampling to the degree that image volumes become excessively large. As shown below, 3-D rendering speed decreases significantly as the size of an image volume increases. In the examples given in RESULTS, image volumes were collected with optical sections spaced 0.4-0.5 µm apart.Image processing.
3-D rendering was performed using Voxx, a voxel-based 3-D rendering
program that we have developed. This program renders sets of images in
back-to-front order, combining them by using alpha blending (a
technique in which images in foreground layers are combined with images
in background layers, and the transparency of the volume is manipulated
by varying image opacity coefficients) or maximum intensity projection
and user-defined transfer functions (1). Voxx supports any
combination of color-indexed (8-bit grayscale or pseudocolor) and
true-color (32-bit RGBA) image stacks. A graph-based editor can be used
to make real-time color and opacity table modifications, permitting one
to employ pseudocolor or contrast enhancement and gamma correction by
using nonlinear intensity mappings (Fig.
1). Currently, only the color-indexed
format permits real-time modification of color and opacity values.
User-defined color and opacity tables and images may be saved and
loaded using several file formats. Voxx can currently import Bio-Rad
PIC, Zeiss LSM, and raw voxel files. In the future we plan to add
support for additional file formats and also to provide 3-D filtering and other graphical tools.
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Biological samples. Images were collected from cultured Madin-Darby canine kidney (MDCK) or pig kidney epithelial (LLC-PK-1) cells, whole microdissected embryonic (day 17) mouse kidneys, or vibratome sections of mouse heart (postnatal day P1) or rat (adult) and mouse (day P5) kidneys, cut between 60 and 150 µm in thickness. Handling, care, and euthanasia of mice and rats conformed to institutional animal care guidelines. Rodent tissues fixed in 4% paraformaldehyde/1× PBS were washed and incubated with fluorescently labeled lectins (Vector Labs, Burlingame, CA) as previously described (3). Nuclei have been labeled with either 4',6-diamidino-2-phenylindole (DAPI) or TO-PRO-3 dye (Molecular Probes, Eugene, OR).
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RESULTS |
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Examples of images rendered using Voxx.
Please refer to the Supplementary
Material1 for this article
(published online at the American Journal of Physiology-Cell
Physiology web site) to view the movies (Movies 2-4)
generated using Voxx. An optical section of a newborn mouse
kidney, labeled with fluorescent peanut agglutinin and then imaged by
two-photon microscopy, is shown in Fig.
2A. Although this image
clearly contains proximal tubules, it is not until the entire set of
215 optical sections is reconstructed that the interconnected nature of
the renal tubular network is apparent. Movie 2A shows the
addition of each focal plane to the volume in a slowed down version of
the back-to-front rendering process. The entire rendered volume is
shown in Fig. 2B, but the 3-D structure of this volume is
more obvious in Movie 2B. Movie 2B, generated by
using a movie capture function in Voxx, is intended to simulate the
experience of interactively using the program, a process in which the
user "grabs" the image volume by using the mouse-controlled cursor
and then changes the orientation and position of the volume in near
real time. The movies presented in the Supplementary Material have been
configured to play at approximately the same frame rates used in
interactive sessions. While the movies themselves are useful for
presentation, we emphasize that the real power of this software lies in
the interactive exploration of the volume provided by fast rendering.
Voxx allows users to explore image volumes just as they would inspect a
3-D object in their hands.
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Recommended system configurations for Voxx. Voxx is freely available to researchers for noncommercial use and can be downloaded (http://nephrology.iupui.edu/imaging/voxx/download). The program currently runs on Microsoft Windows, with development of Mac OS and Linux ports underway. Video boards equipped with NVIDIA GeForce2 GTS graphics processors seem to offer the best cost-performance ratio at this time, although we also use boards equipped with NVIDIA GeForce2 Ultra, Quadro2 Pro, and GeForce3 processors. Future versions of Voxx will include support for some features that are available only on GeForce3-based boards. To maximize the rendering speed, one should use AGP 4x (not PCI) versions of video boards, and the boards should be equipped with at least 64 MB of DDR memory.
The memory requirements of the program depend on the number and size of the image stacks and the number of color channels per stack. Most microscopists seldom collect more than sixty-four 512 × 512 images. We recommend that PCs running Windows 2000 be equipped with at least 256 MB of memory when rendering such a single-channel volume and 384 MB for a three-channel volume. However, image volumes collected using two-photon microscopy may be significantly larger. On occasion, our two-photon volumes contain 256 image planes with up to three channels, requiring up to 768 MB of memory. Voxx renders volumes this large at around 1 frame/s. As shown below (see Fig. 5), typical microscope image volumes are rendered at between 10 and 30 frames/s.
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Voxx performance. We compared the volume rendering performance of three PC-based systems. One was a Dell (Round Rock, TX) Precision 330 equipped with a 1.4 GHz Pentium 4 processor, 1.5 GB of RDRAM memory, and an NVIDIA Quadro2-based video board. The second was a more modest Dell Dimension 4100 equipped with an 800 MHz Pentium III, 512 MB of SDRAM memory, and an NVIDIA GeForce2 GTS board. The third, a Dell Precision 420 with two 933 MHz Pentium III processors, 1 GB of RDRAM memory, and an Oxygen GVX420 video board (3Dlabs, Sunnyvale, CA), was equipped with a special-purpose voxel rendering board (VolumePro 500-2X; TeraRecon, San Mateo, CA). The systems equipped with NVIDIA-based video boards were running Voxx, whereas the VolumePro-based system was running Revli (TeraRecon), a volume-rendering program written for the VolumePro (4).
As shown in Fig. 5, the two NVIDIA-based systems perform nearly identically, which demonstrates that there is no reason to buy the more expensive Quadro2 Pro graphics cards or Pentium 4 and RDRAM-based PCs to run the current version of Voxx. Figure 5 also shows that the rendering speeds of the two NVIDIA-based systems are similar to the rendering speeds of the third system equipped with the more expensive VolumePro voxel processor. The VolumePro-based system outperformed the NVIDIA-based systems for image stacks smaller than 256 × 256 × 256, but the rendering speeds of the systems were very similar for larger stacks containing 512 × 512 images. These results demonstrate that voxel-based rendering using video boards equipped with NVIDIA graphics processors seems to have a major cost-performance advantage over PCs using VolumePro 500 voxel processors. The NVIDIA-based PCs also seem to provide a significant cost-performance advantage over graphics workstations. We find that these PC systems outperform our SGI (Mountain View, CA) Octane SE workstation (data not shown), and others have shown that even the slower GeForce 256 renders small image stacks at speeds similar to the very expensive Reality graphics subsystem used on an SGI Onyx2 workstation (5). ![]() |
DISCUSSION |
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Confocal, multiphoton, and digital deconvolution microscopy have provided biologists with the powerful capability to collect high-resolution image volumes, an approach that is critical to characterizing the complex organization of cells and tissues. However, the proliferation of 3-D microscopy has been slowed by the lack of effective and affordable systems for inspecting and evaluating image volumes.
Our goal was to develop software that would provide near real-time rendering of multichannel image stacks by using computer systems affordable enough so that individuals could purchase systems for use in their offices and labs. In the past, most voxel-based programs had to run on very expensive SGI workstations to achieve near real-time rendering speeds. The recently developed VolumePro voxel processors (2) support volume rendering on PCs, but the current VolumePro 500-2X board is limited to single-channel data and adds thousands of dollars to the cost of each imaging system.
Consequently, we developed Voxx, a program that uses the high-performance 3-D graphics processors already present on many low-cost video boards in PCs. These processors can achieve rendering speeds similar to VolumePro voxel processors and the graphics subsystems used on many SGI workstations. The combination of our free Voxx program and such low-cost video boards should make 3-D image analysis systems available to a larger number of biologists, thus promoting a more thorough exploration of complex biological structures imaged using confocal and multiphoton microscopy systems. We encourage other developers of voxel-based imaging software to add support for these low-cost 3-D graphics processors to their programs as well.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Bacallao for providing data for Fig. 3B.
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FOOTNOTES |
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This work was supported by the Indiana University Strategic Directions Initiative (K. W. Dunn and S. Fang) and a grant (InGen) from the Lilly Foundation to the Indiana University School of Medicine.
Address for reprint requests and other correspondence: K. W. Dunn, Dept. of Medicine, Division of Nephrology, Indiana Univ. School of Medicine, 1120 South Drive, Fesler Hall 115, Indianapolis, Indiana 46202-5116 (E-mail: kwdunn{at}iupui.edu).
1 Supplemental material to this article (Movies 2-4) is available online at http://ajpcell.physiology.org/cgi/content/full/282/1/C213/DC1.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 July 2001; accepted in final form 29 August 2001.
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