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Visual reality: using computer reconstruction and animation to magnify the microscopist's perception.

“The greatest treasure by far, however, was what Grandfather referred to as his ’stereopticon.’” 
 
  Artist Frederick Franck points in this passage to the origin of his singular eye for detail (Franck, 1997 ). Ironically, Franck believes it was a device for viewing space in flat paper images that awakened him as a child to the richness of the actual world. The principle of the stereopticon has also served biologists well, showing them glimpses of the microscopic world from their accustomed binocular point of view, letting them “crawl” into spaces of submicron dimension. Human visual perception has evolved with special emphasis on making sense of a colorful, three-dimensional (3D) reality that we move in and that moves around us. With the development of new imaging and labeling technologies, light microscopy has now evolved so that visual data collected from a single living specimen can go beyond the domain of the stereopticon, to engage the viewer’s full capacity for seeing in space and through time. A challenge arising out of these advances is how to display these data so that the microscopist can sense and actively explore the recording’s full spatial and temporal content, to better understand visually what cannot be seen directly through the microscope eyepiece. Confronted with this opportunity in my own work on live fluorescence imaging of Caenorhabditis elegans development, I have invested some experimental effort in representing multidimensional microscope data interactively on the computer screen. In this essay, I describe my recent experience of trying to make the most of the observer’s ability to see and comprehend microscopic specimens in more than two dimensions. 
 
The basis for 3D visualization in light microscopy is the acquisition of optical sections, 2D images that uniquely portray features found at specific depths of focus within the specimen. A stack of optical sections through a sample defines its 3D structure, and repeated collection of such stacks over time produces a 4D recording. Simultaneous 4D acquisition of separate optical channels, in turn, yields a 5D data set. Software designed to acquire, archive, and play back 4D and 5D recordings has allowed the viewer to animate the data both over time and slice-by-slice through focus, to freely and repeatedly reenact the experience of observing the live specimen on the microscope (Hird and White, 1993 ; Fire, 1994 ; Inoue and Inoue, 1994 ; Thomas et al., 1996 ). This standard “through-focus 4D” representation is quite valuable, especially in transmitted light microscopy, where subtle image contrast prohibits use of volume-rendering techniques. 
 
Yet in many cases a specimen’s full spatial content can be nicely represented in a single image. Fluorescence microscopy, with its high degree of contrast, often lends itself well to volume reconstruction (Minden et al., 1989 ; Chen et al., 1996 ; Amberg, 1998 ; Mohler and White, 1998b ). Computational rendering of 3D structure can be extremely powerful (Drebin et al., 1988 ; Ford-Holevinski et al., 1991 ). It produces real views of the specimen that can never actually be seen on the microscope and allows the viewer to use stereo vision and a sense of spatial motion to discern the position and trajectory of details within the greater space; however, high-resolution fluorescence imaging has been notoriously toxic to the sample, limiting the time dimension of 4D recording. Recently, epifluorescence techniques that spare the viability of the specimen while producing optical sections have become more available. Multiphoton laser-scanning microscopy and low-light, wide-field deconvolution microscopy both minimize the noxious effects of excitation and fluorescence to a point where long 4D recordings of living fluorescent samples can now be obtained (Minden et al., 1989 ; Denk et al., 1990 ; Mohler and White, 1998a ) (J. A. Waddle, personal communication). The increase in use of vital fluorescent probes and green fluorescent protein (GFP) fusion transgenes has combined with these new imaging approaches to bring live time-lapse fluorescence microscopy to new prominence. Thus with low-damage fluorescence imaging we may now fruitfully combine 4D and 5D recording with 3D volume reconstruction to watch the behavior of an entire space-filling fluorescent specimen. 
 
A worthwhile goal then is to present the viewer with a time-animated volume rendering of a such a specimen that can be handled manually and experienced visually on a time scale suitable to the viewer’s intrinsic sensitivity to space and motion. To pursue this goal, I have focused on adapting functions of existing graphics programs to the specific task of portraying these 4D and 5D microscope data sets. Data reconstruction has been performed on either of two different levels of computational sophistication: the inexpensive personal computer and the advanced graphics workstation. In both cases, however, public domain “open source” software is the basis for creating and viewing data reconstructions. My hope is that visualization methods like these may become streamlined enough so that biologists, rather than computer technophiles, may easily use such technology to explore their subjects.