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New Innovations for Biological Imaging and Instrumentation
Summary: Harald Hess leads a group of scientists and engineers in the Applied Physics and Instrumentation Group (APIG). Their goal is to promote and establish advanced technology that will be unique to the Janelia Farm Research Campus and of collective benefit to its researchers.
Scientific insight is often conceived at frontiers where diverse disciplines meet. In biology, several such fields are converging: new ways to label tissues at the molecular level, new techniques in optical and electron microscopy, powerful electronics to collect terabytes of data, and computer algorithms that can reveal complex patterns and relationships. At JFRC, I lead a team that seeks to understand bioimaging challenges, to harness and introduce new disciplines or technologies that are relevant, and to promote a coherent systems approach for optimal benefit to biological research. Three initial projects will characterize this effort: higher resolution novel microscopy, high-throughput microscopy, and correlative microscopy.
Optical Microscopy with Resolution of Electron Microscopy
The detail that can be seen by optical microscopy is limited to ~0.25 microns (about 0.25 percent the diameter of a human hair). Although this level of detail can resolve some of the organelles of a cell, it falls short of seeing an organelle's details and even farther from seeing any nanometer-sized structure that is responsible for so much of molecular biology. Combining three insights has led us to a new kind of optical microscopy that can peer into this regime: (1) Single fluorescent molecules (which might label a protein of interest) can be imaged and their centers can be localized to a fraction of the size of the fuzzy spot that corresponds to their image. (2) Closely spaced, optically overlapping fluorescent molecules can be separated, and each can be localized if there is a distinguishing characteristic. For example, if two molecules light up separately in different image frames, the center of each can be inferred to a fraction of the spot sizes. (3) A new class of activatable fluorescent proteins has been developed in the past several years, and a distinguishing subset of these proteins can be turned into a fluorescing state while the remainder remain dark. This last insight led to a new kind of microscope that my co-inventor, Eric Betzig (HHMI, Janelia Farm Research Campus), and I have dubbed PALM (photoactivated localization microscopy). This microscope can activate, sample, and localize the centers of a very small subset of closely spaced label molecules. After bleaching the first subset, this process is repeated for a new sparse subset to collect new centroid locations, and iterated thousands of times until a significant fraction of fluorescent label molecules have been sampled. (See the movie for an illustration of this principle.)
If we draw only the centers (and not the whole fuzzy ball) of all these fluorescent molecules that have each been imaged individually, we can generate a high-resolution image. The images in Figure 1 show a fluorescent protein–labeled Golgi apparatus in a cell seen by regular microscopy and by PALM.
With colleagues at the National Institutes of Health and Florida State University, we are broadening the utility of this technique and demonstrating ever more compelling applications to biological problems. Developing and streamlining sample preparation techniques, which preserves cell structure and optimizes photophysical properties of fluorescent labels, is key for expanding the application. Furthermore, instead of labeling only one protein, two or even more proteins can be labeled, each with a distinguishing trait such as color. This could paint a picture of how one protein works in relation to another.
PALM is only an initial example of what I hope will be a series of broadly useful innovations in microscopy.
High-Throughput Microscopy
Biological systems are incredibly rich in their information content. A simple tissue such as a fly brain already represents a terabyte of data if sampled at 30-nanometer intervals, and even more if sampled at higher resolution. Current approaches that tap directly into the massive data present in such biological samples are embryonic compared to what is imaginable. For inspiration, we can look at an unusual source, industry. For example, computer chips, with intricate nanometer-sized patterns, are inspected or rendered at giga-sample/sec rates, or even faster. In this way, millions of processors are manufactured daily. Applying this technical approach to the nanoscale structures of biology could pay tremendous dividends for biological researchers.
At JFRC we intend to engineer just such a general purpose capability. If successful it would provide a new level of three-dimensional data of sample tissues. If such data sets can be acquired repeatedly, quickly, and reliably, experiments can be performed on tens or even hundreds of samples to explore, for example, effects of genetics, environment, growth, and toxins. In this way scientists can rapidly design experiments and test hypotheses.
Correlative Microscopy
In addition to resolution and pure number of small sampled volume elements or voxels that comprise images, other dimensions can be added to gain insight into the complex biochemical structure and dynamics of living systems. For example, an electron microscope makes a gray-scale image where the gray level is a measure of how many electrons transmit through different parts of the sample. Figure 2A, taken at the National Institutes of Health, is such an image. The darker sausage-shaped objects are mitochondria, organelles that power much of a cell's activities. A different image of the same area measures the density of a specific labeled protein that resides in the interior of mitochondria (Figure 2B). This PALM image maintains the high resolution of the electron microscope image. Two elements now form an extra dimension of this image data: electron transparency and a specific mitochondrial protein density. The combined data can be rendered as a colorized overlay (Figure 2C).
This simple concept can be extended to other labeled proteins and even to other types of microscopy to expand the voxel information dimension. We intend to engineer systems that can reliably acquire and overlay data to generate higher information content images. This will measure a multitude of values coherently on the same sample. This correlative microscopy will be integrated into the high-throughput system, to maximize the benefits to biologists.
Leveraged Expertise
Such ambitious projects can only succeed with the collective participation of researchers at JFRC. The availability of genetically engineered samples, customized labels, instrumentation support, computation infrastructure, image processing, and theoretical modeling—as well as biologists to give inspiration and direction—are all critical. I hope APIG will bridge diverse fields, nucleate and drive collaborations, and be driven by new research opportunities identified by scientists at JFRC.
Last updated: July 31, 2007
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