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Correspondence to: John M. Robinson, Dept. of Physiology and Cell Biology, Ohio State University, 302 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. E-mail: robinson.21@osu.edu
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Summary |
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Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803808, 2001)
Key Words: correlative microscopy, ultrathin cryosections, immunocytochemistry, fluorescence microscopy, transcription factories, electron microscopy, immunogold
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Introduction |
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THE PAST FEW YEARS have witnessed a renaissance in biological optical microscopy. This has resulted largely from the availability of relatively inexpensive computers, sophisticated electronic cameras, computer software dedicated to imaging and microscopy, and many reagents for labeling cells and tissues. Many advances in the elucidation of structurefunction relationships have relied upon fluorescence labeling. These advances notwithstanding, investigators are often confronted by the question: What is the true size and shape of a structure seen in the light microscope? Often this question can be answered only using an electron microscope. When this has been done, the fluorescence signal has often been collected from one cell or section and the electron microscopy signal collected from another. In some instances adjacent sections have been used. However, in these cases we face an additional question: How do the structures seen with one method relate to those seen with the other method? Obviously, it would be best to image the exact same structures in both microscopes. Such a combination of light and electron microscopy can be referred to as correlative microscopy. Here, we review some applications of correlative microscopy and go on to describe a simple methodology that makes use of ultrathin cryosections and antibody probes labeled with both fluorochromes (for detection by light microscopy) and gold cluster compounds or colloidal gold particles (for electron microscopy).
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Some Examples of Correlative Microscopy |
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Correlative microscopy can provide unique information that is difficult to obtain with a single imaging regimen. A recent example is provided by a study of the cytoskeletal proteins myosin II and tubulin (
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Bridging the Resolution Gap with Ultrathin Cryosections |
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It is often important to section cells and tissues before microscopic examination. However, biological materials are usually fragile and optimal sections cannot be obtained without embedding the sample in a supporting medium. Waxes and resins are often used for this purpose. Alternatively, samples can be frozen before sectioning. Methodology for preparing ultrathin cryosections in which the sample is fixed, frozen, and the vitrified sample is sectioned has been developed (e.g.,
The resolution of the confocal microscope is typically 200 nm in the x- and y-axes and
500 nm in the z-axis (
where no is the refractive index of the immersion medium, is the wavelength, and NA is the numerical aperture. In normal microscopy preparations with a wavelength of 500 nm and an NA of 1.4, Rd is
500 nm.
In addition to confocal and deconvolution microscopy, one can minimize problems associated with z-axis resolution (i.e., 500 nm) by imaging thin sections (100150 nm in thickness) (
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The use of thin cryosections can also reduce the chromatic aberration inevitably associated with the simultaneous collection of multicolor images. Consider, for example, imaging two different antigens in one multiprotein complex with red and green fluorochromes to determine if they co-localize. Because light of different wavelengths is diffracted to different degrees, the red and green images cannot both be exactly in focus. This chromatic aberration becomes acute when objects lying at different depths in cells (or confocal sections) are imaged. However, it can be minimized simply by using cryosections. An in-focus image of one fluorochrome is collected, filters are switched, the microscope refocused, and an image of the second fluorochrome collected (
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Correlative Microscopy with Ultrathin Cryosections: Two Applications |
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Neutrophils display a characteristic range of physiological and biochemical responses on exposure to appropriate stimuli (e.g., phagocytosable particles, chemotactic peptides). Most biochemical studies of stimulus-dependent events (e.g., exocytosis) are carried out on neutrophils in suspension. Lactoferrin has been localized to granules in these rounded cells using rabbit primary antibodies and FNG secondary antibodies (
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The visualization of transcription sites provides a second example (
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Benefits of Ultrathin Cryosections for Correlative Microscopy |
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Immunofluorescence microscopy can allow high throughputs and detection of several different antigens with great sensitivity. The use of ultrathin cryosections brings additional advantages. First, immunolabeling is improved, antigenicity is preserved, and antibodies penetrate more easily deeply into thin sections. Second, images are sharper; z-axis resolution is higher, backgrounds are lower (because out-of-focus flare is eliminated), and one kind of chromatic aberration can be reduced (see above). Third, correlative microscopy is facilitated. This brings additional resolution and, importantly, the ability to confirm that a structure seen by light microscopy is the same as another seen by electron microscopy. The two different preparative procedures we have employed are summarized in Fig 4. Of course, correlative microscopy depends on probes that can be imaged in both light and electron microscopes, and fortunately such probes are now available or can be prepared easily. Although the methods described here are useful for work with single cells, their most important applications may be in the study of tissues, in which correlative microscopy has been more difficult to accomplish.
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Footnotes |
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1 Present address: MRC Clinical Sciences Centre, Imperial College School of Medicine, London, UK.
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Acknowledgments |
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Supported in part by the following grants: NIH HD38764, HD35121, and the American Heart Association (JMR); The Wellcome Trust (PRC), Royal Society (AP), and grants-in-aid for scientific research and project grants of the Center for Molecular Medicine of Jichi Medical School from the Ministry of Education, Science, Sports, and Culture of Japan (TT).
Received for publication March 12, 2001; accepted March 26, 2001.
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