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Are two better than one? Correlative microscopy incorporates both live cell optical microscopy and nano-level observation, using both methodologies in tandem to offer the best of both.

With the explosion of work in genomics and proteomics, scientists can now determine the structures of biomolecules with nano-precision. Understanding nucleic acids, proteins and other biomolecules in detail will ultimately lead us to a particular knowledge of how their structure influences and determines the nature, activities, and vulnerabilities of complex living systems. But just as important as gaining knowledge of static atomic-level structure and function is the ability to observe dynamic events as they occur.

Mix and match

Many scientists today are involved in correlative microscopy, a hybrid methodology that combines the best of both worlds. In some studies, fluorescent dyes are used to identify or track specific cells, cellular components, or pharmacologically active agents in living cells. Various types of fluorescence optical microscopy, with or without deconvolution, are used to observe and record events in real time. Wide field, total internal reflectance fluorescence (TIRF), confocal, multiphoton, near field, and other microscopy techniques are possible singly or in combination. Subsequent observation of the same samples, labeled with nanoparticle markers, is possible at much higher levels of resolution using electron microscopy or atomic force instrumentation. This allows scientists to localize the events that were previously observed under visible, ultraviolet, or infrared light, at molecular and sub-molecular levels.

Equally revealing are studies that use atomic force microscopy (AFM) with fluorescence techniques to observe the intracellular results of biomechanical probes in living tissue. A deeper understanding of complex, multiple-step biochemical reactions--studied in real time and demystified at the molecular level--will help unravel the complex biological functions of the cell and its proteins. Ultimately, the hope is that combining nano-level and optical imaging will enable us to probe molecular abnormalities at the origin of disease, and to tag specific molecules, enabling not only research, but also the kind of molecular targeting that will help deliver therapeutic compounds to specific regions where targeted molecules are found.

Probing for answers

One of the first challenges to researchers doing correlative microscopy is the difficulty of co-labeling a single specimen with both the fluorescent probes necessary for fight microscopy and the gold or other heavy metal labels required for electron microscopy. FluoroNanogold probes from Nanoprobes, Yaphank, NY, have made the process simpler. They include both the 1.4nm Nanogold label and either Fluorescein or Alexa Fluor 488 or 594 probes, combined. The probes permit visualization of targets in fluorescence using the light microscope, and then more detailed structural study via electron microscopy. Since the two reporters are on a single antibody probe, the two answers may be directly correlated. This allows multiple studies of the same area of interest with high accuracy.

One research group developing new methods for labeling and studying epitopes and molecules at multiple levels includes Ralph Albrecht of the Univ. of Wisconsin and Eduardo Rosa-Molinar of the Univ. of Puerto Rico--Rio Piedras. "We are using nano-particles made of gold, silver, platinum, palladium and other metals. These markers are used with specialized transmission electron microscopy (TEM) to localize compounds at molecular and submolecular levels of resolution," says Albrecht. "The nanoparticles vary not only in composition but in shape. We use spheres, pyramids, popcorn, and other dots distinguishable in high resolution scanning electron microscopy as well as TEM. Since the particles are in the 3-to-10-nm size range, we can localize and simultaneously identify a number of different molecules within functional macromolecular complexes."

One of the greatest difficulties of such work in the past was that metal particles used for high-level localization can quench the fluorescent dyes used for light microscopic studies. But, according to Albrecht, the new markers allow fluorescence and TEM probes to be used simultaneously so sequential observation of a single specimen by light microscopy and then by electron microscopy is possible, and the maximal resolution attainable by each type of microscopy is realized. According to Rosa-Molinar, "New probes permit the spatial visualization of expressed gene products in a cell or tissue and the dynamic movements of intracellular organelles. They also permit observation and co-localization of multiple molecular species, first with photon-based imaging and subsequently, at higher spatial resolution, with electron-based imaging technology."

Some researchers use AFM along with TIRF or confocal microscopy to obtain simultaneous measurements of photonic and mechanical signals from single biomolecules. Miklos S.Z. Kellermayer, of the Dept. of Bio physics, Faculty or Medicine at the Univ. of Pecs, Hungary, has used both confocal and TIRF microscopy in combination with AFM to acquire varying temporal and spatial information. Cells loaded with calcium indicator are mechanically manipulated using AFM. Then confocal microscopy is used to image the results. In his laboratory, scientists sometimes also stretch a fluorescently labeled molecule with AFM and then follow the generated forces (using AFM) and the simultaneous changes in fluorescence using TIRF or confocal microscopy.

Most correlative microscopy solutions are custom-built by the individual laboratory to meet individual needs. One commercial solution for an integrated AFM/optical microscope is the Bioscope from Veeco, Woodbury, NY, which was specifically designed for cell biology experiments, and can be mounted on many inverted optical microscopes.

One researcher using this system is Gerald Meininger, Director of the Dalton Cardiovascular Research Center at the Univ. of Missouri-Columbia. His system incorporates an Olympus IX81, Melville, NY., inverted microscope. In his work, the AFM tip is biofunctionalized with an extracellular matrix protein; then the tip interacts with the cell and is retracted. Researchers deliver a mechanical stimulus to the cell and then measure its response in terms of its resistance or "pullback." With optical microscopy, they determine what proteins are involved in that response, and study the calcium wave that sometimes results using a ratio imaging technique. With TIRF and interference reflection microscopy (IRM), they monitor molecular and structural changes in the basal side of the membrane. "With correlative microscopy and multiple labeling at the molecular and submolecular levels of spatial resolution, we have what we need to identify the parts of macromolecular complexes, and see how they fit and how they work together," summarized Albrecht. "By adding fluorescence live cell microscopy at lower resolution, we can view processes on a macro level. This hybrid methodology is very powerful."

--Ilene Semiatin

Ilene Semiatin is a freelance writer

based in White Plains, N.Y.
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Title Annotation:Keywords: * Microscopy * Life Science * Nanotechnology
Author:Semiatin, Ilene
Publication:R & D
Geographic Code:1USA
Date:Oct 1, 2005
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