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BACKGROUND

  1. Top of page
  2. BACKGROUND
  3. ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING
  4. TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING
  5. Acknowledgements
  6. SUGGESTED WEBSITES
  7. LITERATURE CITED

Live-cell imaging is an important analytical tool in laboratories studying biomedical research disciplines, such as cell biology, neurobiology, pharmacology, and developmental biology. Imaging of fixed cells and tissues (for which photobleaching is the major issue) usually requires a high illumination intensity and long exposure time; however, these must be avoided when imaging living cells. Live-cell microscopy usually involves a compromise between obtaining image quality and maintaining healthy cells. Therefore, to avoid a high illumination intensity and long exposure time, spatial and temporal resolutions are often limited in an experiment. Imaging live cells involves a wide range of contrast-enhanced imaging methods for optical microscopy. Most investigations use one of the many types of fluorescence microscopy, and this is often combined with transmitted light techniques, which will be discussed below. Continual advances in imaging techniques and design of fluorescent probes improve the power of this approach, ensuring that live-cell imaging will continue to be an important tool in biology.

ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING

  1. Top of page
  2. BACKGROUND
  3. ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING
  4. TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING
  5. Acknowledgements
  6. SUGGESTED WEBSITES
  7. LITERATURE CITED

Successful live-cell imaging experiments can be a major technical challenge. An important caution is to ensure that cells are in good condition and function normally while on the microscope stage with illumination in the presence of synthetic fluorophores or fluorescent proteins. The conditions under which cells are maintained on the microscope stage, although widely variable, often dictate the success or failure of an experiment.

Imaging Media

Various cell culture media are available based on the particular biochemical requirements of cells. Culture media contain various constituents, including amino acids, vitamins, inorganic salts (minerals), trace elements, nucleic acid constituents (bases and nucleosides), sugars, tricarboxylic acid cycle intermediates, lipids, and co-enzymes. In tissue culture media, an important step is to control oxygen concentration, pH, buffering capacity, osmolarity, viscosity, and surface tension. Commercially available media formulations often include an indicator dye (e.g., phenol red) to visually determine the approximate pH value. A carbon dioxide and bicarbonate buffer system for regulating pH is needed for almost all cell lines. The cells need to be cultured in an atmosphere that contains a small amount of carbon dioxide (usually 5–7%) in incubators to control the dissolved gas concentration. For live-cell imaging, an appropriate atmosphere with carbon dioxide can be difficult to provide, and this usually requires specifically designed culture chambers for a regulated atmosphere. Oxygen requirements can vary among cell lines, but normal atmospheric oxygen tension levels are suitable for most cultures. With regard to osmolarity, most of the cell lines have a large tolerance for osmotic pressure, with good growth at osmolarities between 260 and 320 milliosmolar. When cells are grown in open-plate cultures or Petri dishes, hypotonic medium can be used to cope with evaporation.

Maintenance of the Cellular Environment in Culture

A constant cellular environment is important to maintain. Cells should be grown in culture medium in a carbon dioxide incubator. Mitotic index, viability, and morphological variability should be determined.

Controlling temperature is a critical parameter for most cells in culture. Temperature control is often achieved by peripheral sources of heated air or infrared radiation. Temperature can also be controlled by a heating plate made of metal controlled by a thermistor coupled directly to the chamber, or by transparent thin coatings of electrically conductive metal oxides on the surface of the coverslip for providing efficient conductive heat transfer to the chamber. Temperature fluctuations can occur because the microscope stage, frame, and objectives can act as heat sinks and offset the heating system used. This problem can be solved using commercially available objective lens heaters. For permanent equipment, a box can be constructed around the microscope and heated with warm air.

Choice of Imaging Chamber

Most laboratories that image live cells with light or fluorescence microscopy have their own custom-designed viewing chamber (Khodjakov and Rieder, 2006). These chambers need to keep the specimens viable, while also providing optical properties that are optimal for imaging. The type of chamber that is used depends on various factors, such as the spatial and temporal resolutions necessary for the study, and the study duration. Short-term imaging experiments can be performed by sandwiching of two coverslips, which are separated from each other by a spacer between two plastic or metal plates. Long-term experiments can also be performed for imaging live cells on the microscope stage. The culture cells need to have optimum growth conditions for an extended period of time. In general, imaging chambers include a glass window, usually the thickness of a coverslip (approximately 170 μm), through which the cells are viewed with an objective lens that has a high numerical aperture. Two types of imaging chambers are generally used: (1) open chambers that are exposed to the atmosphere and (2) closed chambers, which are sealed to avoid evaporation of the culture medium. Both types of chambers are available commercially. For some high-resolution studies, the culture medium needs to be replaced frequently, which has led to various chambers being designed where there is a continuous flow of fluid across the surface of cells.

Phototoxicity

Cells are prone to photodamage, especially when fluorophores are present (Stephens and Allan, 2003). When fluorescent molecules are in an excited state, they react with oxygen to produce free radicals that can damage subcellular components and adversely affect the cells. Even when fluorophores are absent, mammalian cells are sensitive to ultraviolet light.

Enhanced power of new imaging techniques can be problematic. For obtaining the maximum signal/noise ratio and resolution, cells need to be illuminated with very high light intensity (Khodjakov and Rieder, 2006). Many methods are used to reduce light-induced damage. An important protective step is to shut off the illuminating light when not required (Stephens and Allan, 2003). Shuttering of the light used in every type of illumination is the most important factor in imaging of living cells. Unwanted wavelengths of light also should be removed. Optimized emission filters should be selected for a maximal signal. Reducing oxygen levels can help limit photobleaching. Additionally, omitting phenol red (used as a pH indicator in media) and serum from the medium can help reduce background fluorescence. Contamination of the illuminating light with even trace amounts of ultraviolet or infrared wavelengths should be avoided.

Establishing Cell Morphology and Conditions in Culture

One of the most important aspects of live-cell imaging experiments is to establish criteria for determining the condition of the cells. These criteria will vary depending upon the type of experiment; however, one of the most important factors to determine is whether there is damage to the cells caused by the imaging process that might affect the results. One of the simplest ways for following an imaging experiment is to compare the morphology and condition of cells that were exposed to light during the study with neighboring cells in the same chamber that were not illuminated. Once the cells are mounted on the microscope stage, they need to be viewed to establish their morphology and condition. In the majority of experiments, particularly when cells have been transiently transfected with fluorescent proteins, a large amount of morphological variability of the cells can be present. Sometimes cells are not transfected or show poor localization of the probe. Some of the transfected cells might overexpress the fluorescent protein, which can lead to toxicity. Cells can show abnormal morphology, such as swollen mitochondria, blebbing, and detachment from the substrate. Cells that do not show normal morphology should not be used for imaging experiments.

TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING

  1. Top of page
  2. BACKGROUND
  3. ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING
  4. TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING
  5. Acknowledgements
  6. SUGGESTED WEBSITES
  7. LITERATURE CITED

When choosing an optical microscopy system for live-cell imaging, the following three variables should be considered: sensitivity of the detector (signal-to-noise), specimen viability (discussed above), and the speed required for image acquisition. The system used should make maximum use of the light and use the fewest optical elements in the light path. To optimize signal-to-noise, the combination of filters selected for imaging of live-cells should closely match the spectral profiles of the fluorophores used for experiments. Advances in detector technology have enabled reduction in illumination levels. Photomultiplier tube cathodes for confocal microscopy have become increasingly sensitive. Sensitivity of the camera is also an important consideration. An intensified camera can be used for this purpose or a sensitive back-illuminated charged couple device (CCD) camera. With regard to speed of acquisition, particularly with simultaneous imaging of multiple fluorophores or radiometric analysis of a single probe, switching between filters or output from a monochromator will reduce the acquisition time.

Currently, there is no all-purpose live-cell imaging system that is suitable for all possible investigations. Researchers must therefore compromise by determining the optimal parameters while minimizing cell damage or death. Researchers also need to understand the pros and cons of different microscopes. Most cellular processes occur in three dimensions over time. Therefore, cells need to be imaged in four dimensions to obtain a complete picture. Most epi-illumination microscopes and confocal systems acquire data series in four dimensions.

Time-lapse imaging (the fourth dimension) is broadly applied to capture events that occur in live-cells over the period of a few seconds to several weeks or months. This technique enables repeated imaging of a cell culture at particular time points (Fig. 1). However, dynamic imaging data are complex and interpretation can be difficult. Therefore, specialized image processing methods for estimation of motion, object detection, visualization, and quantitation can be used (Gerlic et al., 2003).

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Figure 1. Time lapse images of biofilm formation by gfp-tagged Pseudomonas using confocal microscopy. The scale bar indicates 20 μm. The images were kindly provided by Thomas Moninger, Central Microscopy Research Facility of the University of Iowa and Pradeep Singh, University of Washington.

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Live-cell imaging is currently mainly performed by fluorescence microscopy. Some of the more common systems used for imaging live cells are discussed below; the more widely used systems are discussed first and then the less commonly used systems are described. A basic overview of these types of fluorescent microscopy has been published in a previous issue of The Anatomical Record.

Fluorescent Proteins

Many types of fluorescent proteins of different colors are available that are used as fluorescent tags for imaging live-cells. Currently, the most common fluorescent protein used in imaging of cells is green fluorescent protein (GFP; Shimomura et al., 1962; Crivat and Tarasaka, 2012). GFP-like proteins with emission colors ranging from cyan to red have been found in natural sources, including corals, sea anemones, hydrozoans, crustaceans, and even basic chordate animals (Wiedenmann et al., 2009). Table 1 shows some examples of the fluorescent proteins used by researchers, and use and advantages of these proteins.

Table 1. Summary of advantages and uses of fluorescent proteins in live-cell imaging
Fluorescent proteinsAdvantages and uses of fluorescent proteins
Examples of fluorescent proteins used for live cell imaging:Because fluorescent proteins are inherited by all progeny of marked parent cells, they are useful for long-term cell tracking
EGFPFluorescent proteins are useful for live-cell imaging applications because of their unique chemical structure; for example, GFP family proteins are compact, relatively small, and chemically inert
mKO2Fluorescent proteins are minimally disruptive to most proteins when attached to the N or C terminus
mOrange2Fluorescent proteins can mature quickly and remain fluorescent in many subcellular compartments
td-TomatoFluorescent proteins fold well within biological temperature ranges
mRubyFluorescent proteins can be used:
DsRedExpress2-as genetically encoded fluorescence markers
mCherry-to label proteins and subcellular compartments inside living cells
hcRed-track cells in tissue
mPlum-monitor protein–protein interactions
-as biological sensors to monitor biological events and signals
At least five differently colored fluorescent proteins can be imaged at the same time
Red fluorescent proteins are useful for better penetration of cells and tissues because of long wavelength light and decreased cellular autofluorescence in the red emission range; good for whole body imaging
Photoactivatable fluorescent proteins:Fluorescence properties of some fluorescent proteins can be modified by irradiating with light (photoactivatable fluorescent proteins), which creates further opportunities for live-cell imaging; for example:
mEOS2,-green to red photoconvertible proteins (e.g., PA-GFP) are effective for regional optical marking experiments because of high optical contrast and the risk of unintentional photoactivation is considerably decreased
Dronpa, PS-CFP2, PA-mCherry, and PA-GFP-photoactivatable fluorescent proteins have an important role in types of microscopy that allows imaging beyond the diffraction barrier
Fluorescent proteins can be used in live cells using many techniques, such as fluorescence resonance energy transfer, fluorescence correlation spectroscopy and single-molecule imaging

Widefield Systems

Widefield (conventional) systems provide a flexible system for live-cell imaging (Figs. 2 and 3). Fast acquisition can be achieved with flexible excitation and a low cost (Stephens and Allan, 2003). Many experiments using live-cells are best performed by widefield systems and subsequent deconvolution (method for reducing the amount of blurring in fluorescent imaging by reassigning each photon back its point of origin) of the data. However, deconvolution should be carefully and accurately applied to avoid generation of artifacts (Wallace et al., 2001).

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Figure 2. The process of phagocytosis of a ceramic fiber (RCF) by an LLC-MK2 monkey kidney epithelial cell in culture. The plasma membrane undergoes ruffling activity around the fiber as it becomes progressively internalized until it is completely contained within the cytoplasm of the cell. Labeling with DiI demonstrates that during phagocytosis the fiber becomes encased within a portion of the plasma membrane. The speed of incorporation is approximately 0.2 μm/min. Time (in hr:min:sec) is shown in the lower right corner. As an indication of magnification of the time-lapse, the diameter of the cell nucleus is approximately 20 μm. The images were kindly provided by Dr. Cynthia Jensen, the University of Auckland.

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Figure 3. Part of a time sequence of neonatal rat ventricular myocytes plated onto a dish and paced at 1 Hz. The white areas are free calcium ions. The scale is 0.19 μm per pixel. Immunofluorescence microscopy was used. The image was kindly provided by Gregory Bass, the University of Auckland.

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Imaging live-cells using transmitted light is often used with fluorescence microscopy to determine cell shape, motility, and position. This is important for investigating cells that undergo changes in shape, such as mitosis and apoptosis. Differential interference contrast and phase contrast are the most common types of microscopy used. Time-lapse imaging can be used in two spatial dimensions using widefield techniques.

Confocal Microscopy

Time-lapse imaging of living cells is enhanced by the improved resolution in confocal microscopy. However, image collection during time-lapse is limited by the speed of the scanning mirrors. Imaging live tissues using confocal microscopy is more difficult than imaging fixed specimens, and is not always practical because the specimen may not tolerate the particular conditions. Choice of a cell type that is amenable to the conditions of imaging in confocal microscopy is important to avoid experimental problems. Live-cell confocal analysis is a good option because of its increased efficiency of photons and less phototoxic dyes for labeling. Images should be captured as rapidly as possible, using the minimal amount of laser power that enables imaging. Images taken with laser-scanning confocal microscopy are shown in Fig. 4.

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Figure 4. Laser-scanning confocal images of the Wistar rat lens. Two images are shown from the same experiment. (A) A cross-section of Wistar rat lens fiber cells is shown. Scale bar = 10 μm. (B) The summary of fluorescent intensity of 42 slides over a period of 84 sec is shown. The center cell of highest intensity is where the two-photon laser uncaged the fluorescein. Using another 488 nm laser, the fluorescein was excited and imaged. The image was kindly provided by Dr. Nancy Liu, the University of Auckland.

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Confocal microscopy has high irradiance, which may cause photobleaching of specimens (Conchello and Lichtman, 2005). Photobleaching from the laser beam is cumulative over multiple scans and, therefore, exposure to the beam should be reduced as much as possible for image acquisition. Another limitation of confocal microscopy is its inefficient use of light. In-focus light in a confocal microscope needs to pass through a small aperture, and this means that its transmission efficiency is lower than that in widefield systems. This loss can be reduced, but it will never be eliminated. Therefore, high intensity of excitation light is required for confocal microscopy, which increases the possibility of photobleaching to the specimen (Khodjakov and Rieder, 2006).

Confocal microscopy provides control of the area of illumination and, therefore, it is useful for photobleaching techniques, such as fluorescence loss in photobleaching and fluorescence recovery after photobleaching (FRAP; see below).

Spinning-disc confocal microscopy versus laser-scanning confocal microscopy.

Spinning-disc confocal microscopy has some advantages for live-cell imaging compared with laser-scanning confocal microscopy (Stehbens et al., 2012). The frame acquisition rate is high for spinning-disc confocal microscopy because the specimen is scanned by thousands of points of light in parallel. Fast frame rates are advantageous because CCD cameras can be used for direct acquisition of confocal images rather than photomultiplier tubes, which are still the most commonly used light detectors in laser scanning confocal microscopy. Additionally, spinning-disc confocal microscopy is less affected by saturation of fluorescence or ground state depletion compared with laser scanning confocal microscopy. For live-cell imaging, a slight loss of confocality is associated with spinning disc confocal microscopy, but this is compensated for by a better signal-to-noise ratio and a reduced amount of photobleaching.

Multiphoton Microscopy

Multiphoton microscopy has many advantages for imaging living cells. The use of longer-wave excitation greatly reduces photobleaching in contrast to single-photon confocal imaging, and this results in extended viability of living cells during long-term imaging. This type of microscopy allows three-dimensional (3D) fluorescence imaging of live cells that are hundreds of microns deep within thick, strongly scattering samples (Rubart, 2004). Examples of multiphoton fluorescence microscopy include imaging of intracellular calcium dynamics in various tissues, probing functional integration of donor cells after intracardiac transplantation, cellular redox state, and developmental studies, which require viability to be maintained for detecting sequential events in the same specimen over long time periods. The near-infrared laser based approach of multiphoton microscopy is very efficient; therefore, it has many potential uses for noninvasive, noncontact loading of fluorescent probes in various cell types, as well as in studies on cell to cell communication (Konig, 2000).

Total internal reflection fluorescence (TIRF) microscopy

In cell biology, TIRF microscopy is primarily used to observe cell–substrate interactions (Sako and Uyemura, 2002). TIRF microscopy is used for direct imaging of living cells that are within close proximity to the coverslip. TIRF can be used to follow biochemical reactions, structural changes, and movements of biomolecules at the level of single molecules in real time. This technique is useful for observing single living cells in aqueous solution near a glass surface, as well as single molecules on the plasma membrane of live-cells.

FRAP Microscopy

Time-lapse imaging can be used to obtain information on the steady-state distribution of a protein over time but time-lapse imaging cannot determine the kinetic properties of a molecule. To achieve this, a selected pool of fluorescent proteins needs to be distinguished from other fluorescent proteins in cells. This can be accomplished by FRAP, which is a technique that is used to study the motion of cellular processes (Lippincott-Schwartz and Patterson, 2003). This method can be used to determine the mobility and dynamics of proteins that are fluorescently labeled in or associated with cells. In FRAP, fluorescently labeled molecules are intentionally photobleached in the area of interest of the cell, using high-intensity light. The bleached molecules diffuse out of this region and nonphotobleached fluorescently labeled molecules simultaneously move into the bleached region, which results in the fluorescence signal being recovered. The rate and the amount of recovery at the photobleached region is analyzed, revealing information on the dynamic properties of proteins or cytoskeletal structures (e.g., microtubules; Axelrod et al., 1976). FRAP is a time-course experiment in which data must be acquired quickly to successfully record recovery of the bleached area. However, this usually means that spatial resolution is traded for temporal resolution.

Imaging chambers for FRAP experiments are constructed so that the cells remain healthy while optical resolution is maximized. Confocal microscopes are one of the most common types of microscopes used for FRAP experiments (Houtsmuller, 2005). A widefield system with a laser for photobleaching may be a solution to improve temporal resolution.

Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is based on a confocal optical setup and is similar to a laser-scanning microscope. FCS measures dynamics in the time range of 1 μs to 100 ms (Bacia and Schwille, 2003). Autocorrelation measurements evaluate a wide variety of dynamic behavior in vivo. Because FCS has the advantages of high temporal resolution and short analysis time, FCS is useful for studying conformation dynamics, chemical kinetics, and molecular diffusion in solution as well as on membranes (Tian et al., 2011). The FCS focal volume is positioned in a cell's plasma membrane, in the cytoplasm, the nucleus, or outside of the cell. When analyzing dynamic processes on a very fast scale, special precautions are necessary to avoid artifacts. Because FCS temporal resolution is under 100 ns, limitations of FCS for in vivo applications are more likely to be in the slow time range. When slowly moving molecules are analyzed (e.g., nuclear DNA, molecules interacting with the cytoskeleton, or membranes), acquisition times need to be long enough to capture the process. In addition, molecules under examination should not become photobleached during their transit time through the focal volume. The resulting photobleaching decay in the fluorescence trace causes decay in the correlation curve, and this often prevents accurate curve evaluation using standard models.

Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging

Use of fluorescence resonance energy transfer (FRET) increases the spatial resolution of the fluorescence microscope to less than 10 nm. This considerable increase in resolution makes FRET appealing as a technique for studying co-localization events in biological samples, particularly because co-localization can be performed in living cells. FRET can be used to monitor protein–protein interactions inside live-cells (Sun et al., 2012). However, in live-cell studies, a distinct possibility is that FRET measurements will be invalidated by recovery of the acceptor fluorophores when using the method of acceptor photobleaching (similar to FRAP experiments). Therefore, use of acceptor photobleaching in live-cell experiments may be inappropriate.

Fluorescence lifetime imaging (FLIM) is routinely applied for measurements of signaling events inside single living cells. FLIM acquisition is rapid enough (hundreds of milliseconds to seconds) to perform measurements in live cells (Bastiaens and Squire, 1999). The nanosecond fluorescence lifetimes represent an extra spectroscopic dimension. The steady-state fluorescence image also enables the resolution of parameters of the activation state of proteins without disrupting cellular structure. FLIM is an ideal method for ultrahigh throughput screening of drug libraries on living cells because of noninvasiveness of fluorescence, parallel readout, and independence of fluorescence lifetimes on probe concentration.

Fluorescent Quantum Dots

Fluorescent quantum dots are inorganic fluorescent nanocrystals that overcome many limitations of the more commonly used organic fluorophores in live-cell imaging (Jaiswal et al., 2004). Quantum dots can be imaged with any type of fluorescence microscope. They are useful for live-cell imaging because of their enhanced brightness and their resistance to metabolic degradation and photodamage (Jaiswal et al., 2004, Li et al., 2011). Quantum dots are useful in studies of long-term and multicolor imaging of cellular and molecular processes. Quantum dots have been used for imaging of stem cells and cell membrane molecules, including cancer biomarkers expressed on the cell membrane. Quantum dots can penetrate cells and organelles at the subcellular level. Quantum dot/cationic liposome conjugates and electroporation are used to deliver quantum dots to the cytoplasm when there are a large number of cells. Microinjection is used for single cell tracking. Although quantum dots have numerous advantageous optical properties, inorganic semiconductor materials are toxic to live-cells, which can limit their use in live-cell imaging. Nanoparticle toxicity is an area of controversy. However, many experiments have demonstrated that modified quantum dots have limited cytotoxicity. Development of surface coatings has minimized toxicity (Walling et al., 2009).

Coherent Anti-Stokes Raman Scattering Microscopy

Live-cell imaging using fluorescent proteins and fluorescent probes is a powerful tool for cell biology, but a need persists for noninvasive techniques to study tissue and cell dynamics without introducing chemical or genetically encoded probes. Coherent anti-Stokes Raman scattering (CARS) microscopy is used by cell biologists to investigate live-cell dynamics with chemical specificity in a noninvasive, label-free manner (Pope et al., 2012). CARS is a multiphoton process that provides 3D high-resolution. The image contrast is achieved by light that is inelastically scattered by vibrations of endogenous chemical bonds. An advantage of the nonlinear CARS process is that it only occurs in the focal volume where high densities of photons are obtained, enabling intrinsic 3D spatial resolution. A detector pinhole is not necessary.

Most of the research applying CARS microscopy for cell biology has studied lipid dynamics (reviewed by Pezacki et al., 2011) because CARS has a strong signal from lipid C[BOND]H bond stretches. In fluorescence microscopy, obtaining stable, lipid-specific markers is difficult because the labeling process affects lipid function and localization. Therefore, in live cells, a challenge is to achieve rapid time lapse and long time course experiments on lipids and lipid droplets, and accurate quantification is extremely difficult. CARS microscopy has proven to be an excellent method for examining live-cell lipid dynamics with chemical specificity.

Atomic Force Microscopy

Atomic force microscopy is a variant of scanning probe microscopy that was developed for the material sciences. Atomic force microscopy complements light microscopy because atomic force microscopy provides information of the cell not achievable by light microscopy. Various imaging modalities can be used in atomic force microscopy, but only a few (e.g., error signal or amplitude contrast, intermittent contact or tapping mode, and force-distance, and force-volume techniques) are routinely used in live-cell imaging (Dvorak, 2003). Each type of imaging mode achieves unique information on living cells. For example, tapping imaging provides both internal structural and topographic information. The error signal mode is helpful for imaging very fine biological structures, such as “trails” by vertebrate cells moving on a substrate. Atomic force microscopy enables a flexible analytical approach that can provide intracellular detail in live cells, which has distinct advantages over techniques, such as scanning electron microscopy (Francis et al., 2010).

A limitation of atomic force microscopy is considerable differences among cultured cells with regard to their ability to withstand probe scanning. No set protocol exists to ensure successful atomic force microscopy of all cell types. Although successful imaging of live cells using atomic force microscopy still has many obstacles to overcome, no other available method provides dynamic imaging and force spectroscopy at high resolution.

Acknowledgements

  1. Top of page
  2. BACKGROUND
  3. ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING
  4. TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING
  5. Acknowledgements
  6. SUGGESTED WEBSITES
  7. LITERATURE CITED

The author thanks Thomas Moninger, Pradeep Singh, Gregory Bass, Cynthia Jensen and Nancy Liu for providing images, and Andrew Chung for assistance with processing images. Jacqui Ross's (The University of Auckland) advice on confocal microscopy is greatly appreciated.

LITERATURE CITED

  1. Top of page
  2. BACKGROUND
  3. ISSUES OF MAINTAINING CELL VIABILITY DURING IMAGING
  4. TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGING
  5. Acknowledgements
  6. SUGGESTED WEBSITES
  7. LITERATURE CITED