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CBMs have great potential for the labelling of cellulose and hemicellulosic components of the cell wall, and future discovery of CBMs with specificity for pectins and other polymers offers great potential for their use in microscopy. Penium is currently being used for 4-DI of pectin processing in plant cells. This alga can be live labelled with monoclonal antibodies specific for epitopes of homogalacturonans HGs; B, C.

At the isthmus zone of the cell C, arrow , the point of pre-cytokinetic wall expansion, high-esterified HG is released in a narrow band B, arrow. As this HG is displaced outward toward both poles, it is de-esterified, most probably by the enzyme pectin methyl esterase PME.

The development of the wall closely corresponds with the cortical microtubule D and F-actin E networks at the isthmus. Tubulin was identified with an anti-tubulin antibody and F-actin was localized with rhodamine—phalloidin. Labelled cells are placed back in cultures and amounts of new growth can be monitored using CLSM F, arrows. In order to elucidate further the formation of pectin, EM is also employed.

This allowed for structural analysis of the pectin lattice arrow. This story is only just beginning as transformed cells that are expressing FP—PME or FP—cytoskeletal proteins will yield vital dynamic information for the generation of 4-D models of pectin secretion. FPs attached to, and expressed with, a gene of choice or targeting sequences therein have revolutionized direct non-invasive in vivo visualization of live plant cells Chan et al. Here, the genetic sequence for the FP is spliced onto the cDNA of a gene of choice or a partial sequence and inserted into a cell.

Under the proper conditions, the FP—gene construct will be expressed, providing a fluorescence-based emission emanating from where the protein is localized. GFP is small 27 kDa , non-toxic and can be viewed in both live cells or those mildly fixed DeBlasio et al. In plants, the successful expression of GFP was delayed until the removal of a cryptic intron Haseloff et al. Today, a multitude of new FPs are being employed in the study of proteins expressed in transformed plant cells. These have been derived from other marine invertebrates e.

The use of FPs in microscopy is predicated on successful transformation of a cell type and subsequent expression of the FP, feats not especially common nor easy to accomplish. Large libraries of FP-expressing transformed lines are presently available for some model plants e.

However, the microscopist must be aware of several problems that may result in FP-based experiments. These include low fluorescent signals, improper maturation of the FP, unusual or incorrect localization of the FP, and the FP directly affecting the dynamics of the cell under study. Ideally, the use of stable transformants and performing comparative studies of a protein containing different GFP variants or coral-derived proteins provide the best results. For more detailed information concerning the set-up and implementation of an FP study, numerous excellent reviews are available Chalfie and Kain, ; Rizzo et al.

New technologies continue to emerge in LM-based optics and offer new ways to attain images that can be used for 4-DI. In conventional CLSM, intense laser-generated light focused on a specimen may cause significant bleaching or damage, especially when using a UV laser to excite a fluorophore.

In two- or multiphoton microscopy, wavelengths of light twice that of the typically used shorter wavelength are focused on the specimen for short periods of time that are less than the fluorescence decay time of the fluorophore being studied Pawley, This results in fluorescence only at the focused zone Inoue, The advantages of this type of instrument include avoidance of potentially damaging UV light and acquisition of the fluorescent signal from deeper tissues with greater light-gathering efficacy.

This is above the resolution of many cellular components. The specimen is illuminated with multiple interfering beams of light transmitted through a series of diffraction gratings that can produce resolution of nm in the z-plane. Recently, this new technology has been used in plant cell studies including visualizing plasmodesmata and the viral movement protein in tobacco Fitzgibbon et al.

While both these technologies have been around for decades, they provide a direct means for assessing single cell systems. That is, one can directly assess the fate of an injected material in a single cell's developmental or experimental history. Microinjection has been very valuable in various areas of plant cell biology including elucidation of ion gradients, the cytoskeleton and developmental processes Du and Ren, Several technologies that interface LM with spectroscopic or vibrational optics have made recent impacts in plant cell studies.

FTIR microscopy employs LM to focus in on a specific locus of a sample and spectroscopy to measure the vibrations of molecular bonds therein. Recently, this technology has been valuable for analysis of cell wall polymers and specifically for high-throughput screening of cell wall mutants Mouille et al. LEXRF is a recent technology developed for direct visualization of thick tissues that yields imaging data with both topographical and chemical information via X-ray acquisition Kaulich et al. In plant studies, it has been successfully used by Regvar et al.

The ATM probe or microstylus is mounted on a cantilever, is run over a specimen and ultimately provides a direct measurement of the mechanical properties of that specimen Yarbrough et al. ATM requires little or no specimen preparation, but is somewhat limited in that it does not work well for specimens with significant contour i.

The highest resolution that can be acquired in biological microscopy today is via EM. However, due to poor penetrative powers of electrons and exposure to both high vacuum and an irradiating electron beam, living things cannot be visualized with EM. Specimens must be fixed and, in TEM, sectioned before viewing. These limitations restrict the value of EM to static imaging. As important, improved cryo-based specimen preparation technology for TEM and scanning electron microscopy SEM , immunogold cytochemistry, the utilization of electron tomography in rendering high resolution 3-D images, the tremendous potential of new technologies including focused ion beam FIB dissection of cells and the development of EM-specific genetic markers make EM even more valuable for the future.

In TEM, conventional chemical fixation employing aldehyde fixatives e. These processes typically require days to complete and may result in artefact production, cytoplasmic shrinkage and cellular extractions, or may compromise antibody binding to its epitope. To combat these problems, special microwave instrumentation for specimen processing Chebli et al. Likewise, for the elimination of OsO 4 during fixation, the use of new plastics and embedding strategies and the introduction of energy-filtering lenses on many TEMs have significantly enhanced immunogold labelling and the visualization of low contrast structures Zewail and Thomas, The basis of energy filtering is the selection and use of electrons of specific energies that, in turn, enhance contrast of the specimen.

Additionally, these filters may be used in the acquisition of electron energy loss spectra EELS that may be exceptionally useful in determining the elemental composition of a specimen Lutz-Meindl, ; Eder and Lutz-Meindl, ; Darehshouri and Lutz-Meindl, However, cryo-fixation technology has made the greatest impact in TEM-based specimen preparation today.

In many plant studies, specimens are rapidly frozen in a cryogen e. However, these methods produce sufficiently slow freezing rates that result in damaging ice crystal formation occurring in an internal location of a specimen. Likewise, the loss of turgor occurring during specimen excision and trimming i. Once frozen, several options exist for processing the specimen prior to sectioning and TEM viewing. Cryo-sectioning and visualization using a TEM equipped with a special cryo-specimen chamber is the most direct mode of imaging Kuo, , but the instrumentation required is extremely expensive.

More often, a frozen specimen is freeze substituted Staehelin and Kang, ; Takeuchi et al. During this time, the fixatives slowly penetrate and fix the specimen. Freeze substituion is commonly used today in plant cell biology and typically offers outstanding fixation quality. Transmission electron microcopes of kV are the most commonly used instruments in biology laboratories, and 50— nm sections are those that are typically viewed with these instruments. The thinness of these sections is somewhat limiting when considering the dimensions of a cell, but they do afford reasonable views of sub-cellular architecture and specific location identified via immunogold labelling.

Higher kV transmission electron microcopes accommodate thicker sections up to nm at kV or nm sections with kV , but these instruments are considerably more expensive than the kV transmission electron microcope. Recently though, electron tomography ET has proven to be an effective method for generating 3-D imaging or 3-DI.

ET uses multiple 2-D projection images of a 3-D object over a wide range of viewing angles to create a tomogram. ET has been of great value in plant cell biology in the elucidation of the cytokinetic mechanism of higher plants, cell wall development, Golgi vesicle structure and development, and chloroplast architecture Shimoni et al.

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Many general staining protocols have been developed to enhance imaging of specific cellular structures Kuo, ; Nakakoshi et al. Additionally, newly developing labelling methods offer great promise especially in correlative microscopy analyses. These include fluoranogold, metallothionen and the small, genetically encodable protein module, mini-SOG Haas and Otegui, ; Lee et al.

Modern technological advancements in SEM have also helped refine the ultrastructural aspects of the surfaces of plants. SEM visualizes electrons derived from the surface or sub-surface layers of a specimen or captures X-rays generated from the interior that are subsequently used to identify elemental composition or create elemental maps of a specimen.

This type of specimen preparation has been quite adequate for many plant specimens. However, as with TEM, the potential for specimen alteration, in this case extractions or morphological alteration of the surface to be studied, may be of concern. Here, the specimen is kept in a relatively high pressure chamber of the SEM column, the column vacuum is kept low and the working distance is kept short.

The electron detectors of these instruments are also capable of working under the presence of water vapour. Positively charged ions created by interaction of the electron beam with gases in the column neutralize the negative charge on the specimen surface. Image quality is often comparable with conventional SEM preparations. FeSEM uses low voltage but high electron brightness that allows for high magnification, and high-resolution analyses i. FeSEM has been an important imaging tool especially for cell wall studies dealing with cell wall porosity in pollen tubes Derksen et al.

In fact, many of these organisms are, or have the potential to be, model organisms in plant cell research. Model organisms represent well-studied and well-manipulated systems that provide the microscopist with a rich source of proven experimental and technical protocols as well as large pools of pre-existing data from which highly focused hypotheses may be formulated and for which results may be effectively compared.

Additionally, the genomes and transcriptomes of many of these organisms have been or are being sequenced and many have been successfully transformed, allowing, in turn, for the incorporation of FP technology for live cell labelling. This allows for the critical integration of microscopy-based data with molecular data. While several well-known model plants have been used extensively in microscopy-based studies e.

Though certainly not complete, the following represents a description of two model systems and one model group that are or will be especially valuable for 4-DI along with their unique features that may be used in the study of specific cellular phenomena:. When the male gametophyte or pollen of an angiosperm or gymnosperm germinates, a protuberance called the pollen tube emerges and grows toward the egg that is embedded in a female gametophyte and megasporangium Boavida et al.

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To attain such lengths, pollen tubes grow anisotropically at one rapidly expanding polar tip at rates of hundreds of micrometres to millimetres per hour. The basis of unipolar expansion is the precise control of wall polymer secretion and modulation at the tube tip. In the cytoplasm of the growing tip exists a confluence of dynamic sub-cellular activities including Golgi apparatus-derived production of wall precursor-carrying vesicles, cytoskeletal system-mediated membrane trafficking i.

Pollen tubes are popular specimens for cell biology experiments as they are easily germinated and maintained in laboratory cultures and adapt well to microscopy and manipulation in microscopy-based devices e. Pollen tubes may also be studied live with the application of various labels including antibodies and FPs Cheung et al.

As a result of all of these attributes and associated research, detailed models of growth dynamics, including those incorporating 4-DI, have recently emerged Aouar et al. The moss, Physcomitrella patens , represents an emerging model organism for molecular and cell-based studies Cove et al. This moss has a simple haploid phase-dominant life cycle, has an assembled and sequenced genome, and transformed cell lines have been established.

It has become an excellent tool for gene targeting and RNA interference methods in order to study gene function Cove et al. In microscopy-based studies, the protonemata life cycle phase of Physcomitrella has been exceptionally important for the study of fundamental cell and developmental mechanisms as it can be easily grown and experimentally manipulated. The protonemata have been used in studies of wall development Lee et al. The rapid accumulation of microscopy-based and genetic data from this organism make Physomitrella an important and convenient model in interpretation of basic sub-cellular phenomena in plants.

The Charophyceaen green algae or CGA represent the extant group of green algae that are most closely related and ancestral to land plants Becker and Marin, Unique phenotypic features of these algae, such as a unicellular growth habit, large cell size or extraordinary developmental mechanisms, have launched several taxa as potentially important models for various types of cell-based research including 4-DI. For example, the unicellular desmid, Micrasterias , is an exceptional tool for elucidating cellular morphogenesis, the role of the cytoskeleton and the effects of environmental stressors Affenzeller et al.

The large intermodal cells and rhizoids of the Charales, including Chara and Nitella , have served as outstanding models for studying the cellular phenomena of cytoplasmic streaming and cell wall growth mechanics Shimmen and Yakota, ; Proseus and Boyer, , a , b , c ; Shimmen, ; Wei and Lintilac, ; Goldstein et al. The plate-like thallus of Coleochaete has become a highly desirable system for elucidating pattern development is plant cell development Dupuy et al. A recent surge of research has shown that taxa of the late divergent CGA possess cell wall polymers remarkably similar to those of land plants Popper and Fry, ; Domozych et al.

Additionally, a unicellular desmid, Penium margaritaceum , has been shown to be a simple and convenient system for elucidating cell wall dynamics especially those dealing with pectins Domozych et al. This alga is easily grown and manipulated in culture, exhibits a well-defined secretory mechanism during wall development and, most importantly, may be live-labelled with mAbs that bind to specific wall polymer epitopes.

Labelled cells may then be returned to culture where subsequent wall development may be monitored and specific wall events studied via the addition of specific inhibitors or wall-altering enzymes. For example, high esterified homogalacturonans are secreted in a simple band in the cell centre. The next important steps in the study of Penium and other CGA will be gene sequencing and mapping, as well as successful stable transformation so that FP-based imaging may be employed. When coupled with current efforts in functional genomics, 4-DI will yield critical insight into the foundations of plant cell structure, mechanics, developmental modulations and reactions to stress.

These, in turn, may then be used in interpreting the manifestation of macroscopic phenomena, help in understanding how plants survive in our changing biosphere and also contribute to the design of plants and derived products in agriculture, biofuel production and other applied areas. Today, new and refined microscopy-based technologies and methods offer unprecedented imaging possibilities, but careful planning is required to maximize these benefits and minimize inherent limitations of certain approaches of microscopic investigation.

With careful strategy and implementation of new technologies though, the outlook for 4-DI of plant cells is very promising indeed. National Center for Biotechnology Information , U. Journal List Ann Bot v. Published online May Author information Article notes Copyright and License information Disclaimer.

For Permissions, please email: This article has been cited by other articles in PMC. Abstract Background Analysis of plant cell dynamics over time, or four-dimensional imaging 4-DI , represents a major goal of plant science. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility.

Four-dimensional imaging, confocal laser scanning microscopy, fluorophores, transmission electron microscopy, plant cell biology. Open in a separate window. Two- or multiphoton microscopy In conventional CLSM, intense laser-generated light focused on a specimen may cause significant bleaching or damage, especially when using a UV laser to excite a fluorophore. Micromanipulation and microinjection While both these technologies have been around for decades, they provide a direct means for assessing single cell systems. Imaging technologies associated with LM Several technologies that interface LM with spectroscopic or vibrational optics have made recent impacts in plant cell studies.

Low-energy X-ray fluorescence microscopy or LEXRF LEXRF is a recent technology developed for direct visualization of thick tissues that yields imaging data with both topographical and chemical information via X-ray acquisition Kaulich et al. Though certainly not complete, the following represents a description of two model systems and one model group that are or will be especially valuable for 4-DI along with their unique features that may be used in the study of specific cellular phenomena: Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata.

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The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

Morphogenesis of complex plant cell shapes — the mechanical role of crystalline cellulose in growing pollen tubes. Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution. Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens. Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography.


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Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis. Streptophyte algae and the origin of the embryophytes. Plant cell biology in the new millennium: American Journal of Botany. The increase in the backscattering signal by day 7 verifies that the cells have formed a dense, highly scattering layer near the surface over a period of several days.

Mechanical forces are known to affect morphology and genetic expression in cells. To observe the effects of mechanical forces on cells in 3D cultures both micro-topographic substrates and Matrigel cultures see Note 8 were imaged before and after being subjected to uniaxial stretching For these samples, this enables visualization of individual cells via MPM and the local structural microenvironment of the substrates via OCM.

Visualizing the nucleus enables individual cells to be clearly identified while the GFP signal shows the extent of the cell body, the level of adhesion of the cell to the substrate, and interactions with adjacent cells. The flexible polymer PDMS substrate was repeatedly stretched in the direction of the white arrow in b. Mechanical stimulation results in an elongation of the fibroblast cells, roughly along the direction of stretch. The stretching causes an elongation of the cells and an increase in the distribution of the GFP signal indicating that there is an increase in expression of vinculin.

The blue signal is the nuclear stain which identifies individual cells. Scattering in this culture, as seen in the OCM image, is caused by both the cells and the extra-cellular environment collagen secreted by the cells. It is observed that the cells have organized into an interconnected architecture along the fibrous collagen structure of the Matrigel. The 3D reconstruction is shown top left as well as projections along different axes giving a comprehensive view of this cluster of cells. The red channel corresponds to the scattering from the sample OCM while the green and blue channels are fluorescence images MPM.

Three-dimensional reconstruction, rotation, and computational sectioning at arbitrary plane angles allow for a more comprehensive view of a 3D cell culture. Mechanical stimulation in the direction of the arrows in the x - y plane has induced the cells to become more spherical and form clusters, potentially indicating that adhesion sites with the scaffold were broken during mechanical stimulation. Optical sectioning refers to imaging thin sections of the intact specimen. In addition to enabling visualization of samples in 3D, optical sectioning has the advantage of eliminating the need for the harsh processing steps that physically destroy the specimen and may alter some of its structural properties.

The ability to obtain optical sections in live specimens allows one to observe dynamic events in biology. Instead of viewing different specimens at fixed points in time, single specimens can be observed longitudinally over time with minimal disturbance. The optical sectioning techniques discussed in this chapter are particularly well-suited for imaging thick, highly scattering samples that do not allow light to be transmitted.

However, MPM requires focusing ultrashort laser pulses into a sub-femtoliter volume of a sample. Although power levels needed for MPM are moderate 2—20 mW , the high instantaneous intensity of light could possibly damage a specimen through different mechanisms. Exposure to ultrashort pulses of light has been shown to cause cell death through oxidative photodamage 33 , In practice, damage can be minimized by limiting the power levels and reducing the exposure time of the sample to the laser beam. The later can be achieved by rapidly scanning the beam or blocking the beam path to the sample when the beam is not scanning.

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Imaging speed for spectral-domain OCT is limited by the read-out speed of the line camera in the spectrometer. Swept-source OCT using Fourier-domain mode-locked lasers can achieve speeds of several hundred kHz However, these lasers currently operate in the 1. Spectral-domain OCT can be performed with a broad bandwidth laser source centered at any wavelength; however, line cameras sensitive to wavelengths greater than nm use indium-gallium-arsenide detection elements and can be expensive.

Very high imaging speeds are needed when imaging in vivo or when observing fast dynamic events. The chitosin scaffold itself is not visible due to the low scattering properties of the material. Vinculin is a surface adhesion protein and thus the presence of a GFP signal correlates to cell—cell and cell—substrate adhesion.

Cells in the Matrigel culture are in a true 3D microenvironment. The emission spectrum of a dye excited with either one photon or with two simultaneous photons is similar and the same filters can often be used. However, the two-photon excitation spectrum of a fluorescent molecule is not easily predicted from its one-photon spectrum and must therefore be determined experimentally in many cases. The two-photon emission spectra for many commonly used dyes and biomolecules have been characterized 36 , The different responses could potentially be explained by differences in adhesion strength to the PDMS and to the Matrigel, or to the differences in strain experienced at the cellular level in each type of 3D culture.

With this integrated imaging technique, 3D multi-modality information from cultures can be acquired, providing a comprehensive view of the structural and functional characteristics of the sample. National Center for Biotechnology Information , U. Author manuscript; available in PMC Jul 2. Graf and Stephen A. Copyright and License information Disclaimer. The publisher's final edited version of this article is available at Methods Mol Biol. See other articles in PMC that cite the published article.

Abstract Three-dimensional 3D cell cultures are important tools in cell biology research and tissue engineering because they more closely resemble the architectural microenvironment of natural tissue, compared to standard two-dimensional cultures.

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Introduction Culturing cells on two-dimensional 2D substrates has been a standard technique in cell biology research for decades. Confocal Microscopy Confocal microscopy is a high resolution imaging technique that enables optical sectioning of non-transparent samples. Multiphoton Microscopy Multiphoton microscopy is a high-resolution fluorescence imaging technique that has at least a twofold improvement in penetration depth over confocal microscopy Optical Coherence Tomography Optical coherence tomography is a technique which is capable of obtaining high resolution scattering-based images of thick tissue samples 7 , Open in a separate window.

The major components are as follows: The center wavelength is nm with a bandwidth of 20 nm or greater. The major components of the system are as follows: YVO4 laser Coherent with center wavelength at nm with a bandwidth of nm. Custom-built spectrometer for detection of OCM signal. Consists of collimating optics, a blazed diffraction grating having Chitosin Scaffold Cell Culture Preparation Filter chitosan solution and transfer to cylindrical molds.

The micro-incubator is placed in the sample arm of the OCT system allowing the chitosin scaffold to be imaged while maintaining the sterile environment. Three-dimensional OCT images of the same chitosin scaffold are taken at 1, 3, 5, 7, and 9 days after seeding see Note 5. To verify the dynamic changes observed in the OCT images taken on different days, different identical samples are histo-logically prepared after being incubated for the same durations see Note 6. The histological samples are viewed with a standard light microscope and compared to cross-sectional OCT images see Fig.

To more effectively visualize the whole cell population, 3D OCT data images are reconstructed and viewed from different angles see Fig. To visualize the culture at cellular resolution and to observe functional characteristics see Note 7 , CM images of the superficial layers of the chitosin scaffold are taken in fluorescence mode using a commercial system, DM-IRE Leica Microsystems, Bensheim, Germany.

CM images are taken at 1, 3, 5, and 7 days to observe dynamic changes to the culture see Fig. Two MPM channels are obtained by acquiring the same image twice using different emission filters for fluorescence detection see Note 9. Three-dimensional data sets are obtained by acquiring a series of en face planes at different depths in the sample. Using the Analyze software, en face planes are computationally extracted from the 3D data to observe the structure see Fig.

Different projections of the overlaid 3D data set are viewed using the Analyze software see Fig.


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Analysis of the before and after OCM and MPM images allowed the effects of mechanical stimulation on the function and morphology of cells in these two types of cultures to be observed see Note Footnotes 1 Traditional microscopy requires that a specimen be fixed and cut into thin slices and placed on a glass slide for imaging. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

Socializing with the neighbors: Studying cancer in 3 dimensions: Three-dimensional cell culture matrices: Handbook of Biological Confocal Microscopy. Helmchen F, Denk W. Deep tissue two-photon microscopy. Optical coherence tomography OCT: Methods for Analysis of Golgi Complex Function: Volume Franck Perez. Microfluidics in Cell Biology Part C: Microfluidics for Cellular and Subcellular Analysis: Volume Matthieu Piel. Volume H William Detrich. Laser Manipulation of Cells and Tissues: Volume 82 Michael W. Volume 94 Roger D.

Methods in Tau Cell Biology: Volume Stuart Feinstein. Nuclear Mechanics and Genome Regulation: Electron Microscopy of Model Systems: Volume 96 Thomas Mueller-Reichert. About Saurabh Jha P. He is The Robert C. After receiving a B. In , he became Professor and Head of Pharmacology at the University of Iowa College of Medicine, a position he held for eleven years.