Name: atomic force microscopy to study the physical properties of epidermal cells of live arabidopsis roots
Uploaded: 2022-03-31T13:50:00
Description: Watch this Scientific Journal Video about Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots at JoVE.com

This research was funded by CSIC I+D 2018, grant No. 95 (Mariana Sotelo Silveira).; CSIC Grupos (Omar Borsani) and PEDECIBA.

1. Preparation of the plant material and growth conditions
<ol>
	<li>To generate the needed plant material, sterilize the seeds of Arabidopsis wild type and mutant lines of interest. 
	NOTE: In this protocol, we used the following: ttl1: T-DNA insertion lines Salk_063943 (for TTL1; AT1G53300) - Columbia-0 (Col-0) wild-type; the Procuste1 (prc1-1) mutant, which consists of a knock-out mutation (Q720stop) in the CESA6 gene (AT5G64740), as previously descr...

<ol>
	<li>Anderson, C. T., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+construction,+perception,+and+remodeling+of+plant+cell+walls.">Dynamic construction, perception, and remodeling of plant cell walls.</a> Annual Review of Plant Biology. 71, 39-69 (2020).</li><li>Roeder, A. H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifteen+compelling+open+questions+in+plant+cell+biology.">Fifteen compelling open questions in plant cell biology.</a> The Plant Cell. 34 (1), 72-102 (2022).</li><li>Zhang, B., Gao, Y., Zhang, L., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+cell+wall:+Biosynthesis,+construction,+and+functions.">The plant cell wall: Biosynthesis, construction, and functions.</a> Journal of Integrative Plant Biology. 63 (1), 251-272 (2021).</li><li>Hamant, O., Haswell, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+behind+the+wall:+Sensing+mechanical+cues+in+plants.">Life behind the wall: Sensing mechanical cues in plants.</a> BMC Biology. 15 (59), 1-9 (2017).</li><li>Scheres, B., Benfey, P., Dolan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+development.">Root development.</a> The Arabidopsis Book. 1, 0101 (2002).</li><li>Cuadrado-Pedetti, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+arabidopsis+tetratricopeptide+thioredoxin-like+1+gene+is+involved+in+anisotropic+root+growth+during+osmotic+stress+adaptation.">The arabidopsis tetratricopeptide thioredoxin-like 1 gene is involved in anisotropic root growth during osmotic stress adaptation.</a> Genes. 12 (2), 236 (2021).</li><li>Milani, P., Braybrook, S. A., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrinking+the+hammer:+micromechanical+approaches+to+morphogenesis.">Shrinking the hammer: micromechanical approaches to morphogenesis.</a> Journal of Experimental Botany. 64 (15), 4651-4662 (2013).</li><li>Braybrook, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+elasticity+of+plant+cells+with+atomic+force+microscopy.">Measuring the elasticity of plant cells with atomic force microscopy.</a> Methods in Cell Biology. 125, 237-254 (2015).</li><li>Bidhendi, A. J., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+quantify+primary+plant+cell+wall+mechanics.">Methods to quantify primary plant cell wall mechanics.</a> Journal of Experimental Botany. 70 (14), 3615-3648 (2019).</li><li>Desnos, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procuste1+mutants+identify+two+distinct+genetic+pathways+controlling+hypocotyl+cell+elongation,+respectively+in+dark-+and+light-grown+Arabidopsis+seedlings.">Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark- and light-grown Arabidopsis seedlings.</a> Development. 122 (2), 683-693 (1996).</li><li>Fagard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procuste1+encodes+a+cellulose+synthase+required+for+normal+cell+elongation+specifically+in+roots+and+dark-grown+hypocotyls+of+arabidopsis.">Procuste1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of arabidopsis.</a> The Plant Cell. 12 (12), 2409-2423 (2000).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiologia Plantarum. 15 (3), 473-497 (1962).</li><li>Perilli, S., Sabatini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+root+meristem+size+development.">Analysis of root meristem size development.</a> Methods in Molecular Biology. 655, 177-187 (2010).</li><li>Sader, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+virtual+instrument+to+standardise+the+calibration+of+atomic+force+microscope+cantilevers.">A virtual instrument to standardise the calibration of atomic force microscope cantilevers.</a> Review of Scientific Instruments. 87 (9), 093711 (2016).</li><li>Collinsworth, A. M., Zhang, S., Kraus, W. E., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+elastic+modulus+and+hysteresis+of+skeletal+muscle+cells+throughout+differentiation.">Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation.</a> American Journal of Physiology - Cell Physiology. 283 (4), 1219-1227 (2002).</li><li>Mathur, A. B., Collinsworth, A. M., Reichert, W. M., Kraus, W. E., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial,+cardiac+muscle+and+skeletal+muscle+exhibit+different+viscous+and+elastic+properties+as+determined+by+atomic+force+microscopy.">Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy.</a> Journal of Biomechanics. 34 (12), 1545-1553 (2001).</li><li>Sirghi, L., Ponti, J., Broggi, F., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+elasticity+and+adhesion+of+live+cells+by+atomic+force+microscopy+indentation.">Probing elasticity and adhesion of live cells by atomic force microscopy indentation.</a> European Biophysics Journal. 37 (6), 935-945 (2008).</li><li>Peaucelle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM-based+mapping+of+the+elastic+properties+of+cell+walls:+At+tissue,+cellular,+and+subcellular+resolutions.">AFM-based mapping of the elastic properties of cell walls: At tissue, cellular, and subcellular resolutions.</a> Journal of Visualized Experiments: JoVE. (89), e51317 (2014).</li><li>Peaucelle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin-induced+changes+in+cell+wall+mechanics+underlie+organ+initiation+in+Arabidopsis.">Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis.</a> Current Biology. 21 (20), 1720-1726 (2011).</li><li>Fernandes, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+epidermal+cells+of+whole+living+roots+of+Arabidopsis+thaliana:+An+atomic+force+microscopy+study.">Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study.</a> Physical Review E. 85 (2), 21916 (2012).</li></ol>

Cell and cell-wall mechanics are increasingly becoming relevant to gain insight into how mechanics affects growth processes. As physical forces propagate over considerable distances in solid tissues, the study of changes in the physical properties of the cell wall and how they are sensed, controlled, tuned, and impact the plant&#39;s growth are becoming an important field of study2,3,8.
A method is pr...

Plant cells are surrounded by a cell wall that is a complex structure composed of interacting networks of polysaccharides, proteins, metabolites, and water that varies in thickness from 0.1 to several &#181;m depending on the cell type and the phase of growth1,2. Cell wall mechanical properties play an essential role in the growth of plants. Low stiffness values of the cell wall have been proposed as a precondition for cell growth and cell-wall expansion, and there is increasing evidence that all cells sense mechanical forces to perform their functions. However, it is still debated whether changes in the physical properties of the cell wall determines cell fate2,3,4. Because plant cells do not move during development, the final shape of an organ depends on how far and in what direction a cell expands. Thus, Arabidopsis root is a good model to study the impact of cell wall physical properties in cell expansion because different types of expansion occur in different regions of the root. For example, anisotropic expansion is evident in the elongation zone and particularly noticeably in the epidermal cells5.
The method described here was used to characterize the physical properties of the cell wall of epidermal cells at the nanoscale of living Arabidopsis roots using an Atomic Force Microscope (AFM) coupled with an inverted fluorescence phase microscope6. For an extensive revision of the AFM technique, read7,8,9.
This protocol outlines a basic sample preparation method and a general method for AFM-based elasticity measurements of plant cell walls.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63533/63533fig01v2.jpg" /> 
Figure 1: Schematic overview of force-indentation experiment in Arabidopsis roots using atomic force microscopy (AFM). The scheme gives an overview of the steps of a Force-Indentation experiment from the preparation of the substrate to immobilize the root sample firmly (1-2), root viability confirmation through propidium iodide staining (3), cantilever positioning on the surface of an elongated epidermal cell of the primary root (4-5), force curves measurement (6), and force curve processing to calculate the apparent Young&#39;s modulus (7-8). EZ: elongation zone. <a href="https://www.jove.com/files/ftp_upload/63533/63533fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 x Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>Include sodium chloride and phosphate buffer and is formulated to prevent osmotic shock and maintain water balance in living cells.</td>
		</tr>
		<tr>
			<td>AFM software</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic force microscopy (AFM)</td>
			<td>BioScope Catalyst, Bruker, Billerica, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalyst Probe holder-fluid</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>CAT-FCH</td>
			<td>A probe holder for the Bioscope Catalyst, designed for fluid operation in contact or Tapping Mode.&#160; Also compatible with air operation.</td>
		</tr>
		<tr>
			<td>Cryoscopic osmometer; model OSMOMAT 030</td>
			<td>Gonotech, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog Medium</td>
			<td>Duchess Biochemie</td>
			<td>M0221</td>
			<td>Original concentration, (1962)</td>
		</tr>
		<tr>
			<td>Optical inverted microscope coupled to the AFM</td>
			<td>Olympus IX81, Miami, FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEGAMIL</td>
			<td>ANAEROBICOS S.R.L., Buenos Aires, Argentina</td>
			<td>100429</td>
			<td>Neutral, non acidic silicone glue</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Deltalab</td>
			<td>200201.B</td>
			<td>Polystyrene, 55 x 14 mm, radiation sterile.</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td>For root viability test.</td>
		</tr>
		<tr>
			<td>Silicon nitride probe, DNP-10, cantilever A</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>DNP-10/A</td>
			<td>For force modulation microscopy in liquid operation. Probe tip radius of 20-60 nm. 175-&#956;m-long triangular cantilever,&#160; with a spring constant of 0.35 N/m.</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma</td>
			<td>T4537</td>
			<td></td>
		</tr>
	</tbody>
</table>

atomic force microscopy to study the physical properties of epidermal cells of live arabidopsis roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Force-Indentation experiments 
The following text presents some results expected when a force-indentation experiment is conducted to show the typical output to expect when the protocol is well executed.
Force-displacement curves 
Representative force indentation plots that were obtained indenting live samples at a position placed in the center of the cell of the root elongation zone are presented in Figure 2. When the AF...

Watch this Scientific Journal Video about Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots at JoVE.com

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

This method allows to quantify changes in the physical properties of the plant cell wall during development and relate this microscopic changes to the growth of an entire organ. The main advantage of this technique is that it's not invasive and it does not require any treatment that allows the in vivo quantification of physical properties of the cell wall add subcellular resolution relatively rapid. To begin, spread a thin layer of silicone glue into the Petri dish with a cover slip and leave it in the air for 45 seconds.Using tweezers, place the seedling on the glue and orient its direction to avoid contact between the seedling's protruding parts, and the cantilever. Then press the root gently to the silicone glue layer to bind it firmly. Leave it for 45 seconds, and add the 1X PBS solution.Mount the standard silicon nitride cantilever with a pyramidal tip into the AFM probe holder for fluid, and align the laser on the cantilever close to the position of the tip. Then move the photo diode to place the laser spot at the center of the detector. Calibrate the deflection sensitivity by performing an indentation with a ramp size of 3 micrometers, an indentation and retraction rate of 0.6 micrometers per second and a trigger threshold of 0.5 volts.Ensure that the probe is not interacting with the sample and calibrate the cantilever's spring constant. Using the thermal tune utility, click on calibrate followed by thermal tune or on the thermal tune icon in the nano scope toolbar and enter the cantilever temperature. After selecting a frequency range, click on the sample harmonic oscillator fluid button.Next, click on acquired data in the thermal tune panel. Now, adjust median filter width to three. Adjust PSD binwidth to reduce the noise in the acquired data by averaging and set fit boundaries around the first resonance peak.Click on calculate spring constant K, and then click on yes in the pop-up window, asking if the user wants to use this value. Using the inverted optical microscope at 10, 20 and 40 times eye piece magnification, position the AFM probe on the surface of the fourth elongated epidermal cell of the primary root ensuring to position it in the center of the cell. With the previously calculated spring constant value obtained force curves with a ramp size of 3 micrometers, a trigger threshold of 11 nanonewton, and an indentation and retraction rate of 0.6 micrometers per second at selected points.Obtain force curves from three cells per root for each treatment and capture at least 150 force curves for each root. This plot shows an expected result when a force indentation experiment is conducted on live samples positioned at the center of the cell of the root elongation zone. When the AFM tip starts to indent, the surface of the cell wall, the force begins to increase because of the cell wall opposition to the defamation.The increase of force continues until the maximum force value is reached. After this point, the unloading part of the indentation begins. The force grows following a parabola in the indentation part, which is important to fit each curve to the prediction model for pyramidal indenters used for calculations.As a fitting parameter, the contact point position should correspond to the cell wall surface before indentation and is considered the origin of the AFM tip displacement. Force curves in which it is impossible to detect the point of contact before the indentation should be discarded. Further, the loading and unloading curve of the force indentation experiment must be devoid of noise.After fitting each force curve to the model, histograms were obtained that showed the distribution of the frequency of the obtained values of the apparent Young's modulus. This histogram shows the frequency obtained with a set of 201 successful indentations on nine different cells of three different plants of Columbia Zero grown in control conditions. Indentation could be difficult for some genotypes due to the morphology of the root.For example, the TTL 1 PRC 1-1 double mutant grown in severe osmotic stress. These histograms show the probability distribution of the obtained values of the apparent Young's modulus from nine different cells. Only histogram H that corresponds to one cell could be fitted to a Gaussian distribution.The metal presented here could be combined with other techniques such as grow rate studies or analysis of the chemical composition of the cell wall. These other techniques will further complement information to understand how the chemical composition on the physical properties of the cell wall affect the growth of an organ.

atomic-force-microscopy-to-study-physical-properties-epidermal-cells

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...