We thank the Biological Imaging Development Center (BIDC) University of California, San Francisco for the use of the Imaris, Imaris XT and Matlab. We thank V. Kharazia for the technical assistance and A.T. Henry, L.K. Floren, L. Daitch for their contributions to the editing of the manuscript. This work was supported by funding from the State of California Medical Research on Alcohol &amp; Substance Abuse through UCSF to SEB, the National Institutes of Health: 1R21DA029966-01 and NIH Fast Track award to screen the MLSMR collection to SEB, UCSF School of Pharmacy (Dean's Office and Clinical Pharmacy) and the School of Medicine (Clinical Pharmacology &amp; Experimental Therapeutics) to CLHK.

1. Three-dimensional Morphometric Analysis of Single-cell Phenotypic Changes
<ol>
	<li>Human embryonic Kidney (HEK293) cells were transfected with hemagglutinin (HA)-tagged corticotropin releasing factor receptor-2 (CRF-R2), a G protein-coupled receptor (GPCR) as described previously4, 5.</li>
	<li>The cells were left untreated (no treatment, NT), stimulated with the CRF-R2 endogenous ligand, corticotropin releasing factor, CRF (1 &mu;M, 30 min), or pretreated with a selective CRF-R2 antagonist, anti-sauvagine 30 (AS-30, 1 &mu;M, 30 min) prior to agonist treatment.</li>
	<li>The cells were then fixed, permeabilized and treated with anti-HA. CRF-R2 was visualized using Alexa 594 nm conjugate anti-mouse (IgG1) antibody. DAPI was used to visualize the nuclei mitotic stage.</li>
	<li>To limit the experimenter subjectivity, the experimental conditions were not known until after the images were acquired and analyzed.</li>
	<li>We acquired images from fixed HEK293 cells using a Plan-apochromat 63x/1.4 oil DIC objective and Zeiss LSM 510 META confocal microscope connected to a Coherent integrated two-photon laser system comprised of a Verdi-V5 laser and a Mira 900-F laser system.</li>
	<li>During the data acquisition process, the cells were compartmentalized both by multispectral sectioning, 488 nm, and 790 nm (~350 nm 2ph Ex.) and z-partitioning (0.5 &mu;m increments) to include data from the nuclear membrane to the outer extracellular receptor extremities.</li>
	<li>The fluorescence data were first processed using Imaris, which allows the visualization and segmentation of 3D microscopy dataset, and a 3D model composed of cubic voxels was created for morphometric analysis. 
	Then, Imaris XT module was utilized to interface Imaris with MATLAB computer program language to determine the spot coordinates of the GPCR extensions. 
	To take into account cellular variability, we obtained and analyzed fluorescence images taken from 22 cells: no treatment (NT) (n = 7), agonist (CRF) treatment (n = 8) and pre-treatment with antagonist (AS-30) prior to agonist treatment (n = 7) (Figure 2).
	<ol>
		<li>The region of Interest (ROI) should include one cell that is not in active mitotic stage and not close to other cells. In this manner, the analysis will include cells with only one nucleus and the receptor extensions are not perturbed by proximity of other cells.</li>
		<li>The cellular 3D structure was first reconstructed from multispectral fluorescence data using Imaris (v.7.1.1).</li>
		<li>Following the algorithm designed by Imaris, first the Surface rendering was utilized to represent the nuclear membrane. Imaris will determine if there is more than one nucleus in the ROI.</li>
		<li>Then the spots-creation algorithm was utilized to locate the CRF-R2 extension. Spots detection was utilized because it compensates for background noise and irregular intensity of the complex network of amorphous-shaped cells.</li>
		<li>To maximize the inclusion of each unit of fluorescence detection of CRF-R2, the spots' diameter was set to 0.2 &mu;m, which is the smallest unit within the image to extrapolate distinct information in the form of a measured intensity using a Gaussian filter. Spot filtering was incorporated in the spots automated creation process. The software, however, gives the user the flexibility to use filters to define the parameters.</li>
		<li>To avoid data truncation, the data set was converted from 8-bit (unsigned) fixed point, to 32-bit decimal.</li>
		<li>The voxel intensities data were exchanged to spot coordinates data using Imaris XT module interfaced with MATLAB and the exact spatial location of each spot was determined by performing a distance transformation using the nuclear membrane as a reference point (Figure 3).</li>
	</ol>
	</li>
	<li>The resulting data can be quantified and presented in graphical format for statistical analysis. Comparisons between groups were performed using Two-way ANOVA and Bonferroni posttest. Data are presented as mean &plusmn; SD. Differences are considered significant at *p &lt; 0.05. Calculations were made with GraphPad Prism 5.02 (Figure 4).</li>
</ol>
2. Representative Results
To demonstrate the power of our approach, we quantified the cellular changes that result from the interaction of G protein-coupled receptors (GPCRs) and corticotropin releasing factor receptor-2 (CRF-R2) with its endogenous ligand CRF in transfected HEK293 cells.
We show that CRF-R2 receptors are located in the plasma membrane and project from finite regions of the membrane of the cells (Figure 2A and Movie 1). Using conventional 2D analysis, it is possible to detect this subset of extracellular CRF-R2 receptors only if we analyze the receptor adhesion points on the glass covers. Consequently we lose any other information derived from z-stacked multispectral data (Figure 5).
When the cells are treated with CRF, the extracellular receptors are greatly reduced, as shown by the decrease in distance of the spots from the plasma membrane. They are also redistributed from primarily finite locations into a number of discrete locations (Figure 2B and Movie 2).
The effect of CRF on receptor membrane distribution is prevented by pretreatment with the CRF-R2 specific antagonist, antisavagine 30 (AS-30) and we find that the CRF-R2 extensions do not change (Figure 2C and Movie 3).
The distal distribution of spots, plotted in the 5 &mu;m spectrum-color coded intervals, is utilized to visualize the distance of the voxels from the nuclear membrane. No treatment and antagonist pretreatment (AS-30, 1 &mu;M, 30 min) before agonist treatment (CRF, 1 &mu;M, 30 min) show no significant (n.s.) difference in GPCR contraction. Treatment of the cells with the agonist (CRF, 1 &mu;M, 30 min) progressively reduces the number of CRF-R2-containing voxels when compared to no treatment, 0-5 &mu;m (n.s.), 6-15 &mu;m (**p&lt;0.01) and &gt;15 &mu;m (***p&lt; 0.005), or compared to AS-30 treatment, 0-10 &mu;m (n.s.), 11-15 &mu;m (**p&lt;0.01) and &gt;15 &mu;m (***p&lt; 0.005) (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/4233/4233fig1.jpg" /> 
Figure 1 . Diagram of the current available techniques and their limitation to analyze fluorescence images. <a href="https://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4233/4233fig2.jpg" /> 
Figure 2. 3D multispectral fluorescence images of HEK293 transfected cells with HA-CRF-R2 before and after agonist and antagonist treatment. Merged images showing HA-CRF-R2-probed using anti-HA and visualized using Alexa 488 conjugated anti-mouse (IgG1) secondary antibody; DAPI was used to visualize the nuclei. The images were acquired with confocal laser scanning (CLS) microscope. Scale bar 5 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/4233/4233fig3.jpg" /> 
Figure 3. 3D model of HEK293 transfected cells with HA-CRF-R2 reconstructed from CLS images using Imaris software. Surface rendering of the nucleus and spots creation describing GPCR extensions converted to small vesicles. The fluorescence data were first processed using Imaris which allows the visualization and segmentation of 3D microscopy data set. Then, Imaris XT was utilized to interface Imaris with MATLAB. The voxel intensities were exchanged into spot coordinates. The spots spectrum-coded color (blue 0-5 &mu;m, green, 6-10 &mu;m, yellow 11-15 &mu;m and red &gt;15 &mu;m) represent the distance form the nuclear membrane. Scale 5 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/4233/4233fig4.jpg" /> 
Figure 4. Graphic representation of the distal distribution of spots plotted in the spectrum-color coded in 5 &mu;m intervals is utilized to visualize the distance of the voxels from the nuclear membrane. No treatment and pretreatment with an antagonist (AS-30, 1 &mu;M, 30 min) before agonist treatment (CRF, 1 &mu;M, 30 min) show no significant (n.s.) difference in GPCR contraction. Treatment of the cells with agonist (CRF, 1 &mu;M, 30 min) progressively reduces the distance of the number of CRF-R2-containing voxels when compared to no treatment 0-5 &mu;m (n.s.), 6-15 &mu;m (**p&lt;0.01) and &gt;15 &mu;m (***p&lt; 0.005); or AS-30 treatment 0-10 &mu;m (n.s.), 11-15 &mu;m (**p&lt;0.01) and &gt;15 &mu;m (***p&lt; 0.005).
<img alt="Figure 5" src="/files/ftp_upload/4233/4233fig5.jpg" /> 
Figure 5. Limitation of the 2D morphometric analysis of HEK 293 transfected cells with HA-CRF-R2. The mid-plane section of cells (3-4&mu;m above the glass coverslip) showing the center of the nuclei visualized with DAPI and HA-CRF-R2 probed using anti-HA and visualized using Alexa 488nm conjugated anti-mouse (IgG1) secondary antibody and acquired with CLS shows no difference between (A) no treatment (NT) and (B) agonist (CRF, 1 &mu;M, 30 min), while the receptor adhesion points are dramatically different.
Movie 1. Freely rotating 3D model in the "Surpass" mode of HEK 293 transfected cells with HA-CRF-R2, no treatment, to evaluate cellular phenotype differences among receptor proteins. Scale bar from 5 to 20 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4233/4233movie1.avi" target="_blank"> Click here to view movie.</a>
Movie 2. Freely rotating 3D model in the "Surpass" mode of HEK 293 transfected cells with HA-CRF-R2, agonist treatment, CRF (CRF, 1 &mu;M, 30 min) to evaluate cellular phenotype differences among receptor proteins. Scale bar from 5 to 20 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank"> Click here to view movie.</a>
Movie 3. Freely rotating 3D in the "Surpass" mode of HEK 293 transfected cells with HA-CRF-R2, pretreatment with an antagonist (AS-30, 1 &mu;M, 30 min) before agonist treatment (CRF, 1 &mu;M, 30 min) to evaluate cellular phenotype differences among receptor proteins. Scale bar from 5 to 20 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank"> Click here to view movie.</a>

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No conflicts of interest declared.

We showed that CRF treatment induced a significant change in the morphology and location of CRF-R2. The change in CRF-R2 was inhibited by selective antagonist treatment. We showed that receptor modifications were not detected and cannot be measured using the standard 2D multispectral techniques. The ability to study complex 3D images is critical to incorporate the complexity of biological parameters for morphometric analysis. We were able to make 3D measurements of cells without a pre-defined shape with inconsistent fluo...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments (optional) </td>
 </tr>
 <tr>
 <td>Human Embryonic Kidney (HEK293) </td>
 <td>American Type Culture Collection</td>
 <td>CRL-1573 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Invitrogen</td>
 <td>11965118 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Invitrogen</td>
 <td>SH30070.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AlexaFluor-488 (IgG2b) </td>
 <td>Invitrogen</td>
 <td>A-11001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>monoclonal anti-HA.11 (IgG1)</td>
 <td>Covance</td>
 <td>16B12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Vector Laboratories</td>
 <td>H-1200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CRF</td>
 <td>Sigma</td>
 <td>C2917</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antisauvagine-30 (AS-30)</td>
 <td>Sigma</td>
 <td>A4727 </td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

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JoVE Journal

Biology

14.5K 조회수.  University of California, San Francisco. 우리는 단일 세포의 세 차원 공 촛점 형광에서 가져 정의되지 않은 형태의 형태학의 변화를 측정하기 Imaris 신경 과학, ImarisXT 및 MATLAB을 사용하는 소프트웨어 플랫폼을 개발했습니다. 이 새로운 접근 방식은 수용체 활성화에 따라 세포 모양의 변화를 수량화하는 데 사용되므로 약물 발견을위한 가능한 추가 도구를 나타냅니다 할 수 있습니다.

비디오: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...