We would like to thank Gaby Martins and the organizers of the EMBO2010 3D Developmental Imaging workshop where this work began and all of the contributors to the Fiji and ImageJ projects.

1.&#160;4D Time-lapse Imaging Experiments
The settings used for image acquisition will differ depending on the equipment used. The ability of confocal microscopy to optically section a sample depends on a number of factors: the wavelength of excitation, pinhole size, numerical aperture of the objective, the refractive index of the sample and the medium in which the sample is embedded. The size of the confocal pinhole selected will determine the thickness of the optical section collected. A small...

<ol>
	<li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 42-42 (2004).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+Microscopy.">ImageJ for Microscopy.</a> BioTechniques. 43 (S1), 25-30 (2007).</li><li>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE transactions on image processing : a publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Kuglin, C. D., Hines, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+correlation+image+alignment+method.">The phase correlation image alignment method.</a> Proceedings of the IEEE, International Conference on Cybernetics and Society. , 163-165 (1975).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+motility+and+actin+cytoskeleton+of+individual+neurons+and+neural+crest+cells+in+zebrafish+embryos.">Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.</a> J VIs. Exp. (36), (2010).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+extrusion+from+the+epidermis+of+developing+zebrafish.">Live imaging of cell extrusion from the epidermis of developing zebrafish.</a> Journal of Visualized Experiments: JoVE. (52), (2011).</li><li>Benard, E. L., vander Sar, A. M., Ellett, F., Lieschke, G. J., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments: JoVE. (61), (2012).</li><li>Nguyen-Chi, M. 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=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

Our ability to use post-processing software to correct sample drift of datasets derived from extended time-lapse microscopy experiments is restricted by a number of factors. The ability to discern drift versus migratory movement of a sample is dependent on the cellular markers used. Cellular markers that are either widely expressed within a sample or are not involved in migratory events during image acquisition provide the best source for drift correction. The plugin uses a single channel to register the movement between...

Confocal imaging is widely used in cell and developmental biology to follow cell movements and changes in morphology. Capturing a series of optical sections at different focal planes allows the generation of a three-dimensional (3D) model of a sample, which can then be extended into four-dimensions (4D) by creating a time-lapse series of 3D datasets. The generation of 4D datasets allows detailed measurement of cell movements and behaviors. In long-term time-lapse experiments it is common to observe sample movement. This can be caused by slight inaccuracies in the hardware controlling stage and focal positions. While in others cases, drift is a result of movements induced by sample growth or flexibility within the sample mounting media. Methods exist to compensate or limit these movements including improvements to hardware focusing systems and increased rigidity of the mounting medium. However, these approaches cannot be applied in many cases due to the imaging set up required to provide suitable conditions for the samples maintenance and growth. Open source software solutions do exist for the correction of movement in 2D over time, through the use of the StackReg and TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/) 5 plugins in ImageJ or Fiji, but these cannot be applied to 4D datasets.
To correct for the sample drift we have developed a plug-in (Correct 3D drift) to utilize the open-source imaging-processing platform, Fiji 1. Our plug-in is able to perform phase correlation registration to correct movement that occurs as a result of sample drift in three-dimensional time-lapse experiments. Phase correlation 6 is a computational efficient method to determine translation between images. The plug-in described here utilizes the phase-correlation library developed by Preibisch et al.&#160;7. In multi-channel experiments, the plug-in utilizes one channel to determine the required correction. This correction is then applied to any additional channels resulting in registration of the 4D dataset. &#160;
In the zebrafish model system it is possible to carry out time-lapse imaging over a period of many hours, or even several days 8. A common method for mounting the zebrafish is to embed the anaesthetized live embryo in low melting point agarose (0.8-1.5%), restricting its movement 9-11. Whilst movement is restricted growth of the sample still occurs, resulting in the cells within the field of view shifting position. In order to follow movement of the cells within the embryo it is necessary to first correct for movement of the entire sample. This protocol was developed with zebrafish specimens, and has been utilized to image somite development12 but can be applied to any 4D confocal dataset.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

In the developing zebrafish, fast muscle cells fuse into multinucleated fibers from 19 hours post-fertilization (20- somite stage) 13. In order to visualize the movement of nuclei and fusion of muscle cells we carried out 4D confocal time-lapse imaging using a transgenic strain that expresses green fluorescent protein (GFP) under the control of the skeletal &#945;-actin promoter to label all of the muscle cells 14 and injected RNA encoding the red fluorescent protein mCherry tagged with a nuclear lo...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.2K ビュー.  Monash University. タイムラプス顕微鏡は、発生過程の可視化を可能にする。画像取得中に成長または試料のドリフトが正確に従うと開発中の細胞の動きを測定する能力を低下させる。我々は、時間をかけて、三次元試料ドリフトを補正するためのオープンソースの画像処理ソフトウェアの使用を記載している。 

ビデオ: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over...

intravital subcellular microscopy of the mammary gland

Intravital Subcellular Microscopy of the Mammary Gland

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton...

using in vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

preparation of murine submandibular salivary gland for upright intravital microscopy

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

three and four-dimensional visualization and analysis approaches to study vertebrate axial elongation and segmentation

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

visualizing the developing brain in living zebrafish using brainbow and time-lapse confocal imaging

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous...

confocal time lapse imaging as an efficient method for the cytocompatibility evaluation of dental composites

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental...

time-lapse 3d imaging of phagocytosis by mouse macrophages

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic...

time-lapse imaging of mitosis after sirna transfection

Time-lapse Imaging of Mitosis After siRNA Transfection

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

time-lapse imaging of mouse macrophage chemotaxis

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune...

a layered mounting method for extended time-lapse confocal microscopy of whole zebrafish embryos

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different...

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell...

long-term time lapse imaging of mouse cochlear explants

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five...

analyzing craniofacial morphogenesis in zebrafish using 4d confocal microscopy

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant...

time-lapse live imaging and quantification of fast dendritic branch dynamics in developing drosophila neurons

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

a versatile method for mounting arabidopsis leaves for intravital time-lapse imaging

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time...

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...