The authors would like to thank Florian Gaertner (Institute of Science and Technology Austria, Klosterneuburg, Austria) for his expert advice on two-photon microscopy experiments at the time when we established the technique in the lab, and Yves Lutz at the Imaging Center IGBMC /CBI (Illkirch, France) for his expertise and help with the two-photon microscope. We also thank Jean-Yves Rinkel for his technical help and Ines Guinard for the drawing of the schema in Figure 1. We thank ARMESA (Association de Recherche et D&#233;veloppement en M&#233;decine et Sant&#233; Publique) for its support in the acquisition of the two-photon microscope. AB was supported by post-doctoral fellowships from Etablissement Fran&#231;ais du Sang (APR2016) and from Agence Nationale de la Recherche (ANR-18-CE14-0037-01).

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S., 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=Membrane+budding+is+a+major+mechanism+of+in+vivo+platelet+biogenesis.">Membrane budding is a major mechanism of in vivo platelet biogenesis.</a> Journal of Experimental Medicine. 217 (9), 20191206 (2020).</li><li>Scott, M. K., Akinduro, O., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+4-dimensional+tracking+of+hematopoietic+stem+and+progenitor+cells+in+adult+mouse+calvarial+bone+marrow.">In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.</a> Journal of Visualized Experiments: JoVE. (91), e51683 (2014).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li><li>Tiedt, R., Schomber, T., Hao-Shen, H., Skoda, R. 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D., 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=Lineage+tracing+of+Pf4-Cre+marks+hematopoietic+stem+cells+and+their+progeny.">Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny.</a> PLoS One. 7 (12), 51361 (2012).</li><li>Stegner, D., 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=Thrombopoiesis+is+spatially+regulated+by+the+bone+marrow+vasculature.">Thrombopoiesis is spatially regulated by the bone marrow vasculature.</a> Nature Communications. 8 (1), 127 (2017).</li><li>Drew, P. J., Blinder, P., Cauwenberghs, G., Shih, A. Y., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+particle+velocity+from+space-time+images+using+the+Radon+transform.">Rapid determination of particle velocity from space-time images using the Radon transform.</a> Journal of Computational Neuroscience. 29 (1-2), 5-11 (2010).</li><li>Nakagawa, 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=Ketamine+suppresses+platelet+aggregation+possibly+by+suppressed+inositol+triphosphate+formation+and+subsequent+suppression+of+cytosolic+calcium+increase.">Ketamine suppresses platelet aggregation possibly by suppressed inositol triphosphate formation and subsequent suppression of cytosolic calcium increase.</a> Anesthesiology. 96 (5), 1147-1152 (2002).</li><li>Kim, S., Lin, L., Brown, G. A. J., Hosaka, K., Scott, E. 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(113), e542205 (2016).</li><li>Guesmi, 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=Dual-color+deep-tissue+three-photon+microscopy+with+a+multiband+infrared+laser.">Dual-color deep-tissue three-photon microscopy with a multiband infrared laser.</a> Light Science &amp; Applications. 7, 12 (2018).</li><li>Wang, 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=Three-photon+imaging+of+mouse+brain+structure+and+function+through+the+intact+skull.">Three-photon imaging of mouse brain structure and function through the intact skull.</a> Nature Methods. 15 (10), 789-792 (2018).</li><li>Kim, J., Bixel, M. 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The authors have no conflicts of interest to declare.

The mechanisms of platelet formation are highly dependent on the bone marrow environment. Hence, intravital microscopy has become an important tool in the field to visualize the process in real-time. Mice with fluorescent megakaryocytes can be obtained by crossing mice expressing the Cre recombinase in megakaryocytes with any floxed reporter mice containing a conditional fluorescent gene expression cassette. Here, mTmG reporter mice were used (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo10) crossed with Pf4-cre mice11, allowing intense green fluorescence labeling in megakaryocytes and platelets12. It is important to note that some leakage has been reported with Pf4-cre mice in leukocytes, including monocytes/macrophages and fibroblasts12,13.
However, megakaryocytes are easily recognized by their large size and nucleus. Experimenters new to the field could compare observations in [mTmG; Pf4-cre] mice with those obtained after megakaryocyte-specific labeling. This is possible through intravenous administration of an Alexa-fluor-labeled antibody directed against the platelet-specific protein, GPIX (CD42a)3,14. This latter approach is also beneficial for imaging fluorescent megakaryocytes from any genetically engineered mouse without performing tedious and time-consuming mouse crossings with fluorescent reporter mice. Here, 5-7-week-old mice were used as younger mice present with a thinner bone that is more translucent and facilitates imaging compared to older mice. However, mice that are too young may present difficulties in the installation of the cranial ring without leakage.
In this study, intravital imaging was performed using a Leica microscope equipped with a 25x water objective with a numerical aperture of 0.95 (Table of Materials). The objective should be conical so that it can enter the cup formed by the cranial ring with an optimal working distance. Images were recorded with a resonant scanner (12 or 8 kHz). The overall settings will depend on the type of scientific question to be answered and should be optimized depending on the magnification, resolution, and acquisition speed needed. For instance, with a 12 kHz resonant scanner, a 25x objective would be better suited to measure platelet velocity, whereas an 8 kHz scanner and a lower magnification could be sufficient to record proplatelet elongation.
The most critical step of the protocol is the correct attachment of the ring to the skull bone so that no leakage occurs, and so that it does not detach during recording. For that, it is essential that the skull bone be dry just before applying the ring. Once the ring is glued, rapidly re-humidify the bone. Furthermore, due to the force exerted by the weight of the head on the ring, the ring is subjected to a tension that may cause it to detach partially during the recording. This may cause leakage and thus, dryness of the bone, leading to both poor-quality images and undesirable inflammatory reactions. This problem can be easily avoided by adding a folded compress under the nose of the mouse to support the head (Figure 3C). Another critical point is to minimize bleeding and the presence of blood inside the ring as it may lead to blurred images. This must be kept in mind when studying mice that present defective hemostasis and are prone to bleeding. Some tracers, such as dextran, may present leakage in the bone marrow cavity if injected in a concentrated form, which worsens vessel imaging, though not the green fluorescence of megakaryocytes. Qtracker-655 does not leak much but is cleared from the circulation within 30 min so that new injections of the tracer are required to visualize vessels over longer periods.
One limitation of this method described here is the necessity to stop acquisition every 35 min to re-inject anesthetics. This limitation can be overcome if an anesthesia machine is available. In that case, anesthesia can be induced similarly by i.p. injection of a mixture of ketamine and xylazine, which is the easiest way to perform the insertion of the catheter and the minor surgery. Once positioned below the microscope objective, anesthesia is then maintained by inhalation of gases (mixture of oxygen, anesthetics such as isoflurane and ambient air). In this way, observations can be made over several hours without interruption. However, for ethical reasons, observations were limited to 3 h in this study. Another limitation in using ketamine/xylazine mixture could be its potential deleterious impact on platelet functions16 that could require alternative anesthesia schemes.
Overall, the principal merit of this well-established protocol is its noninvasive aspect with only minimal surgery. Other protocols have been developed to perform time-lapse intravital imaging in long bones. These rely on either invasive surgery that requires muscle tissue removal and bone abrasion17,18, implanted endoscopic probes close to the femur head19,20, or installing a window chamber in the femur21. All these procedures result in major trauma and varying degrees of inflammation. Inflammation is not only detrimental to the mouse, but it has also been reported to modify the process of platelet formation6 so that uncontrolled inflammatory conditions might lead to discrepancies and misinterpretation of the data. That is why this procedure was chosen to avoid inflammatory reactions. However, depending on the scientific question, experimenters may prefer to have a deeper observation field and higher resolution (less scattering), which is possible by using a skull-thinning-based approach at the expense of a low degree of inflammatory reaction.
Because of its noninvasive nature, the major limitation of this method is the maximal depth that can be reached. Due to the density of the bone and its scattering properties, it is possible to image regions within a depth of only a few hundred micrometers, preventing observations of the whole calvarial marrow. The development of three-photon microscopy, which relies on even higher infrared excitation wavelengths, seems to be a promising approach with superior deep-tissue resolution. It has already been successfully used to image deep into the brain even through the bone22,23, opening up the exciting possibility to image bone marrow within long bones without having to thin the bone or implant a surgical window.
In summary, this method is increasingly used to study the behavior of megakaryocytes in the bone marrow and visualize the extension of proplatelets.In addition, by allowing the visualization of the outer calvarial compact bone as well as part of the underlying marrow, this method has already been used in many applications outside the platelet field. It has been used for the study of both bone and marrow cell dynamics in their environment, including osteoblasts and osteoclasts, leukocyte trafficking and marrow exit, endothelial cells and microvasculature architecture, blood flow dynamics in marrow microvessels, or cell homing24.

Platelets are produced by megakaryocytes-specialized cells located in the bone marrow. The precise way megakaryocytes release platelets in the circulation has long remained unclear owing to the technical challenge in observing real-time events through the bone. Two-photon microscopy has helped overcome this challenge and led to major advances in understanding the platelet formation process. The first in vivo megakaryocyte observations were made by von Andrian and colleagues in 2007, with the visualization of fluorescent megakaryocytes through the skull1. This was possible because the bone layer in the frontoparietal skull of young adult mice has a thickness of a few tens of microns and is sufficiently transparent to allow visualization of fluorescent cells in the underlying bone marrow2.
Ensuing studies applied this procedure to evaluate proplatelet formation under various conditions and to decipher the underlying mechanisms3,4,5,6. These studies provided definitive evidence that megakaryocytes dynamically extend protrusions, called proplatelets, through the endothelial barrier of the sinusoid vessels (Figure 1). These proplatelets are then released as long fragments that represent several hundred platelets in volume. The platelets will be formed after the remodeling of the proplatelets in the microcirculation of downstream organs, notably in the lungs7. To date, however, the precise process and molecular mechanisms remain subject to debate. For instance, the proposed role of the cytoskeletal proteins in the elongation of proplatelets differs between in vitro and in vivo conditions3, and differences in proplatelet formation have been demonstrated under inflammatory conditions6. Complicating things further, a recent study disputed the proplatelet-driven concept and proposed that in vivo, platelets are essentially formed through a membrane-budding mechanism at the megakaryocyte level8.
This paper presents a protocol for the observation of megakaryocytes and proplatelets in the bone marrow from the skull bone in living mice, using a minimally invasive procedure. Similar approaches have been previously described to visualize other marrow cells, notably hematopoietic stem and progenitor cells9. The focus here is on the observation of megakaryocytes and platelets to detail some parameters that can be measured, notably proplatelet morphologies and platelet velocity. This protocol presents how to insert a catheter into the jugular vein to inject fluorescent tracers and drugs and observe through the skull bone. The calvarial bone is exposed using minor surgery so that a ring is glued to the bone. This ring serves to immobilize the head and prevent movements due to breathing and form a cup filled with saline as the immersion medium of the lens. This technique is well suited to i) observe in real time the sinusoid geometry and megakaryocytes interacting with the vessel wall; ii) follow megakaryocytes in the process of proplatelet formation, elongation, and release; and iii) measure platelet movements to monitor the complex sinusoid blood flow. Data obtained using this protocol have been recently published3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GNU Octave software</td>
			<td>GNU Project</td>
			<td></td>
			<td>https://www.gnu.org/software/octave/</td>
		</tr>
		<tr>
			<td>&#160;Histoacryl 5 x 0, 5 mL</td>
			<td>Braun</td>
			<td>1050052</td>
			<td>injectable solution of surgical glue</td>
		</tr>
		<tr>
			<td>HyD hybrid detectors Leica Microsystems 4tunes</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>GNU project</td>
			<td></td>
			<td>Minimum version required</td>
		</tr>
		<tr>
			<td>Imalgene/Ketamine 1000 fl/10 mL</td>
			<td>Boehring</td>
			<td>03661103003199</td>
			<td>eye protection</td>
		</tr>
		<tr>
			<td>Leica SP8 MP DIVE microscope equipped with a 25x water objective, numerical aperture of 0.95</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>simultaneous excitation of AlexaFluor-488 and Qtracker-655</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>https://www.mathworks.com/</td>
		</tr>
		<tr>
			<td>Ocrygel 10 g</td>
			<td>Laboratoires T.V.M.</td>
			<td>03700454505621</td>
			<td>Silicon dental paste blue and yellow</td>
		</tr>
		<tr>
			<td>Picodent twinsin speed</td>
			<td>Rotec</td>
			<td>13001002</td>
			<td></td>
		</tr>
		<tr>
			<td>Qtracker 655 vascular label</td>
			<td>Invitrogen</td>
			<td>Q21021MP</td>
			<td>injectable solution</td>
		</tr>
		<tr>
			<td>Resonant scanner, 8 or 12 kHz</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun Xylazine 2% fl/25 mL</td>
			<td>Bayer</td>
			<td>04007221032311</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue gel</td>
			<td></td>
			<td></td>
			<td>to glue the ring to the bone</td>
		</tr>
		<tr>
			<td>Surflo IV catheter - Blue 22 G</td>
			<td>Terumo</td>
			<td>SR-OX2225C1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Saph pulsing laser (Coherent) (femtosecond)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon imaging of megakaryocytes and proplatelets in the mouse skull bone marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

Watch this Scientific Journal Video about In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow at JoVE.com

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

in-vivo-two-photon-imaging-megakaryocytes-proplatelets-mouse-skull

Nanyang Technological University

Research

JoVE Journal

Biology

4.1K Views.  Université de Strasbourg. We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy. 

Video: In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

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

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

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.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...