The studies were conducted at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science.
We are grateful to Orna Yeger for her help in designing and running this protocol.

1. Sample preparation
<ol>
<li>After extracting the tissue which is to be examined, mineralized tissues can be positioned in the instrument and imaged. To image the mouse femur one should follow the following steps:<ol>
			<li>Remove post mortem leg from a C57/Bl6 embryo 18.5 days postceutus (E18.5).</li>
			<li>Seal the narrow end of a polystyrene pipette tip (20-200 &mu;l) using epoxy resin or another glue, and fill the tip with the working buffer (PBS or other).</li>
			<li>Fit tightly the leg into the tip and seal the other end with parafilm sheet.</li>
			<li>Place the pipette tip into a suitable holder and follow the protocol from chapter 3. For visualizing the femur from the mouse leg embryo, the instrument is set 40 KV and 200 &mu;A. For 8&mu; resolution 1000 projection images with 4x magnification have to be acquired.</li></ol></li>
		<li>Non-mineralized tissues have to be initially fixed and stained in order to increase the X-ray attenuation of the tissue of interest by using one of the many available protocols8,9. For the rat lungs and similar samples, the preparation protocol is:<ol>
		<li>Orthotopical implantation of non small cell lung carcinoma (NSCLC) NCI-H460 on nude rat lungs</li>
		<li>Lung cancer nodules start being detectable 4 weeks from implantation</li>
		<li>Sacrifice the rat and immediately infuse it with saline solution mixed with heparin</li>
		<li>Inject it with of a diluted solution of Microfil (Flowtech), (2 ml of compound solution, 3 ml of diluent and 0.3 ml of curing agent) into the left ventricle to stain the bronchial circulation</li>
		<li>Extract the lungs and heart of the rat</li>
		<li>Immobilize the sample (see chapter 2 of the protocol) by fitting it tightly into a 50 ml plastic test tube</li>
		<li>Create a saturated ethanol atmosphere by placing on the bottom of the tube a cloth damped in ethanol</li>
		<li>Glue or screw the tube in a holder of the instrument</li>
		<li>Proceed with setting the imaging parameters (chapter 3). For complete imaging of the rat lungs the source is set at 40KV and 100 &mu;A. In order to reach 16&mu; resolution one has to acquire 2500 projection images at a magnification of 0.5x.</li></ol></li> 
</ol>
2.	Sample immobilization
At high resolution, it is important to avoid any change in the sample position during the measurement. For this, the sample is tightly fixed into a plastic recipient that fits its size. Polystyrene pipette tips, plastic Pasteur pipettes or specially built plastic holders are used in this respect. According to the experimental requirements, the sample can be examined in air or immersed in ethanol or buffer solutions. Typical immobilization and final positioning of the leg of the mouse embryo in the instrument is shown in Fig 1. 
<img src="/files/ftp_upload/2688/2688fig1.jpg" alt="Figure 1"/> Figure 1. Final positioning of the embryonic mouse leg in the micro CT instrument.
3.	Setting acquisition parameters: x-ray voltage and current, CCD exposure time
<ol>
<li>Placed into a holder, the sample is put in the rotation stage of the instrument</li>
<li>A first x-ray image is taken with the voltage and current arbitrarily set.</li>
<li>If the image is too dark, one should increase the number of photons first, so increase a little bit the current. If this is not enough, one should increase slightly the energy of the x-ray photons, i.e. the voltage on the x-ray tube.</li> 
<li>If the image is too bright, one should first decrease the voltage, then the current.</li> 
<li>The brightness of the image can be increased by binning. Binning of 1 takes into account the intensity of each pixel in the image, whereas binning of 2 takes the sum of each matrix of 2x2 pixels. The image will be about 4 times brighter than in the case of binning 1, but will have half the resolution.</li>
<li>After setting the optimum brightness, one has to optimize the exposure time of the camera to compromise between the best contrast on one side and a reasonable duration of the experiment on another side.</li>
<li>The contrast of images, especially of the low absorbing samples, can be improved by using filters, which reduce the photon flux, mainly that of the lower energy photons.</li>
</ol>
4.	Sample positioning 
<ol>
<li>Choose the working magnification. Possible choices are 0.5x,4x,10x,20x and 40x. The field of view decreases with increasing magnification.</li>
<li>Get the optimal resolution and field of view by setting the distances between the x-ray source and the sample and between the sample and the detector. Increasing the source to sample distance decreases the field of view and increases the resolution. Sample to detector distance has the opposite effect.</li>
</ol>
The whole field to be viewed in 3D should be present in the projection image at all angles. One should check this by rotating the sample at different angles and by bringing the sample as close as possible to the rotation axis. For this, one should follow the following steps:
<ol start="3">
<li>Take an image at 0 deg, and then rotate the sample at -20 deg. If the desired volume has shifted laterally, one should correct its position by repositioning the rotation axis.</li> 
<li>After correction, the sample is to be rotated at another angle and the position corrected again, until the field of interest is inside the image at all angles from -90 to 90 deg.</li> 
</ol>
5.	High resolution tomogram
<ol>
<li>During the measurement, the sample is rotated by a small angle at a time and at each angle one projection image is taken. The total number of images is always a compromise between the desired resolution on one side and the time of the measurement and the file size on another side. As shown in Fig.2, every single projection includes all slices in the sample superposed one over another, and therefore cannot reveal the 3D structure of the sample.</li> 
</ol>
<img src="/files/ftp_upload/2688/2688fig2.jpg" alt="Figure 2"/> Figure 2. Projection images of the lung of rat at 0&deg;(A), 45&deg; (B) and 90&deg; (C) rotation angle.
<ol start="2">
<li>Only after taking projection images at least between -90 and 90 deg, one can proceed to the reconstruction of the sample volume. Reconstruction takes between 10 min and 2 hours, depending of the software used and of the number of projections. Again, the final quality of the 3D image is a compromise between the desired resolution and the time one wants to spend and the size of the resulting file. </li>
</ol>
6.	Image scale calibration
The pixel level (value) in a reconstructed image is unique for that image. In order to compare two different images, a unique intensity scale has to be imposed on each image. For this 
<ol>
<li>Run a tomography with a standard phantom using the same experimental conditions as for the sample</li>
<li>Recalibrate the sample image using the values obtained for the phantom. The most common scale is the Hounsfield (or CT) scale. For 4x magnification the background value of 15,000 (for water or PBS) was replaced with 0 and the maximum value of 35,000 for the bone was replaced with the standard Hounsfield value of 3000. Other pixel values resulted from linear interpolation or extrapolation based on those limits.</li>
</ol>
7.	Image processing and analysis
 After obtaining high resolution images, one has to extract relevant information by using image analysis software. The software package to be used has to be designed to work with very large files (up to 20Gb).
8. Representative Results
A representation of a femur from a C57/Bl6 mouse at embryonic day 18.5 (E18.5) - four days after the initiation of the mineralization process is shown in Fig 3. The mineral layers are clearly visible (white), while soft tissues are not visible in this preparation. We took 1000 projection images with a linear magnification of 4x. The final resolution is 8 microns. A careful analysis of the volume rendering shown in Fig.1, shows that the bone volume fraction (the fraction of the bone volume that is occupied by mineralized tissue) is 0.18, and the bone mineral density is 723 mg/cm3. Those values allow us to compare this structure with bones in other stages of development.
<img src="/files/ftp_upload/2688/2688fig3.jpg" alt="Figure 3"/> 
Figure 3. Different representations of a 3D image of a mouse femur embryo. The transversal (cross section) (A), the sagital (medio-lateral) section (B) and a snapshot of the volume rendering (C) are shown. 
Figure 4 shows a 3D image of the lungs of a female nude rat (RNU), 12 weeks old, implanted orthotopically with non small cell lung carcinoma (NSCLC) NCI-H460. 2500 projection images were taken with a linear magnification of 0.5x, ensuring a final resolution of 16 microns. The image shows the Microfil stained blood vessels (down to 20 micron diameter).The image analysis shows that 4 weeks after implantation, multiple cancer nodules are formed. They are covering a significant fraction of the lung volume (17%). Most of the pulmonary staining was found in the peripheral areas of the tumors. Significantly, as shown in the fig 4B, several blood vessels are present also inside the nodules, covering, according to preliminary analysis some 3% of their volume.
<img src="/files/ftp_upload/2688/2688fig4.jpg" alt="Figure 4"/> Figure 4. 3D image of cancer nodules growing in a rat lung. A snapshot of the volume rendering (A) and a section through the volume (B) are shown. The cancer nodules are marked with arrows. 
Movie 1. Volume rendering of the mouse femur in Figure 1. <a href="https://www.jove.com/files/ftp_upload/2688/2688movie1.mpg">Click here to watch the movie.</a>
Movie 2. Volume rendering of the rat lungs in Figure 2. <a href="https://www.jove.com/files/ftp_upload/2688/2688movie2.mpg">Click here to watch the movie.</a>
Movie 3. Serial sections through the lungs. The nodules appear as grey areas in most of the slices. <a href="https://www.jove.com/files/ftp_upload/2688/2688movie3.mpeg">Click here to watch the movie.</a>

<ol>
	<li>Schambach, S. J., Bag, S., Schilling, L., Groden, C., Brockmann, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+micro-CT+in+small+animal+imaging.">Application of micro-CT in small animal imaging.</a> Methods. 50, 2-13 (2010).</li><li>Bauer, J. S., Link, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+osteoporosis+imaging.">Advances in osteoporosis imaging.</a> Eur J Radiol. 71, 440-449 (2009).</li><li>Chappard, D., Retailleau-Gaborit, N., Legrand, E., Basle, M. F., Audran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+insight+bone+measurements+by+histomorphometry+and+microCT.">Comparison insight bone measurements by histomorphometry and microCT.</a> J Bone Miner Res. 20, 1177-1184 (2005).</li><li>Muller, R., Van Campenhout, H., Damme, B. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+human+bone+biopsies:+a+quantitative+structural+comparison+of+histological+sections+and+micro-computed+tomography.">Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography.</a> Bone. 23, 59-66 (1998).</li><li>Mueller, K., Yagel, R., Wheller, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Aliased+3D+Cone-Beam+Reconstruction+Of+Low-Contrast+Objects+With+Algebraic+Methods.">Anti-Aliased 3D Cone-Beam Reconstruction Of Low-Contrast Objects With Algebraic Methods.</a> IEEE Transactions on Medical Imaging. 18, 519-537 (1999).</li><li>Kachelriess, M., Schaller, S., Kalender, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+single-slice+rebinning+in+cone-beam+spiral+CT.">Advanced single-slice rebinning in cone-beam spiral CT.</a> Med Phys. 27, 754-772 (2000).</li><li>Endo, M., Komatsu, S., Kandatsu, S., Yashiro, T., Baba, 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+combination-weighted+Feldkamp-based+reconstruction+algorithm+for+cone-beam+CT.">A combination-weighted Feldkamp-based reconstruction algorithm for cone-beam CT.</a> Phys. Med. Biol. 51, 3953-3965 (2006).</li><li>Marxen, M., Thornton, M. M., Chiarot, C. B., Klement, G., Koprivnikar, J., Sled, J. G., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Med Phys. 31, 305-313 (2004).</li><li>Plourabou&#233;, F., Cloetens, P., Fonta, C., Steyer, A., Lauwers, F., Marc-Vergnes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+high-resolution+vascular+network+imaging.">X-ray high-resolution vascular network imaging.</a> J Microsc. 215, 139-148 (2004).</li></ol>

No conflicts of interest declared.

C57/Bl6 mouse at embryonic day 18.5 (E18.5) is four days after the initiation of the mineralization process. At this stage of development, the future bone is made of many layers of mineralized osteoids, clearly seen in Fig 3. At this point, one should stress that mineralized tissues can be visualized at lower resolution with different instruments which require less sample handling. The present protocol (and the micro CT instrument used in it) besides providing higher resolution, offers the highest flexibility for the user to choose the best geometrical parameters for the measurement.
Results in Fig 4 show that in the orthotopic lung cancer animal models, human non small cell lung cancer can induce recruitment of blood vessels and neovascularization. We consider that the lung tissue was neither moved, nor has its shape changed during the measurement. The user should take special precautions to avoid such changes during a tomography. For some samples, especially for the softer tissue, one has to build special holding devices that immobilize perfectly the sample during the measurement. Unfortunately the presence of high leakages of contrast agent in the surroundings of the tumors prevented reliable quantification of the peripheral blood vessels. As a result the images are tainted by some staining agent especially at the edges, which is clearly present in movies 2 and 3. We could not prevent this spill, but the useful information about the cancer nodules, including their size, shape and the presence of inner blood vessels was not affected. We could clearly conclude that at least for the bronchial circulation which was studied here, peripheral blood supply participates in tumor perfusion, with some perfusion present also inside the tumor.

For image acquisition we have used a MICRO XCT-400 microfocussed X-ray tomographic system produced by Xradia, Concord, USA.
Images were processed and analyzed using ImageJ (NIH, USA), Avizo (VSG, France) and MicroView (General Electric, USA) software packages. Any available image analysis software can be used instead

high resolution 3d imaging of ex-vivo biological samples by micro ct

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

Watch this Scientific Journal Video about High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT at JoVE.com

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

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18.5K vues.  Weizmann Institute of Science. Visualisation de volume non destructifs peuvent être atteints que par des techniques tomographiques, dont le plus efficace est la tomographie aux rayons X micro ordinateur (TAO).

Vidéo: High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

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Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

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Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...