The authors would like to acknowledge Caitlin Kowalewski for aiding in the editing and formatting of this publication.

1. Schlieren Photography
<ol>
	<li>Establishment of the Test Region 
	Note: The test region exists directly in front of the mirror.
	<ol>
		<li>Clamp a spherical concave mirror (150 mm diameter, focal length 1,500 mm) in a ring stand clamp large enough to support the mirror. Attach the ring stand clamp with the mirror to a ring stand perpendicular to the floor. The current study used a 3 foot ring stand, but any height can be used as long as it is tall enough to be able to center the mirror in the viewing window of the source.</li>
		<li>Place the ring stand and mirror to the side of the mass spectrometer source. Make the face of the mirror parallel to, and at the same height, as the source.</li>
		<li>Position the mirror so its center is aligned with the center source region of the mass spectrometer. Some overlap of the instrument will occur.</li>
	</ol>
	</li>
	<li>Cutoff, Camera and Light Source
	<ol>
		<li>Cutoff
		<ol>
			<li>Attach a metal plate to the top of the tripod. The plate will serve as a platform to hold both the razor blade and the light source. The razor blade acts as what is known as the &#34;cutoff&#34; in schlieren photography.</li>
			<li>Attach the razor blade to the metal plate using a magnet so that the sharp edge is vertical.</li>
			<li>Place the tripod in line with the mirror at twice the focal length of the mirror, 3,000 mm. Align the razor blade orthogonal to the path of light reflected from the mirror.</li>
			<li>Manually adjust the height of the tripod so that the sharp edge of the razor blade is approximately aligned with the center of the mirror. 
			NOTE: Fine adjustment will happen later.</li>
		</ol>
		</li>
		<li>Camera
		<ol>
			<li>Mount a digital camera with 300 mm telephoto lens on a separate tripod.</li>
			<li>Position the camera so the lens (when at full zoom) is 4 cm directly behind and at the same height as the razor blade. Do not remove the lens cap at this time.</li>
		</ol>
		</li>
		<li>Optional Monitor
		<ol>
			<li>Connect the video output of the camera to a computer monitor or TV to easily view the schlieren phenomenon in real time. 
			NOTE: This is a recommended process. This procedure may vary depending on the type of camera used.</li>
		</ol>
		</li>
		<li>Pinhole Light Source
		<ol>
			<li>Drill a small hole (approximately 0.6 mm in diameter) into the center of a cover (in this case, a vial cap the same diameter of the flashlight was used) which can be attached/taped to the light source. Ensure that the cover has sufficient diameter to completely cover the flashlight lens.</li>
			<li>Attach the cover over a 200 lumen LED flashlight using foil tape. 
			NOTE: The flashlight will get warm and a high temperature tape is recommended.</li>
		</ol>
		</li>
		<li>Light Source Positioning
		<ol>
			<li>First use a laser pointer to align the light source with the mirror, razor blade, and camera, to ensure proper positioning of the light source.</li>
			<li>Place the laser pointer on the metal plate next to the razor blade.</li>
			<li>Manually move the laser pointer so the beam is hitting the center of the mirror. Adjust as necessary to ensure the reflected beam intersects orthogonally to the razor blade so that approximately half of the beam is blocked.</li>
			<li>Manually adjust the mirror&#39;s position to aim the beam of the laser pointer directly at the razor blade if the beam alignment was not achieved in 1.2.5.3. 
			CAUTION! Do not look directly into the laser pointer or the reflected beam.</li>
			<li>Ensure that the laser beam is centered on the lens while keeping the lens cap on the camera.</li>
			<li>Replace the laser pointer with the covered flashlight while everything is aligned. Ensure that the flashlight is in the same orientation as the laser pointer.</li>
			<li>Turn on the flashlight and, using a piece of white paper, observe the reflected light at the cutoff. Ensure that the beam is a small focused spot at the cutoff.</li>
			<li>Make any vertical adjustments necessary to block approximately half of the reflected light beam with the cutoff.</li>
			<li>Remove the lens cap on the camera and focus on the mirror. 
			NOTE: It is recommended that the camera/lens be used in manual focusing mode.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Example Test Object: Mass Spectrometry Ionization Source
<ol>
	<li>Manually align the mass spectrometry ion source within the test region, with a distance of 10 mm between the end of the nozzle and the inlet of the mass spectrometer.</li>
	<li>Manually open the needle valve to the ambient source allowing nitrogen to flow through the source.</li>
	<li>Open the software used to control the mass spectrometer. For this study, the software used was &#34;SQ driver&#34;. Click on file -open- then select the appropriate tune file.</li>
	<li>Apply all voltages and temperatures to the ambient source once the manual tune is opened. Each mass spectrometer will have its own software for this step. For the current study, once the manual tune is open, click the button &#34;Source Voltage is off&#34; and the button &#34;All gas and heaters are off&#34; to perform this task.</li>
	<li>Observe the appearance of the flow exiting the nozzle with the schlieren apparatus on the view screen of the digital camera as the temperature increases. Observe the gas stream (see description in &#34;Results&#34; section) coming out of the end of nozzle. The gas stream can be viewed on the back of the camera, or it can be viewed directly on a LCD monitor.</li>
	<li>Collect the image by either recording a video from the camera, or taking a picture of the gas stream, once desired images are visualized live on the camera.</li>
	<li>Transfer the picture(s) collected to a computer with the camera memory card or USB connection and view the image with software of your choosing.</li>
</ol>
3. Determination of Spray Half Angle from a Collected Image
<ol>
	<li>Open the collected image using an image viewing software and print out the collected image(s).</li>
	<li>Draw a line on the printed image(s) defining the center axis of the gas stream parallel to the direction of flow using a ruler.</li>
	<li>Draw a line along the edge of the visualized gas stream on the printed image(s) using a ruler. This can be visualized better from a recorded video due to a shimmer that is present in video format; use this to help identify the edge in the printed images. Mark the outer edges of the gas streams to obtain a range for the spray half angle.</li>
	<li>Measure the angle produced between the center axis and the line drawn in 3.2 using a protractor.</li>
</ol>

<ol>
	<li>Settles, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Schlieren and Shadowgraph Techniques: Visualization Phenomena in Transparent Media. , (2001).</li><li>Strawa, A. W., Chapman, G. T., Arnold, J. O., Canning, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+range+and+aerothermodynamic+testing.">Ballistic range and aerothermodynamic testing.</a> J. Aircraft. 28 (7), 443-449 (1991).</li><li>Settles, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gas+leaks+by+using+schlieren+optics.">Imaging gas leaks by using schlieren optics.</a> Pipeline &amp; Gas Journal. 226 (9), 28-30 (1999).</li><li>Takagi, T., Kubota, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+schlieren+optics+for+detection+of+protein+bands+and+other+phenomena+in+polyacrylamide+gel+electrophoresis.">The application of schlieren optics for detection of protein bands and other phenomena in polyacrylamide gel electrophoresis.</a> Electrophoresis. 11 (5), 361-366 (1990).</li><li>Clark, I. G., Cruz, J. R., Huges, M. F., Ware, J. S., Madlangbayan, A., Braun, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerodynamic+and+Aeroelastic+Characteristics+of+a+Tension+Cone+Inflatable+Aerodynamic+Decelerator.">Aerodynamic and Aeroelastic Characteristics of a Tension Cone Inflatable Aerodynamic Decelerator.</a> , (2009).</li><li>Froome, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Refractive+Indices+of+Water+Vapour,+Air,+Oxygen,+Nitrogen+and+Argon+at+72+kMc/s.">The Refractive Indices of Water Vapour, Air, Oxygen, Nitrogen and Argon at 72 kMc/s.</a> Proc. Phys. Soc. B. 68, 833-835 (1955).</li><li>Winter, G. T., Wilhide, J. A., LaCourse, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Direct+Sample+Analysis+(DSA)+Ambient+Ionization.">Characterization of a Direct Sample Analysis (DSA) Ambient Ionization.</a> J. Am. Soc. Mass Spectrom. 26 (9), 1502-1507 (2015).</li><li>Pfeuffer, K. P., Schaper, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halo-Shaped+Flowing+Atmospheric+Pressure+Afterglow:+A+Heavenly+Design+for+Simplified+Sample+Introduction+and+Improved+Ionization+in+Ambient+Mass+Spectrometry.">Halo-Shaped Flowing Atmospheric Pressure Afterglow: A Heavenly Design for Simplified Sample Introduction and Improved Ionization in Ambient Mass Spectrometry.</a> Anal. Chem. , 7512-7518 (2013).</li><li>Pfeuffer, K. P., Shelley, J. T., Ray, S. J., Hieftje, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Mass+Transport+and+Heat+Transfer+in+the+FAPA+Ambient+Ionization+Source.">Visualization of Mass Transport and Heat Transfer in the FAPA Ambient Ionization Source.</a> J. Anal. At. Spectrom. 28 (379-387), 379-387 (2013).</li><li>Pfeuffer, K. P., Ray, S. J., Hieftje, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+Visualization+of+Mass+Transport+for+the+Flowing+Atmospheric+Pressure+Afterglow+(FAPA)+Ambient+Mass-Spectrometry+Source.">Measurement and Visualization of Mass Transport for the Flowing Atmospheric Pressure Afterglow (FAPA) Ambient Mass-Spectrometry Source.</a> J. Am. Soc. Mass Spectrom. 25 (5), 800-808 (2014).</li><li>Keelor, J. D., Dwivedi, P., Fern&#225;ndez, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Effective+Approach+for+Coupling+Direct+Analysis+in+Real+Time+with+Atmospheric+Pressure+Drift+Tube+Ion+Mobility+Spectrometry.">An Effective Approach for Coupling Direct Analysis in Real Time with Atmospheric Pressure Drift Tube Ion Mobility Spectrometry.</a> J. Am. Soc. Mass Spectrom. 25 (9), 1538-1548 (2014).</li></ol>

There are no competing financial interests with this article.

There are several considerations which must be addressed prior to attempting this protocol. In addition to the space around the mass spectrometer for the source and mirror, enough open space must be available to accommodate the distance of twice the focal point of the mirror. Furthermore, the size of the mirror is ultimately decided by the size of the source that is under study. If the mirror is too small, the source will not be fully visualized. It is important to note that some, if not all, of the source covers must be...

Mass Spectrometry, an analytical tool available for molecular mass identification, has become one of the most powerful analytical techniques to date. Over the last decade a whole host of new ambient ionization sources have become available for mass spectrometry detection. For the data collected in this manuscript, the Direct Sample Analysis (DSA) source was utilized. Although these sources are extremely versatile, a more detailed knowledge of the physical ionization process is needed for its optimization and extension of purpose. The aim of this experiment is to gain a better understanding of the ionization process within the ambient sources through visualization of the nitrogen stream on the device using a technique called schlieren photography.
Scientific study often initiates through observation, which is difficult if the object of study is transparent to the naked eye. Schlieren photography is a technique that allows the invisible to become visible through relying on changes in the refractive index within transparent media1. The inhomogeneity of the refractive indices causes a distortion of the light allowing for visualization. The schlieren technique has been routinely used in a variety of specialty fields including ballistics modeling, aerospace engineering, general gas detection and flow monitoring, and at times to visualize protein bands in gel electrophoresis2-5.
Most ambient ionization sources use a stream of gas in order to facilitate the ionization. A wide range of conditions can exist for source options, however the parameters of this experiment must involve the utilization of a gas with a refractive index that differs from the surrounding lab air. This specific study utilizes hot nitrogen. It should be noted that only a small difference in refractive index is observed between pure nitrogen from the gas stream and air at RT6, mainly because air is composed mostly of nitrogen. This issue is overcome in this instance due to the high temperatures of the pure nitrogen in the gas stream which produces a significant enough change in refractive index for the gas to be observed.
Other mass spectrometry sources such as a Desorption Atmospheric Chemical Ionization (DAPCI)7, Flowing Atmospheric Pressure Afterglow (FAPA)8-10, and Direct Analysis in Real Time (DART)11 ionization sources have used schlieren photography. The intention of this protocol is to discuss how to study ambient ionization using a basic schlieren photography configuration. This technique, however, is applicable to any number of different analytical techniques that involve gaseous streams.

<table><tbody><tr><td>Flashlight</td><td>EAGTAC</td><td>D25A Ti</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Spherical Concave Mirror</td><td>Anchor Optics</td><td>27633</td><td></td></tr><tr><td>Rebel EOS T2i</td><td>Canon</td><td>4462B001</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>300 mm telephoto lens</td><td>Canon</td><td>6473A003</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Direct Sample Analysis (DSA) Ionization Source</td><td>PerkinElmer</td><td>MZ300560</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Sq 300 MS with SQ Driver Software</td><td>PerkinElmer</td><td>N2910801</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Ring Stand</td><td>Fisher Scientific</td><td>11-474-207</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Laser Pointer</td><td>Apollo</td><td>MP1200</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>razor blade</td><td>Blue Hawk</td><td>34112</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>small drill bit #73</td><td>CML Supply</td><td>503-273</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Protractor</td><td>Sterling&amp;nbsp;</td><td>582</td><td>or equivalent&amp;nbsp;</td></tr><tr><td>Hose Clamp</td><td>Trident</td><td>720-6000L</td><td>or equivalent&amp;nbsp;</td></tr></tbody></table>

visualization of ambient mass spectrometry with the use of schlieren photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

A schematic of the schlieren setup including the mass spectrometry ionization source can be found in Figure 1. When all schlieren components are properly aligned, gases within the test region can be seen as contrasting dark and light regions. Figure 2 illustrates how this contrast can be used to observe how the shape of the nitrogen jet flow from the mass spectrometry source changes as nozzle size decreases.
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Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

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Chemistry

8.2K Views.  University of Maryland, Baltimore County. This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

Video: Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation

Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy and dynamics1-4. Fundamental studies of photoionization processes of biomolecules provide information about the electronic structure of these systems. Furthermore determinations of ionization energies and other properties of biomolecules in the gas phase are not trivial, and these experiments provide a platform to generate these data. We have developed a thermal vaporization technique coupled with supersonic molecular beams that provides a gentle way to transport these species into the gas phase. Judicious combination of source gas and temperature allows for formation of dimers and higher clusters of the DNA bases. The focus of this particular work is on the effects of non-covalent interactions, i.e., hydrogen bonding, stacking, and electrostatic interactions, on the ionization energies and proton transfer of individual biomolecules, their complexes and upon micro-hydration by water1, 5-9.
We have performed experimental and theoretical characterization of the photoionization dynamics of gas-phase uracil and 1,3-dimethyluracil dimers using molecular beams coupled with synchrotron radiation at the Chemical Dynamics Beamline10 located at the Advanced Light Source and the experimental details are visualized here. This allowed us to observe the proton transfer in 1,3-dimethyluracil dimers, a system with pi stacking geometry and with no hydrogen bonds1. Molecular beams provide a very convenient and efficient way to isolate the sample of interest from environmental perturbations which in return allows accurate comparison with electronic structure calculations11, 12. By tuning the photon energy from the synchrotron, a photoionization efficiency (PIE) curve can be plotted which informs us about the cationic electronic states. These values can then be compared to theoretical models and calculations and in turn, explain in detail the electronic structure and dynamics of the investigated species 1, 3.

Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet VUV Synchrotron Radiation

A molecular beam coupled to tunable vacuum ultraviolet photoionization mass spectrometer at a synchrotron provides a convenient tool to explore the electronic structure of isolated gas phase molecules and clusters. Proton transfer mechanisms in DNA base dimers were elucidated with this technique.

Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy ...

Engineering

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Bioengineering

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

Biochemistry

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Biology

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Schlieren Imaging: A Technique to Visualize Supersonic Flow Features

Source: Jose Roberto Moreto, Jaime Dorado, and Xiaofeng Liu, Department of Aerospace Engineering, San Diego State University, San Diego, CA
Military jet fighters and projectiles can fly at incredible speeds that exceed the speed of sound, which means they are traveling at a supersonic speed. The speed of sound is the speed at which a sound wave propagates through a medium, which is 343 m/s. Mach numbers are used to gauge the flight speed of an object relative to the speed of sound.
An object traveling at the speed of sound would have a Mach number of 1.0, whereas an object traveling faster than the speed of sound has a Mach number greater than 1.0. The compressibility effects of air must be accounted for when traveling at such speeds. A flow is considered compressible when the Mach number is greater than 0.3. In this demonstration, Mach 2.0 supersonic flow over a cone will be analyzed by visualizing the formation of shock waves and compression waves in compressible flow using a Schlieren system.

Schlieren Imaging: Visualization of Supersonic Flow Features

Source: Jose Roberto Moreto, Jaime Dorado, and Xiaofeng Liu, Department of Aerospace Engineering, San Diego State University, San Diego, CA
Military jet ...

Education

JoVE Science Education

Aeronautical Engineering

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.