Name: photoelectron imaging of anions illustrated by 310 nm detachment of f&#8722;
Uploaded: 2018-07-27T14:45:00
Description: Watch this Scientific Journal Video about Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of Fâˆ' at JoVE.com

This material is based upon work supported by the National Science Foundation under CHE - 1566157

NOTE: A general experimental protocol is presented here, specific to the WUSTL instrument. Specific instrument settings for the F&#8722; image presented in Figure 4a can be found in Table 1-2.
1. Ion Generation
<ol>
	<li>To generate anions, apply a backing gas or gas mixture (for F&#8722;, 40 psig. of O2) behind the pulsed nozzle and operate the nozzle at 10 Hz.
	<ol>
		<li>Set the nozzle duration on digital dela...

Two factors are particularly critical to the success of the described protocol. The best possible velocity mapping conditions must be determined and more crucially, a sufficient and relatively time invariant yield of the desired anion must be produced. Regarding the VMI focusing steps, steps 5.2 and 5.3 should be repeated in tandem with image analysis to determine the condition which gives the sharpest (narrowest) image features. Fine tuning of the electrode voltages (V5 and V6) is influenced by the size and location of ...

Anion photoelectron imaging1 is a variant on photoelectron spectroscopy and represents a powerful probe of atomic/molecular electronic structure and the interactions between electrons and neutral species. The information obtained is essential in developing the understanding of bound and metastable (electron-molecule scattering resonances) negative ion states, doorway states for chemical reduction, dissociative attachment processes and ion-molecule interactions. Furthermore, the results provide vital tests of high level ab initio theoretical methods, particularly those designed to deal with highly correlated systems and/or non-stationary states.
The technique combines ion production, mass spectrometry and charged particle imaging2,3,4 to sensitively probe electronic (and for small molecules, vibrational) structure. Working with anionic species allows good mass selectivity via time of flight mass spectrometry (TOF-MS). Visible/near ultraviolet (UV) photons are sufficiently energetic to remove the excess electron, allowing the use of table top laser sources. An additional benefit of the use of anions is the ability to photoexcite low-lying, unstable anionic states which represent energy regimes under which the electrons and neutral atoms/molecules strongly interact. The use of velocity mapped imaging5 (VMI) affords uniform detection efficiency, even at low electron kinetic energies, monitors all ejected photoelectrons and simultaneously reveals the magnitude and direction of their velocities.
The experimental results are photoelectron images which contain photoelectron spectra (details of parent anion internal energy distributions and the energies of daughter neutral internal states) and photoelectron angular distributions (related to the electron orbital prior to the detachment). A particularly interesting application of the technique is found in fs time-resolved studies. An initial ultrafast laser pulse (pump) excites to a dissociative anion electronic state, and a second temporally delayed ultrafast pulse (probe) then detaches electrons from the excited anion. The control of the pump-probe time difference follows the evolution of energy states of the system and the changing nature of the orbitals of the system on the timescale of the atomic motion. Examples include the photodissociation of I2&#8722; and other interhalogen species6,7,8,9, the fragmentation and/or electron accommodation in I&#8722;&#183;uracil10,11,12,13, I&#8722;&#183;thymine13,14, I&#8722;&#183;adenine15, I&#8722;&#183;nitromethane16,17 and I&#8722;&#183;acetonitrile17 cluster anions and the revelation of the hitherto unexpectedly long timescale for the production of Cu&#8722; atomic anions after the photoexcitation of CuO2&#8722;18.
Figure 1 shows the Washington University in St. Louis (WUSTL) anion photoelectron imaging spectrometer19. The instrument consists of three differentially pumped regions. Ions are produced in the source chamber which operates at a pressure of 10&#8722;5 Torr and contains a discharge ion source20, and electrostatic ion extraction plate. Ions are separated by mass in a Wiley-McLaren TOF-MS21 (the pressure in the TOF-Tube is 10&#8722;8 Torr). Ion detection and probing takes place in the detection region (pressure of 10&#8722;9 Torr) which contains a VMI lens5 and a charged particle detector. The main components of the instrument are schematically illustrated in Figure 1b where the shaded region represents all the elements contained within the vacuum system. Gas is introduced through the pulsed nozzle into the discharge. To offset the high inlet pressure, the source chamber is maintained under vacuum using an oil-based diffusion pump. The discharge region is illustrated in more detail in Figure 2a. A high potential difference is applied between the electrodes, which are insulated from the face of the nozzle by a series of Teflon spacers. In fact, the Teflon acts as the source of fluorine atoms for the results shown later.
The discharge produces a mixture of anions, cations and neutral species. The ion extraction plate, ion acceleration stack, potential switch and microchannel plate (MCP) detector (Figure 1b) form the 2 m long Wiley McLaren TOF-MS. Ions are extracted by the application of a (negative) voltage pulse to the ion extraction plate and then all ions are accelerated to the same kinetic energy. Variation of the extraction pulse magnitude focuses the arrival time in the VMI lens while the einzel lens reduces the spatial cross section of the ion beam. Anions are re-referenced to ground using a potential switch22, the timing of which acts as a mass discriminator. Anion selection is achieved by synchronizing the arrival of a visible/near uv photon pulse with the arrival time of the anion in the VMI lens. The ion separation and detection regions use oil free turbopumps to protect the imaging detector.
Anions and photons interact to produce photoelectrons throughout the spatial volume of the Steinmetz solid, representing the overlap between the ion and laser beams. The VMI lens (Figure 2b) consists of three open electrodes, the purpose of which is to ensure that all photoelectrons reach the detector and that the momentum space distribution of the photoelectrons is maintained. To achieve this, different voltages are applied to the extractor and repeller such that, regardless of the spatial point of origin, electrons with the same initial velocity vector are detected at the same point on the detector. The detector consists of a set of chevron-matched MCPs which act as electron multipliers. Each channel has a diameter on the order of a few microns, localizing the gain and preserving the initial impact position. A phosphor screen behind the MCPs indicates the position via the amplified electron pulse as a flash of light which is recorded using a charge coupled device (CCD) camera.
The timing and duration of the various voltage pulses required are controlled using a pair of digital delay generators (DDG, Figure 3). The whole experiment is repeated on a shot by shot basis with a repetition rate of 10 Hz. For each shot, several ions and photons interact producing a few detection events per camera frame. Several thousand frames are accumulated into an image. The image center represents the momentum space origin and hence the distance from the center (r) is proportional to the speed of an electron. Angle &#952;, (relative to the photon polarization direction) represents the direction of an electron&#39;s velocity. An image contains the distribution of detection event densities. Thus, it can also be viewed as representing the probability density for detection (at a given point) of an electron. Invoking the Born interpretation of the wave function (&#968;) an image represents |&#968;|2 for the photoelectron23.
The 3D electron probability density is cylindrically symmetric about the polarization of the electric vector (&#949;p) of the radiation with consequent scrambling of information. Reconstruction of the original distribution is achieved mathematically24,25,26,27. The radial distribution (of electrons) in the reconstruction is the momentum (velocity) domain photoelectron spectrum which is converted into the energy domain via application of the appropriate Jacobian transformation.
The anion photoelectron imaging spectrometer (Figure 1) used in these experiments is a custom-built instrument28. The settings in Table 1 and Table 2 for the protocol are specific to this instrument for the production of F&#8722; and imaging of its photoelectron distribution. Several similar versions of the design are used in various research laboratories6,29,30,31,32,33,34,35,36,37,38,39,40,41,42, but no two instruments are exactly alike. Additionally, instrument settings are strongly interdependent and highly sensitive to small changes in conditions and instrument dimensions.

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			<td style="width:244px;">Berkeley Nucleonics Corp.</td>
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			<td style="width:244px;">Berkeley Nucleonics Corp.</td>
			<td style="width:119px;">577-8c</td>
			<td>DDG2</td>
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			<td height="21" style="height:21px;width:216px;">HV Power Supplies</td>
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			<td height="21" style="height:21px;width:216px;">HV Power Supplies</td>
			<td style="width:244px;">Stanford Research Systems</td>
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			<td>V2</td>
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			<td height="21" style="height:21px;width:216px;">HV Power Supplies</td>
			<td style="width:244px;">Stanford Research Systems</td>
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			<td>V5</td>
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			<td height="21" style="height:21px;width:216px;">HV Power Supplies</td>
			<td style="width:244px;">Burle Inc.</td>
			<td style="width:119px;">PF1053</td>
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			<td height="21" style="height:21px;width:216px;">HV Power Supplies</td>
			<td style="width:244px;">Burle Inc.</td>
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			<td>V9,V11</td>
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			<td style="width:244px;">Bertan</td>
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			<td height="21" style="height:21px;width:216px;">HV Pulsers</td>
			<td style="width:244px;">Directed Energy Inc.</td>
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			<td>V2</td>
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			<td height="21" style="height:21px;width:216px;">HV Pulsers</td>
			<td style="width:244px;">Directed Energy Inc.</td>
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			<td>V1</td>
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			<td height="21" style="height:21px;width:216px;">HV Pulsers</td>
			<td style="width:244px;">Directed Energy Inc.</td>
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			<td height="21" style="height:21px;width:216px;">HV Pulsers</td>
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			<td>V3</td>
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			<td height="21" style="height:21px;width:216px;">Pulsed Nozzle Driver</td>
			<td style="width:244px;">Parker Hannifin (General Valve)</td>
			<td style="width:119px;">Iota-One</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Pulsed Nozzle</td>
			<td style="width:244px;">Parker Hannifin (General Valve)</td>
			<td style="width:119px;">Series 9</td>
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			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:244px;">Imperx</td>
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			<td height="21" style="height:21px;width:216px;">Imaging Detector</td>
			<td style="width:244px;">Beam Imaging Systems</td>
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			<td height="21" style="height:21px;width:216px;">2&#215;Rotary Pump</td>
			<td style="width:244px;">Leybold</td>
			<td style="width:119px;">D16B</td>
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			<td height="21" style="height:21px;">Oxygen Gas</td>
			<td>Praxair</td>
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			<td style="width:119px;">INDI-10</td>
			<td></td>
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photoelectron imaging of anions illustrated by 310 nm detachment of f&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

By centroiding43 the data recorded on the 640&#215;480 pixel CCD array of the camera, a grid resolution of 6400&#215;4800 is possible. However, extraction of the spectra and angular distributions involves inverse Abel transformation of the data which requires the image intensity to vary relatively smoothly. As a compromise, the centroided data is &#34;binned&#34; by summing n&#215;n blocks of points. Similar treatment is also necessary for the display of imaging re...

Watch this Scientific Journal Video about Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of Fâˆ' at JoVE.com

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

This method can help answer key questions in the electron scattering molecular spectroscopy and reaction dynamics field, such as the nature of molecular orbitals, molecular electronic structure, vibrational energy levels, and the nature of scattering and auto-detachment resonances. The main advantage of this technique is that it's highly efficient, recording an entire photoelectron spectrum and angular distribution in a single image. To begin generating anions, apply a backing gas or gas mixture behind the pulsed nozzle.Operate the nozzle at 10 hertz. Set the nozzle duration on the digital delay generator Channel A.Then, trigger the pulsed nozzle driver to inject the gas into discharge. Using Channel C on the digital delay generator, apply the high voltage discharge pulse V1.To monitor the anion mass spectrum, first put the instrument into ion mode.Connect the detector voltage divider into the imaging detector microchannel plates. Apply voltage V11 to the detector anode. After this, connect the ion detector voltage divider output to the oscilloscope channel one input.Connect the microchannel plate power supply to the voltage divider, and gradually increase the voltage as outlined in Table 2. In order to separate the anions by time of flight mass spectrometry, set the acceleration stack voltage to V3 as outlined in the text protocol. Use Channel E on the digital delay generator to set the timing and duration for the potential switch high voltage pulse.First, reduce the voltage applied to the ion detector voltage divider to zero. Switch the spectrometer into imaging mode. Then, disconnect the ion detector voltage divider from the microchannel plates.Connect the microchannel plate power supply and the imaging power supply to the imaging high voltage pulse, and connect the imaging high voltage pulse to the imaging microchannel plates. Turn on the HV pulser. After this, apply a permanent voltage to the phosphor screen and the microchannel plates.Connect the fast-photo diode to oscilloscope channel two. Using Channels H and G, externally trigger the ak laser flash lamps and key switch. Adjust the timing of the laser trigger until the photodiode output is close to, but preceding, the ion signal of interest.Next, apply voltage to the imaging repeller and extractor electrodes. Set the camera to long exposure. Using Channel H, adjust the laser trigger timing to maximize the number of electron detection events observed on the PC screen.Use Channel F to set the pulse timing and duration such that the imaging pulse is centered on the arrival time of the photon pulse. To begin, use Channel E to trigger the charge coupled device camera to open at the start of an experimental cycle. Collect several frames with the laser pulse coincident with the anion of interest.Then collect several frames with the laser pulse not coincident with any anion. Subtract the frames collected off coincidence from the frames collected on coincidence. Repeat this process of collecting and subtracting frames to accumulate a background subtracted image.After this, adjust the imaging repeller and extraction electrode voltages. Repeat the frame collection and subtraction process again to generate a new background subtracted image. To begin image collection, switch the camera to centrated collection.Repeat the frame collection and subtraction process at the optimum focusing condition to accumulate a sub-pixel resolution image. Here is a representative image resulting from the photo detachment at fluoride ions at a photon energy of four electron volts. The left half of this image is the experimentally measured image, while the right half is an inverse able transformation of the data displayed at the same resolution.The two concentric circles correspond to the two narrow transitions seen in the photoelectron spectrum. The integrated intensities over all angles for each radial distance from the center are scaled by the appropriate Jacobian transformation to generate the photoelectron spectrum. The two transitions reflect the existence of two low lying electronic states of neutral fluorine.The difference in the transition kinetic energies reveals that the first excited state is just 50 milli electron volts higher than the ground state, the measure of the strength of the spin orbit interaction. The photoelectron angular distributions for each transition show that the electron distribution is polarized perpendicular to the electric vector of the radiation. These angular distributions can be seen to be almost identical when scaled relative to their respective maxima.The optimal focusing condition is seen to be a ratio of 0.700 between the repeller and extractor. Even small alterations to this ratio are seen to be detrimental to the velocity resolution. The photoelectron imaging technique has paved the way for researchers in the field of chemical reaction dynamics, photoelectron spectroscopy, and electron scattering.This allows for a researcher to probe the nature of electron wave functions from both stable ions and from dynamic systems, such as the evolution of a chemical reaction from reactants to products. In addition, interaction between electrons and neutral species can be examined, ranging from the simple atom all the way to the more complex molecular clusters. Remember, working with high-pressured gases, high voltages, and Class 4 laser radiation is hazardous, and precautions must be taken.Be careful to avoid leaks from gas lines and do not exceed recommended pressure ratings. Switch off voltage supplies when exchanging cables. Also, never look directly into the laser beam, and avoid special reflections.These may result in a permanent loss of vision. Appropriate eye protection is required, but will not protect from looking directly into the laser beam.

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In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
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X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb explosion imaging setup in which a hitherto-impractical camera angle is realized. In our imaging technique, diatomic molecules are irradiated with a circularly polarized strong laser pulse. The ejected atomic ions are accelerated perpendicularly to the laser propagation. The ions lying in the laser polarization plane are selected through the use of a mechanical slit and imaged with a high-throughput, 2-dimensional detector installed parallel to the polarization plane. Because a circularly polarized (isotropic) Coulomb exploding pulse is used, the observed angular distribution of the ejected ions directly corresponds to the squared rotational wave function at the time of the pulse irradiation. To create a real-time movie of molecular rotation, the present imaging technique is combined with a femtosecond pump-probe optical setup in which the pump pulses create unidirectionally rotating molecular ensembles. Due to the high image throughput of our detection system, the pump-probe experimental condition can be easily optimized by monitoring a real-time snapshot. As a result, the quality of the observed movie is sufficiently high for visualizing the detailed wave nature of motion. We also note that the present technique can be implemented in existing standard ion imaging setups, offering a new camera angle or viewpoint for the molecular systems without the need for extensive modification.

We present a protocol for creating a real-time movie of a molecular rotational wave packet using a high-resolution Coulomb explosion imaging setup.

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2&#039;,3&#039;-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...