We thank Robert Berger (Philipps-Universit&#228;t Marburg, Germany) for inspiring discussions about the interpretation of our data and molecular chirality in general. We are grateful to Julia Kiedrowski, Alexander Schie&#223;er and Michael Reggelin from TU Darmstadt (Germany), as well as Benjamin Spenger, Manuel Mazenauer and J&#252;rgen Stohner from ZHAW W&#228;denswil (Switzerland) for providing the sample.
The project was supported by the Hessen State Initiative for Scientific and Economic Excellence under the focus ELCH (Electron dynamics of chiral systems) and the Federal Ministry of Education and Research (BMBF). MS acknowledges financial support by the Adolf Messer foundation.

<ol>
	<li>G&#252;bitz, G., Schmid, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+Separation+by+Chromatographic+and+Electromigration+Techniques.+A+Review.">Chiral Separation by Chromatographic and Electromigration Techniques. A Review.</a> Biopharm. Drug Disposition. 22, 291-336 (2001).</li><li>Berova, N., Polaravapu, P. L., Nakanishi, K., Woody, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comprehensive Chiroptical Spectroscopy. , (2012).</li><li>Bijvoet, J. M., Peerdeman, A. F., van Bommel, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+Absolute+Configuration+of+Optically+Active+Compounds+by+means+of+X-rays.">Determination of the Absolute Configuration of Optically Active Compounds by means of X-rays.</a> Nature. 168 (4268), 271-272 (1951).</li><li>Pitzer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Determination+of+Absolute+Molecular+Stereochemistry+in+Gas+Phase+by+Coulomb+Explosion+Imaging.">Direct Determination of Absolute Molecular Stereochemistry in Gas Phase by Coulomb Explosion Imaging.</a> Science. 341 (6150), 1096-1100 (2013).</li><li>Pitzer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Configuration+from+Different+Multifragmentation+Pathways+in+Light-Induced+Coulomb+Explosion+Imaging.">Absolute Configuration from Different Multifragmentation Pathways in Light-Induced Coulomb Explosion Imaging.</a> Chem Phys Chem. 17 (16), 2465-2472 (2016).</li><li>Vager, Z., Naaman, R., Kanter, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coulomb+Explosion+Imaging+of+small+molecules.">Coulomb Explosion Imaging of small molecules.</a> Science. 244 (4903), 426-431 (1989).</li><li>Herwig, P. H., 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=Imaging+the+Absolute+Configuration+of+a+Chiral+Epoxide+in+the+Gas+Phase.">Imaging the Absolute Configuration of a Chiral Epoxide in the Gas Phase.</a> Science. 342 (6162), 1084-1186 (2013).</li><li>D&#246;rner, R., 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=Cold+Target+Recoil+Ion+Momentum+Spectroscopy:+a+'momentum+microscope'+to+view+atomic+collision+dynamics.">Cold Target Recoil Ion Momentum Spectroscopy: a 'momentum microscope' to view atomic collision dynamics.</a> Physics Reports. 330, 95-192 (2000).</li><li>Ullrich, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recoil-ion+and+electron+momentum+spectroscopy:+reaction-microscopes.">Recoil-ion and electron momentum spectroscopy: reaction-microscopes.</a> Rep. 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Phys. B.: At. Mol. Opt. Phys. 49 (23), 234001 (2016).</li><li>Hine, J., Dowell, A. M., Singley, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Dihalides+as+Intermediates+in+the+Basic+Hydrolysis+of+Haloforms:+IV+Relative+Reactivities+of+Haloforms.">Carbon Dihalides as Intermediates in the Basic Hydrolysis of Haloforms: IV Relative Reactivities of Haloforms.</a> J. Am. Soc. Chem. 78, 479-482 (1956).</li><li>Wales, B., 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=A+coincidence+detection+algorithm+for+improving+detection+rates+in+coulomb+explosion+imaging.">A coincidence detection algorithm for improving detection rates in coulomb explosion imaging.</a> Nucl. Instr. Meth. in Phys. Res. A. 667, 11-15 (2012).</li><li>Kastirke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Konstruktion und Aufbau einer UHV-tauglichen COLTRIMS-Kammer. , (2014).</li><li>Calegari, F., 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=Charge+migration+induced+by+attosecond+pulses+in+bio-relevant+molecules.">Charge migration induced by attosecond pulses in bio-relevant molecules.</a> J. Phys. B.: At. Mol. Opt. Phys. 49 (14), 142001 (2016).</li><li>Gaie-Levrel, F., Garcia, G. A., Schwell, M., Nahon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VUV+state-selected+photoionization+of+thermally-desorbed+biomolecules+by+coupling+an+aerosol+source+to+an+imaging+photoelectron/photoion+coincidence+spectrometer:+case+of+the+amino+acids+tryptophan+and+phenylalanine.">VUV state-selected photoionization of thermally-desorbed biomolecules by coupling an aerosol source to an imaging photoelectron/photoion coincidence spectrometer: case of the amino acids tryptophan and phenylalanine.</a> Phys. Chem. Chem. Phys. 13, 7024-7036 (2010).</li><li>Ablikim, U., 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=Identification+of+absolute+geometries+of+cis+and+trans+molecular+isomers+by+Coulomb+Explosion+Imaging.">Identification of absolute geometries of cis and trans molecular isomers by Coulomb Explosion Imaging.</a> Sci. Rep. 6, 38202 (2016).</li></ol>

The authors declare no competing interests.

Due to the variety of components, a COLTRIMS setup requires a rather high level of technical expertise, especially in the areas of vacuum technique, particle detection, fast electronics and data analysis. Before turning to the investigation of complex species, it should thus be thoroughly checked if the setup is running properly, e.g. by performing and analyzing a measurement on a diatomic or triatomic species.
Optimizing the intensity and duration of the laser pulses and the overlap with the molecular jet is essential to achieve as many multiple ionization events as possible. As the momentum distribution might broaden due to sequential ionization during different cycles of the laser pulse, the pulse duration should ideally not exceed 40 fs. During the measurement, it is crucial to obtain sufficient statistics. On the positive side, the determination of absolute configuration does not require a particularly high precision compared to other coincidence experiments, i.e. the procedure is rather robust to fluctuations in the laser or jet intensity and to electric field distortions in the spectrometer.
The most fundamental limitation of the technique concerns its applicability to larger molecules. One must keep in mind that the results represent the momenta of fragments, not the structure of the molecules in real space. For complex bio-molecules, the relation between measured momenta and molecular structure is not expected to be as straightforward as for the molecules presented here. In addition, complex molecules can produce many break-up channels that do not carry information on the configuration, possibly decreasing the yield of relevant channels. Theoretical modeling of the fragmentation, control of break-up patterns and more sophisticated analysis procedures will be needed if the technique is to be extended to molecules with three or more carbon atoms. At the current stage, it seems not possible to investigate the configuration of proteins or molecules of similar complexity, but the actual limitations have still to be determined.
Another limitation of the current setup is the relatively high sample consumption due to the molecular jet. It can be reduced by implementing a recycling mechanism (e.g. cold traps in the vacuum foreline). Nevertheless, it would be beneficial to test other sample preparation methods such as effusive jets, thermodesorption18 or laser desorption techniques19 that have been successfully applied for studying bio-molecules in the gas phase.
Coulomb Explosion Imaging is a destructive method, i.e. molecules that have been fragmented for determination of configuration cannot be used further. However, only a small fraction is actually ionized (which is one of the reasons for the high sample consumption mentioned in the previous paragraph). It might thus be possible to use the recycled molecules for subsequent application.
As the measurement of momenta allows to create an &#39;aligned&#39; data set of the molecules&#160;and to select certain spatial directions, the coincidence technique opens new perspectives for the investigation of asymmetry effects in chiral molecules This is in particular the case if the momenta of the electrons are measured in coincidence which can be achieved by using a complete COLTRIMS setup. Pump-probe techniques additionally allow to study the structural dynamics of chiral species.
Very recently, Coulomb Explosion Imaging has also been used to determine the absolute geometries of cis and trans-isomers,20 adding a new class of possible species and questions to be addressed. As the investigation of stereochemistry with coincidence spectroscopy is still in its infancy, the authors hope that this article helps to inspire researchers working in the directions outlined in the preceding paragraphs to new experiments.

Chirality is a feature of our nature that has been fascinating researchers for more than 150 years. In the 19th century, Pasteur, van&#39;t Hoff and others discovered that molecules can occur in two mirror image structures that are not super-imposable - like our left and right hands. This property was termed &#39;chiral&#39;, from the Greek word for &#39;hand&#39;.
So far, no difference in thermodynamic properties or in energy levels of left- and right-handed forms (the two &#39;enantiomers&#39;) has been found. In order to analyze the handedness of a given sample and to separate the enantiomers, interaction with other chiral molecules can be used, as is for example done in various chromatographical approaches.1 Chiroptical methods such as (vibrational) circular dichroism, (V)CD, and optical rotatory dispersion, ORD, are routinely employed to distinguish between enantiomers.2
When it comes to the determination of the microscopic structure, these techniques require additional information, e.g. from quantum-chemical calculations. The only technique that is widely accepted to directly determine the absolute configuration is anomalous X-ray diffraction.3
It has recently been shown that the absolute configuration of simple chiral species can be determined by Coulomb Explosion Imaging.4,5 In this approach, molecules in the gas phase are multiply ionized so that the remaining cores strongly repel each other. This repulsion leads to fast fragmentation (&#39;explosion&#39;) of the molecules. The direction and the magnitude of the fragment momenta correlate to the structure of the molecule &#8211; for small molecules, the momentum directions correspond surprisingly well to the bond axes. Coulomb Explosion for molecular structure determination has been pioneered using molecular ion beams from an accelerator.6 This beam foil technique has recently also been applied for chiral recognition.7
Contrary to anomalous X-ray diffraction, the sample must not be crystalline but provided in the gas phase. This makes the Coulomb Explosion approach ideal for volatile species and thus complementary to X-ray diffraction. In certain cases, the handedness can even be determined for individual molecules.
In practice, the exact reconstruction of the molecular structure has proven difficult even for methane derivatives, e.g. molecules with a central carbon and different substituents. This is attributed to the fact that the interaction between the fragments is not exactly Coulombic and that not all bonds break simultaneously. In order to obtain stereochemical information, especially to distinguish between enantiomers, this reconstruction is fortunately not necessary. Instead, the momentum vectors of different fragments can be correlated to yield a quantity that is distinct for the left- and right-handed molecules. To get reliable results, at least four fragment momenta have to be recorded.
In order to measure this momentum information, the fragments from one &#8211; and only one &#8211; molecular break-up have to be detected in a single measurement step. This condition is usually referred to as &#39;coincident detection&#39;. In addition, the emission directions have to be analyzed, which amounts in practice to record the time and the position of the fragment impact in a list-mode data format.
In atomic and molecular physics, techniques have been developed that implement this approach of measurement by employing electrostatic spectrometers for mass separation and time- and position-sensitive multi-hit detectors. The most prominent example is the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) setup &#8211; also known as Reaction Microscope.8,9 A sketch for this kind of experiment is given in Figure 1. Contrary to a standard COLTRIMS that can record electrons as well, Coulomb Explosion Imaging requires only the ion detector.
Spectrometer and detector are mounted under ultra-high vacuum (&#60;1 x 10-9 hPa) to avoid creation of ions from residual gas. Single molecules of the sample are provided via a gaseous free molecular jet created by supersonic expansion: By virtue of the vapor pressure, the molecules expand through a small nozzle (around 50 &#181;m diameter) into the vacuum. This part of the experiment, the source chamber, is separated from the interaction region by usually two skimmers and differentially pumped stages. An additional differentially pumped section is located behind the interaction region to dump the gas jet and thus avoid background gas in the interaction region.
The ionizing radiation intersects with the molecular jet under 90&#176;. Most laboratories nowadays use femtosecond laser pulses, although synchrotron radiation, fast ions or electron impact are possible &#39;projectiles&#39; to induce Coulomb Explosion.
The following protocol makes the assumption that a running setup for coincident imaging of ions and a femtosecond laser are available in the lab. The peak intensity needed to induce Coulomb Explosion into four or even five fragments must be on the order of 6 x 1014 W/cm2. To avoid exceedingly long measurements, the repetition rate of the laser should be 10 kHz or more. This is crucial because, on the one hand, coincident detection can only be ascertained if the probability for fragmentation in the laser focus is significantly below 1 per laser pulse (ideally not more than 10%). The total fragmentation rate, on the other hand, should not be lower than a few kHz because the share of relevant multifragmentation pathways is usually less than 10-4. As encouraging fact, it should be mentioned that in principle already a single fragmentation event is sufficient to identify the configuration of an enantiopure sample, and that detection of a few hundred allows to determine the abundance of the enantiomers in a sample of unknown enantiomeric composition.

<table><tbody><tr><td>CHBrCl2</td><td>SigmaAldrich</td><td>139181-10G</td><td>or other suitable sample</td></tr><tr><td>femtosecond laser system</td><td>KMLabs</td><td>Wyvern500</td><td></td></tr><tr><td>High-reflective mirrors</td><td>EKSMA</td><td>042-0800</td><td></td></tr><tr><td>mirror mounts</td><td>Newport</td><td>U100-A-LH-2K&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>focusing mirror (protected silver, f = 75 mm)</td><td>Thorlabs&amp;nbsp;</td><td>CM254-075-P01</td><td>(if available: f = 60 mm)</td></tr><tr><td>COLTRIMS spectrometer, including electronics and data acquisition system</td><td>RoentDek</td><td>custom</td><td>contrary to the standard COLTRIMS, only one detector is needed</td></tr></tbody></table>

coulomb explosion imaging as a tool to distinguish between stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

In this part, we show results obtained for halomethanes. These species are ideal for proof-of-principle experiments due to their simplicity and high vapor pressure. In the meantime, the more complex species halothane has been investigated using single soft-x-ray photons from a synchrotron source to induce multiple ionization.14
CHBrClF
Bromochlorofluoromethane (CHBrClF) is a textbook example for chiral molecules with a stereogenic carbon atom. It is also the ideal candidate for Coulomb Explosion Imaging due to its simple structure and the high vapor pressure (around 600 hPa at room temperature). Unfortunately, the species is not available commercially; for the experiment presented here, a racemic mixture was synthesized by reacting CHBr2Cl with HgF2 according to reference15. Enantio-enriched samples are difficult to obtain in the quantities needed so that only results for racemates have been obtained so far.
For the results presented here the sample was cooled to around 240 K to obtain an appropriate target density with the given nozzle (10% of ionization probability per pulse). The peak intensity of the laser was estimated to be 6 x 1014 W/cm2. The measurement at 100 kHz laser repetition rate took 11 h.
In order to distinguish R and S-enantiomers, a normalized triple product is calculated from the momentum vectors of the three halogens fluorine, chlorine, and bromine. Geometrically, this quantity can be interpreted as the cosine of the angle between the fluorine momentum and the plane of the chlorine and bromine momenta.
<img alt="Equation 3" src="/files/ftp_upload/56062/56062eq3.jpg" />
Figure 3 shows cos&#952; for the isotope CH79Br35ClF, together with the geometric definition. Two clear peaks are visible, indicating the enantiomers. The position of the peaks is consistent with a classical molecular dynamics simulation. As almost no background is present, the assignment of handedness works on a single molecule level.
CHBrCl2
The chirality of CHBrCl2 occurs only if the both isotopes 35Cl and 37Cl are present in the same molecule. A sample with natural abundance of isotopes thus contains chiral and achiral molecules. Two additional complications arise here: Firstly, the time-of-flight distributions of the chlorine and bromine isotopes overlap respectively due to the small mass difference. This is particularly relevant for chlorine as the determination of handedness depends on the correct assignment of the isotopes. Secondly, the chiral species CH79Br35Cl37Cl has (within the setup&#39;s accuracy) the same total mass as the achiral species CH81Br35Cl2. The investigation of this species can thus be seen as a benchmark test for the method.
With the spectrometer used (spectrometer length s = 60.5 mm, electric field strength E = 57.1 V/cm), the data for the chiral isotope CH79Br35Cl37Cl could be selected via the total momentum, using an algorithm suggested by reference16 to assign which of the hits belongs to which isotope.
Geometrical considerations lead to the conclusion that there can be orientations of the molecule in space where the two chlorine isotopes have the same time-of-flight; in this case, they cannot be distinguished as a matter of principle. A procedure to sort out these events has been described in the supplementary materials reference4. As a result, the configuration even of isotopically chiral molecules can be determined with high reliability.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56062/56062fig1.jpg" /> 
Figure 1: View into a COLTRIMS Setup. Molecules enter the setup through the nozzle and pass through a pair of skimmers. In the interaction chamber, the laser pulses cross with the molecular jet under 90&#176;. Ions are guided by the electric field of the spectrometer to the detector (top). For better visibility, not all spectrometer plates are shown. The remaining molecules are dumped in a differentially pumped section (jet dump) to keep the background pressure in the interaction region as low as possible. Figure modified from reference17 with permission by G. Kastirke. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56062/56062fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56062/56062fig2.jpg" /> 
Figure 2: Four-Particle Coincidence Spectrum. This histogram is an extension of a time-of-flight mass spectrum to four particles: The sum of the time-of-flights for the first and second hit on the detector are plotted on the x-axis, the sum for the third and fourth hit on the y-axis. The center of the peaks allows to identify the masses of the four detected fragments. The shape of the structures contains additional information: If the momenta of the fragments add up to zero, the events are contained in a narrow line (H, CF, Cl, Br). If an undetected fragment carries momentum, the non-zero total momentum of the measured particle leads to a broadening of the features. For illustration purposes, data from synchrotron, not laser, measurements are used here due to higher statistics. Figure reproduced from reference5 with permission by Wiley-VCH. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56062/56062fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56062/56062fig3.jpg" /> 
Figure 3: Distinction of enantiomers in the five-particle break-up of CHBrClF via the chirality parameter cos &#952; as defined in the text. The peak at positive values corresponds to the R-enantiomer, the peak at negative values to the S-enantiomer. The inset illustrates cos &#952; geometrically. The low background allows for an assignment of handedness for individual molecules. Figure reproduced from reference4 with permission by AAAS. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56062/56062fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers at JoVE.com

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

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9.0K Views.  Goethe-Universität Frankfurt am Main. Voor kleine chirale soorten biedt Coulomb explosie Imaging een nieuwe benadering om te bepalen van de handigheid van individuele moleculen.

Video: Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Engineering

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 ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Biology

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 ...

Stereoisomers

On the basis of mirror symmetry, stereoisomers of an organic molecule can be further classified into diastereomers and enantiomers. Diastereomers are stereoisomers that are not mirror images of each other. Substituted alkenes, such as the cis and trans isomers of 2-butene, are diastereomers, as these molecules exhibit different spatial orientations of their constituent atoms, are not mirror images of each other,&#160;and do not interconvert. Here, the interconversion is suppressed due to restricted rotation around the &#960; bond. Another class of diastereomers &#8212; those without &#960; bonds &#8212; are molecules that have non-superposable spatial orientations and exhibit different configurations of their substituent groups at some, but not all, stereocenters. For example, cis-1,2-dimethylcyclohexane and trans-1,2-dimethylcyclohexane are diastereomers, as these molecules are not superposable and have a different configuration of the methyl and hydrogen groups at only one out of their two stereocenters.
Enantiomers are stereoisomers that are mirror images of each other. As only chiral molecules can have non-superposable mirror images, enantiomers are chiral molecules. For example, the chiral molecule 2-butanol and its mirror image are enantiomers, as these molecules exhibit non-superposable spatial orientations of their constituent atoms and are mirror images of each other. A chiral molecule and its mirror image are collectively referred to as an enantiomeric pair, or a pair of enantiomers. The enantiomer of a chiral molecule can be drawn by taking its mirror image from any position or&#160;exchanging the positions of two of the substituents at each stereocenter of the molecule.

Stereoisomers: Diastereomers and Enantiomers

On the basis of mirror symmetry, stereoisomers of an organic molecule can be further classified into diastereomers and enantiomers. Diastereomers are ...

KEY TERMS AND CONCEPTS

<h3>Isomerism in Complexes</h3>
Isomers are different chemical species that have the same chemical formula.
Transition metal complexes often exist as geometric isomers, in which the same atoms are connected through the same types of bonds but with differences in their orientation in space. Coordination complexes with two different ligands in the cis and trans positions from a ligand of interest form isomers. For example, the octahedral [Co(NH3)4Cl2]+ ion has two isomers (Figure 1) In the cis configuration, the two chloride ligands are adjacent to each other. The other isomer, the trans configuration, has the two chloride ligands directly across from one another.
<img alt="Image1" src="/files/ftp_upload/11459/11459_Image1.svg" style="text-align: center; height: 200px;" />
Figure 1. The cis and trans isomers of [Co(H2O)4Cl2]+ contain the same ligands attached to the same metal ion, but the spatial arrangement causes these two compounds to have very different properties.
Different geometric isomers of a substance are different chemical compounds. They exhibit different properties, even though they have the same formula. For example, the two isomers of [Co(NH3)4Cl2]NO3 differ in color; the cis form is violet, and the trans form is green. Furthermore, these isomers have different dipole moments, solubilities, and reactivities. As an example of how the arrangement in space can influence the molecular properties, consider the polarity of the two [Co(NH3)4Cl2]NO3 isomers. Remember that the polarity of a molecule or ion is determined by the bond dipoles (which are due to the difference in electronegativity of the bonding atoms) and their arrangement in space. In one isomer, cis chloride ligands cause more electron density on one side of the molecule than on the other, making it polar. For the trans isomer, each ligand is directly across from an identical ligand, so the bond dipoles cancel out, and the molecule is nonpolar.
Another important type of isomers are optical isomers, or enantiomers, in which two objects are exact mirror images of each other but cannot be lined up so that all parts match. This means that optical isomers are non-superposable mirror images. A classic example of this is a pair of hands, in which the right and left hand are mirror images of one another but cannot be superposed. Optical isomers are very important in organic and biochemistry because living systems often incorporate one specific optical isomer and not the other. Unlike geometric isomers, pairs of optical isomers have nearly identical properties (boiling point, polarity, solubility, etc.). Optical isomers differ only in the way they affect polarized light and how they react with other optical isomers. For coordination complexes, many coordination compounds such as [M(en)3]n+ [in which Mn+ is a central metal ion such as iron(III) or cobalt(II)] form enantiomers, as shown in Figure 2. These two isomers will react differently with other optical isomers. For example, DNA helices are optical isomers, and the form that occurs in nature (right-handed DNA) will bind to only one isomer of [M(en)3]n+ and not the other.
<img alt="Image2" src="/files/ftp_upload/11459/11459_Image_2.svg" style="text-align: center; height: 200px;" />
Figure 2. The complex [M(en)3]n+ (Mn+ = a metal ion, en = ethylenediamine) has a non-superposable mirror image.
The [Co(en)2Cl2]+ ion exhibits geometric isomerism (cis/trans), and its cis isomer exists as a pair of optical isomers (Figure 3).
<img alt="Image3" src="/files/ftp_upload/11459/11459_Image_3.svg" style="text-align: center; height: 200px;" />
Figure 3. Three isomeric forms of [Co(en)2Cl2]+ exist. The trans isomer, formed when the chlorines are positioned at a 180&#176; angle, has very different properties from the cis isomers. The mirror images of the cis isomer form a pair of optical isomers, which have identical behavior except when reacting with other enantiomers.
This text is adapted from <a href="https://openstax.org/books/chemistry-2e/pages/19-2-coordination-chemistry-of-transition-metals">Openstax, Chemistry 2e, Chapter 19.2 Coordination Chemistry of Transition Metals.</a>

Stereoisomerism: Geometric &amp; Optical Isomers

Isomerism in Complexes
Isomers are different chemical species that have the same chemical formula.
Transition metal complexes often exist as geometric ...

&sup1;H NMR Chemical Shift Equivalence: Enantiotopic and Diastereotopic Protons

Replacing each alpha-hydrogen in chloroethane by bromine (or a different functional group) yields a pair of enantiomers. Such protons are called prochiral or enantiotopic and are related by a mirror plane. Enantiotopic protons are chemically equivalent in an achiral environment. Because most proton NMR spectra are recorded using achiral solvents, enantiotopic hydrogens yield a single signal.
In chiral compounds such as 2-butanol, replacing the methylene hydrogens at C3 produces a pair of diastereomers. Such protons are called diastereotopic, and they perceive different environments in all conformations. They cannot be interchanged by rotation or reflection and have slightly different chemical shifts.

1H NMR Chemical Shift Equivalence: Enantiotopic and Diastereotopic Protons

Replacing each alpha-hydrogen in chloroethane by bromine (or a different functional group) yields a pair of enantiomers. Such protons are called prochiral ...

Analytical Chemistry

Interpreting Nuclear Magnetic Resonance Spectra

Stereoisomerism of Cyclic Compounds

In this lesson, we delve into the role of ring conformation and its stability, which determines the spatial arrangement and, consequently, the molecular symmetry and stereoisomerism of cyclic compounds. 1,2-Dimethylcyclohexane is used as a case study to evaluate the possible number of stereoisomers. Here, given the multiple (n = 2) chiral centers, there are 2n = 4 possible configurations that lack a plane of symmetry, as the ring skeleton exists in a non-planar chair conformation. In addition, the potential for ring-flipping in a cyclohexane ring entails that each of these four possible configurations could further exist as a mixture of two or more conformations.
The effect of conformational flexibility in a ring system on the number of possible stereoisomers is shown using a case study of cis and trans configurations of 1,2-dimethylcyclohexane. While the cis configurations are chiral molecules (non-superposable mirror images) with the enantiomers as potential distinct stereoisomers, the rapid ring-flipping at room temperature renders these configurations interconvertible and inseparable. Accordingly, they represent conformations of the same molecule. On the other hand, the trans isomers are chiral molecules that cannot be superposed by rotation of the molecule or ring-flipping and exist as unique compounds. This proves the presence of three stereoisomers for the chosen example&#8212;the cis isomer and the pair of trans enantiomers.
This is further elucidated using another ring structure with a difference of substitutional position: 1,3-dimethylcyclohexane. The cis configuration is achiral due to a molecular plane of symmetry. Consequently, the system with two chiral centers exhibits three stereoisomers&#8212;the two trans non-interconvertible enantiomers and an achiral cis configuration. In essence, when a ring structure is evaluated, the two aspects that need to be studied are the ring-flipping and the plane of symmetry to determine the possible number of stereoisomers.

In this lesson, we delve into the role of ring conformation and its stability, which determines the spatial arrangement and, consequently, the molecular ...

Photochemical Electrocyclic Reactions: Stereochemistry

The absorption of UV&ndash;visible light by conjugated systems causes the promotion of an electron from the ground state to the excited state. Consequently, photochemical electrocyclic reactions proceed via the excited-state HOMO rather than the ground-state HOMO. Since the ground- and excited-state HOMOs have different symmetries, the stereochemical outcome of electrocyclic reactions depends on the mode of activation; i.e., thermal or photochemical.
Selection Rules: Photochemical Activation
Conjugated systems containing an even number of &#960;-electron pairs undergo a disrotatory ring closure. For example, photochemical activation of (2E,4E)-2,4-hexadiene, a conjugated diene containing two &#960;-electron pairs, gives cis-3,4-dimethylcyclobutene.
<img alt="Figure1" src="/files/ftp_upload/12414/12414_PT_Figure_1.svg" style="text-align: center; height: 100px;" />
Conjugated systems with an odd number of &#960;-electron pairs undergo a conrotatory ring closure. For example, (2E,4Z,6E)-2,4,6-octatriene, a conjugated diene containing three &#960;-electron pairs, forms trans-5,6-dimethyl-1,3-cyclohexadiene.
<img alt="Figure2" src="/files/ftp_upload/12414/12414_PT_Figure_2.svg" style="text-align: center; height: 100px;" />

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

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