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Modern Spectroscopy

Rapid and accurate Gram differentiation is paramount as the first step of pathogen identification and antibiotics administration. However, the current method requires additional reagents, is time-consuming, and is operator dependent. Here we show the principle of tip enhanced Raman spectroscopy (TERS) can differentiate between Gram negative and positive species, by detecting the changes in tip-enhancement in the Raman scattering from the bacteria's lipid-bilayer membrane, which specifically enhances Gram negative bacteria.

Modern spectroscopy

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The preliminary analysis of bacterial clinical samples is Gram stain differentiation and has been so for over a century1. Currently, initial antimicrobial treatment is administered according to direct microscopic morphology and Gram staining, hours ahead of final biochemical identification of bacteria in culture2. Although Gram staining diagnostics can be highly specific, its sensitivity to detect the presence of bacteria is relatively low, requires additional reagents, is time consuming, and can depend on operator technique3, 4. Few attempts have been made to improve the sensitivity of this method5, 6. Previous efforts to explore spectroscopy as a tool to replace Gram staining have yielded mixed results7,8,9. These methods required complicated procedures and provided relatively low specificity.

Raman spectroscopy is a branch of vibrational spectroscopy that uses the spectrum of inelastically scattered light for interpretation and highly sensitive structural identification of chemicals based on their unique vibrational fingerprints. Raman spectroscopy is an invaluable analytical tool for monitoring changes in molecular bond structure10.

Surface-enhanced Raman scattering (SERS) can provide strongly increased Raman signals from molecules which have been deposited onto nanometer sized metallic structures and/or nanostructured metallic surfaces. When the incident laser light in an experimental set-up strikes the metallic surface, the surface plasmons associated with the electronic resonances of the metal localize the electric field on to the adjacent molecules, thereby greatly increasing their efficiency for Raman scattering11. Several reports were published on bacterial identification using the SERS spectroscopic technique12, 13. In Tip-enhanced Raman spectroscopy (TERS), SERS-like enhancement is produced when a metallized AFM probe amplifies local electromagnetic field that is experienced by the molecules underneath the probe. In addition to the enhancement, TERS can provide Raman spectra with nanoscale spatial resolution14, 15. In TERS, the local field enhancement is enabled when the incident laser light strikes the nanosize tip-apex16. TERS on bacteria17,18,19 and for other biochemical applications was reported before20, 21.

CHEM 560 Introduction to Modern Spectroscopy3 semester hours Theory, instrumentation principles, methods for data collection, results interpretation, and contemporary applications of modern spectroscopic methods including Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-Mass Spectrometry (LC-MS), Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), and Nuclear Magnetic Resonance Spectroscopy (NMR). The course will include hands-on use of instrumentation in the laboratory setting.

Modern spectroscopy in the Western world started in the 17th century. New designs in optics, specifically prisms, enabled systematic observations of the solar spectrum. Isaac Newton first applied the word spectrum to describe the rainbow of colors that combine to form white light. During the early 1800s, Joseph von Fraunhofer conducted experiments with dispersive spectrometers that enabled spectroscopy to become a more precise and quantitative scientific technique. Since then, spectroscopy has played and continues to play a significant role in chemistry, physics and astronomy. Fraunhofer observed and measured dark lines in the Sun's spectrum,[1] which now bear his name although several of them were observed earlier by Wollaston.[2]

Throughout the early 1800s, a number of scientists pushed the techniques and understanding of spectroscopy forward.[13][17] In the 1820s, both John Herschel and William H. F. Talbot made systematic observations of salts using flame spectroscopy.[18][19][20]

In 1835, Charles Wheatstone reported that different metals could be easily distinguished by the different bright lines in the emission spectra of their sparks, thereby introducing an alternative mechanism to flame spectroscopy.[21][22] In 1849, J. B. L. Foucault experimentally demonstrated that absorption and emission lines appearing at the same wavelength are both due to the same material, with the difference between the two originating from the temperature of the light source.[23][24] In 1853, the Swedish physicist Anders Jonas Ångström presented observations and theories about gas spectra in his work Optiska Undersökningar (Optical investigations) to the Royal Swedish Academy of Sciences.[25] Ångström postulated that an incandescent gas emits luminous rays of the same wavelength as those it can absorb. Ångström was unaware of Foucalt's experimental results. At the same time George Stokes and William Thomson (Kelvin) were discussing similar postulates.[23] Ångström also measured the emission spectrum from hydrogen later labeled the Balmer lines.[26][27] In 1854 and 1855, David Alter published observations on the spectra of metals and gases, including an independent observation of the Balmer lines of hydrogen.[28][29]

The systematic attribution of spectra to chemical elements began in the 1860s with the work of German physicists Robert Bunsen and Gustav Kirchhoff,[30] who found that Fraunhofer lines correspond to emission spectral lines observed in laboratory light sources. This laid way for spectrochemical analysis in laboratory and astrophysical science. Bunsen and Kirchhoff applied the optical techniques of Fraunhofer, Bunsen's improved flame source and a highly systematic experimental procedure to a detailed examination of the spectra of chemical compounds. They established the linkage between chemical elements and their unique spectral patterns. In the process, they established the technique of analytical spectroscopy. In 1860, they published their findings on the spectra of eight elements and identified these elements' presence in several natural compounds.[31][32] They demonstrated that spectroscopy could be used for trace chemical analysis and several of the chemical elements they discovered were previously unknown. Kirchhoff and Bunsen also definitively established the link between absorption and emission lines, including attributing solar absorption lines to particular elements based on their corresponding spectra.[33] Kirchhoff went on to contribute fundamental research on the nature of spectral absorption and emission, including what is now known as Kirchhoff's law of thermal radiation. Kirchhoff's applications of this law to spectroscopy are captured in three laws of spectroscopy:

In the 1860s the husband-and-wife team of William and Margaret Huggins used spectroscopy to determine that the stars were composed of the same elements as found on earth. They also used the non-relativistic Doppler shift (redshift) equation on the spectrum of the star Sirius in 1868 to determine its axial speed.[34][35] They were the first to take a spectrum of a planetary nebula when the Cat's Eye Nebula (NGC 6543) was analyzed.[36][37] Using spectral techniques, they were able to distinguish nebulae from stars.

In 1895, the German physicist Wilhelm Conrad Röntgen discovered and extensively studied X-rays, which were later used in X-ray spectroscopy. One year later, in 1896, French physicist Antoine Henri Becquerel discovered radioactivity, and Dutch physicist Pieter Zeeman observed spectral lines being split by a magnetic field.[46][13]

This branch of spectroscopy deals with radiation related to atoms that are stripped of several electrons (multiply ionized atoms (MIA), multiply charged ions, highly charged ions). These are observed in very hot plasmas (laboratory or astrophysical) or in accelerator experiments (beam-foil, electron beam ion trap (EBIT)). The lowest exited electron shells of such ions decay into stable ground states producing photons in VUV, EUV and soft X-ray spectral regions (so-called resonance transitions).

In the same period the laboratory spectroscopy of MIA becomes relevant as a diagnostic tool for hot plasmas of thermonuclear devices (see Nuclear fusion) which begun with building Stellarator in 1951 by Spitzer, and continued with tokamaks, z-pinches and the laser produced plasmas.[76][77] Progress in ion accelerators stimulated beam-foil spectroscopy as a means to measure lifetimes of exited states of MIA.[78] Many various data on highly exited energy levels, autoionization and inner-core ionization states were obtained.

A new page in the spectroscopy of MIA may be dated as 1986 with development of EBIT (Levine and Marrs, LLNL) due to a favorable composition of modern high technologies such as cryogenics, ultra-high vacuum, superconducting magnets, powerful electron beams and semiconductor detectors. Very quickly EBIT sources were created in many countries (see NIST summary[83] for many details as well as reviews.)[84][85]

Many early scientists who studied the IR spectra of compounds had to develop and build their own instruments to be able to record their measurements making it very difficult to get accurate measurements. During World War II, the U.S. government contracted different companies to develop a method for the polymerization of butadiene to create rubber, but this could only be done through analysis of C4 hydrocarbon isomers. These contracted companies started developing optical instruments and eventually created the first infrared spectrometers. With the development of these commercial spectrometers Infrared Spectroscopy became a more popular method to determine the "fingerprint" for any molecule.[39] Raman spectroscopy was first observed in 1928 by Sir Chandrasekhara Venkata Raman in liquid substances and also by "Grigory Landsberg and Leonid Mandelstam in crystals".[60] Raman spectroscopy is based on the observation of the raman effect which is defined as "The intensity of the scattered light is dependent on the amount of the polarization potential change".[60] The raman spectrum records light intensity vs. light frequency (wavenumber) and the wavenumber shift is characteristic to each individual compound.[60] 041b061a72

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