direct-comparison-hyperspectral-stimulated-raman-scattering-coherent

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 ...

Purdue University

In vivo vibrational spectroscopic imaging is inhibited by relatively slow spectral acquisition on the second scale and low photon collection efficiency for a highly scattering system. Recently developed multiplex coherent anti-Stokes Raman scattering and stimulated Raman scattering techniques have improved the spectral acquisition time down to microsecond scale. These methods using a spectrometer setting are not suitable for turbid systems in which nearly all photons are scattered. We demonstrate vibrational imaging by spatial frequency multiplexing of incident photons and single photodiode detection of a stimulated Raman spectrum within 60 μs. Compared to the spectrometer setting, our method improved the photon collection efficiency by two orders of magnitude for highly scattering specimens. We demonstrated in vivo imaging of vitamin E distribution on mouse skin and in situ imaging of human breast cancerous tissues. The reported work opens new opportunities for spectroscopic imaging in a surgical room and for development of deep-tissue Raman spectroscopy toward molecular level diagnosis. 

Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons.

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in . The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution.

Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipid-laden plaques, as it provides simultaneous morphological and lipid-specific chemical information of an artery wall. Real-time imaging and display at video-rate speed are critical for clinical utility of the IVPA-US imaging technology. Here, we demonstrate a portable IVPA-US system capable of imaging at up to 25 frames per second in real-time display mode. This unprecedented imaging speed was achieved by concurrent innovations in excitation laser source, rotary joint assembly, 1 mm IVPA-US catheter size, differentiated A-line strategy, and real-time image processing and display algorithms. Spatial resolution, chemical specificity, and capability for imaging highly dynamic objects were evaluated by phantoms to characterize system performance. An imaging speed of 16 frames per second was determined to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. The translational capability of this system for the detection of lipid-laden plaques was validated by ex vivo imaging of an atherosclerotic human coronary artery at 16 frames per second, which showed strong correlation to gold-standard histopathology. Thus, this high-speed IVPA-US imaging system presents significant advances in the translational intravascular and other endoscopic applications.

Real-time intravascular photoacoustic-ultrasound imaging of lipid-laden plaque in human coronary artery at 16 frames per second.

Graphene grain boundaries (GBs) and other nanodefects can deteriorate electronic properties. Here, using transient absorption (TA) microscopy we directly visualized GBs by TA intensity increase due to change in density of state. We also observed a faster decay due to defect-accelerated carrier relaxation in the GB area. By line-illumination and parallel detection, we increased the TA intensity imaging speed to 1000 frames per second, which is 6 orders of magnitude faster than Raman microscopy. Combined with a resonant optical delay tuner which scans a 5.3 ps temporal delay within 92 μs, our system enabled spectroscopic TA imaging, at a speed of 50 stacks per second, to probe and characterize graphene nanodefects based on the TA decay rate. Finally, we demonstrate real-time nondestructive characterization of graphene at a rolling speed of 0.3 m/min, which matches the fastest roll-to-roll manufacturing process reported.

High-Speed Spectroscopic Transient Absorption Imaging of Defects in Graphene.

Triboluminescence (TL) is shown to enable selective detection of trace crystallinity within nominally amorphous solid dispersions (ASDs). ASDs are increasingly used for the preparation of pharmaceutical formulations, the physical stability of which can be negatively impacted by trace crystallinity introduced during manufacturing or storage. In the present study, TL measurements of a model ASD consisting of griseofulvin in polyethylene glycol produced limits of detection of 140 ppm. Separate studies of the particle size dependence of sucrose crystals and the dependence on polymorphism in clopidogrel bisulfate particles are both consistent with a mechanism for TL closely linked to the piezoelectric response of the crystalline fraction. Whereas disordered polymeric materials cannot support piezoelectric activity, molecular crystals produced from homochiral molecules adopt crystal structures that are overwhelmingly symmetry-allowed for piezoelectricity. Consequently, TL may provide a broadly applicable and simple experimental route for sensitive detection of trace crystallinity within nominally amorphous materials.

Triboluminescence from Pharmaceutical Formulations.

Quadrupole ion traps (QITs) are versatile platforms for performing experiments with gas-phase ions due to their abilities to store ions of both polarities and to conduct MS experiments. The QIT is particularly useful as a reaction cell for ion/ion reactions. In the case of an ion/ion reaction experiment in a QIT, multiply charged reactant ions may initially be of relatively low m/z (e.g., m/z < 1000) whereas the product ions can be one or more orders of magnitude higher in m/z (e.g., m/z > 100,000). Several factors can limit the m/z range over which an ion/ion reaction experiment can be conducted. These include (1) the efficiency of the detector, (2) the m/z range over which oppositely charged ions can be mutually stored, and (3) the m/z range over which ions can be mass selectively ejected into an external detector. High-frequency waveforms provide larger m/z trapping ranges for mutual storage of oppositely charged ions whereas low-frequency waveforms provide better trapping for very high m/z product ions. Presented here is a method that switches from a high-frequency sine wave prior to and during an ion/ion reaction to a low-frequency square wave to eject low m/z reagent ions and improves confinement of the product ions before mass-selective ejection by scanning the frequency of the square wave. This approach addresses the third issue, which is the primary limiting factor with QITs operated at high RF (e.g., > 900 MHz). Graphical Abstract.

Increasing the Upper Mass/Charge Limit of a Quadrupole Ion Trap for Ion/Ion Reaction Product Analysis via Waveform Switching.

Ion isolation was achieved via selective pulsing of the entrance and exit ion mirrors in an electrostatic linear ion trap mass spectrometer (ELIT). Mirror switching has been described previously as a method for capturing injected ions in ELIT devices. After ion trapping, mirror switching can be used as a method for ion isolation of successively narrower ranges of mass-to-charge (/) ratio. By taking advantage of the spatial separation of ions in an ELIT device, pulsing of the entrance and/or exit mirrors can release unwanted ions while continuing to store ions of interest. Furthermore, mirror switching can be repeated multiple times to isolate ions of very similar / values with minimal loss of the stored ions, as is demonstrated by the isolation of protonated l-glutamine and l-lysine (Δ / = 0.0364) from a mixture of the two amino acid ions and the isobaric mixture of [PC P-18:0/22:6] and [PC 19:0/19:0] (Δ / = 0.0575). As isolation is accomplished due to the spatial/temporal separation of ion packets within the ELIT, multiple reflection-time-of-flight (MR-TOF) mass spectra are shown to demonstrate separation in the ELIT at the time of isolation. An isolation resolution of greater than 35 000 fwhm is demonstrated here using a 5.25 in. ELIT. This resolution corresponds to the fwhm resolution necessary to reduce contaminant overlap of an equally abundant adjacent ion to 1% or less of the isolated ion intensity.

Mirror Switching for High-Resolution Ion Isolation in an Electrostatic Linear Ion Trap.

Simultaneous isolation of ions of disparate mass-to-charge (/) ratios is demonstrated via appropriately timed pulsing of entrance and exit ion mirrors in an electrostatic linear ion trap (ELIT) mass spectrometer. Manipulation of the voltages of the entrance and exit mirrors, referred to as "mirror switching", has been demonstrated as a method in which ions can be both captured and isolated. High resolution isolation (>35 000) was previously demonstrated by selective gating of trapping electrodes to avoid ion lapping while closely spaced ions could continue to separate [ Johnson et al.  2019 , 91 , 8789 ]. In this work, we demonstrate that advantage can be taken of the ion lapping phenomenon in an ELIT to enable the simultaneous isolation of ions of disparate / ratios using mirror switching. This process is demonstrated with minimal ion loss using isotopologues of three carborane compounds ranging in / from 320 to 1020. Simultaneous isolation is demonstrated with the isolation of two and three peaks in separate isotopic distributions as well as with the isolation of alternating isotopologues within the same distribution. Such simultaneous isolation experiments are particularly useful when conducting experiments in which a mass calibrant is needed or when multiplexing in a tandem MS workflow.

Simultaneous Isolation of Nonadjacent / Ions Using Mirror Switching in an Electrostatic Linear Ion Trap.

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high  ions. The additional tandem-MS (MS) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at  > 50,000.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions.

Native mass spectrometry (MS) focuses on measuring the masses of large biomolecular complexes and probing their structures. Large biomolecular complexes are readily introduced into mass spectrometers as gas-phase ions using electrospray ionization (ESI); however, the ions tend to be heavily adducted with solvent and salts, which leads to mass measurement errors. Various solution clean-up approaches can reduce the degree of adduction prior to introduction to the mass spectrometer. Gas-phase activation of trapped ions can provide additional adduct reduction, and charge reduction ion/ion reactions increase charge state separation. Together, gas-phase activation and charge reduction can combine to yield spectra of well separated charge states for improved mass measurements. A simple gas-phase collisional activation technique is to apply a dipolar DC (DDC) field to opposing electrodes in an ion trap. DDC activation loses its efficacy when ions are trapped at low  values, which is true of the high  ions generated by charge reduction ion/ion reactions. Digital ion trapping (DIT) readily traps high  ions at higher  values by varying trapping frequency rather than amplitude, but the low frequencies used to trap high  ions also decreases the efficacy of DDC activation. We demonstrate here using ions derived from GroEL that IR activation of ions shows no discrimination against high  ions trapped with DIT, because they can be focused equally well to the trap center to interact with the IR laser beam. Following pump out of excess background gas, IR activation can also induce efficient dissociation of the GroEL complex. This work demonstrates that IR activation is an effective approach for ion heating in native MS over the unusually wide range of charge states accessible via gas-phase ion/ion reactions.

Gregory Eakins

Department of Chemistry

Purdue University

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

Gregory Eakins

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry

Purdue University

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

Department of Chemistry