skip to main content

Title: Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy

Being able to image chemical bonds with high sensitivity and speed, stimulated Raman scattering (SRS) microscopy has made a major impact in biomedical optics. However, it is well known that the standard SRS microscopy suffers from various backgrounds, limiting the achievable contrast, quantification and sensitivity. While many frequency-modulation (FM) SRS schemes have been demonstrated to retrieve the sharp vibrational contrast, they often require customized laser systems and/or complicated laser pulse shaping or introduce additional noise, thereby hindering wide adoption. Herein we report a simple but robust strategy for FM-SRS microscopy based on a popular commercial laser system and regular optics. Harnessing self-phase modulation induced self-balanced spectral splitting of picosecond Stokes beam propagating in standard single-mode silica fibers, a high-performance FM-SRS system is constructed without introducing any additional signal noise. Our strategy enables adaptive spectral resolution for background-free SRS imaging of Raman modes with different linewidths. The generality of our method is demonstrated on a variety of Raman modes with effective suppressing of backgrounds including non-resonant cross phase modulation and electronic background from two-photon absorption or pump-probe process. As such, our method is promising to be adopted by the SRS microscopy community for background-free chemical imaging.

Authors:
; ; ; ; ;
Award ID(s):
1904684
Publication Date:
NSF-PAR ID:
10149198
Journal Name:
Optics Express
Volume:
28
Issue:
10
Page Range or eLocation-ID:
Article No. 15663
ISSN:
1094-4087; OPEXFF
Publisher:
Optical Society of America
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10−30 cm2sr−1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.

  2. One of the top priorities in observational astronomy is the direct imaging and characterization of extrasolar planets (exoplanets) and planetary systems. Direct images of rocky exoplanets are of particular interest in the search for life beyond the Earth, but they tend to be rather challenging targets since they are orders-of-magnitude dimmer than their host stars and are separated by small angular distances that are comparable to the classicalλ<#comment/>/Ddiffraction limit, even for the coming generation of 30 m class telescopes. Current and planned efforts for ground-based direct imaging of exoplanets combine high-order adaptive optics (AO) with a stellar coronagraph observing at wavelengths ranging from the visible to the mid-IR. The primary barrier to achieving high contrast with current direct imaging methods is quasi-static speckles, caused largely by non-common path aberrations (NCPAs) in the coronagraph optical train. Recent work has demonstrated that millisecond imaging, which effectively “freezes” the atmosphere’s turbulent phase screens, should allow the wavefront sensor (WFS) telemetry to be used as a probe of the optical system to measure NCPAs. Starting with a realistic model of a telescope with an AO system and a stellar coronagraph, this paper provides simulations of several closely related regression models that take advantagemore »of millisecond telemetry from the WFS and coronagraph’s science camera. The simplest regression model, called the naïve estimator, does not treat the noise and other sources of information loss in the WFS. Despite its flaws, in one of the simulations presented herein, the naïve estimator provides a useful estimate of an NCPA of∼<#comment/>0.5radian RMS (≈<#comment/>λ<#comment/>/13), with an accuracy of∼<#comment/>0.06radian RMS in 1 min of simulated sky time on a magnitude 8 star. Thebias-corrected estimatorgeneralizes the regression model to account for the noise and information loss in the WFS. A simulation of the bias-corrected estimator with 4 min of sky time included an NCPA of∼<#comment/>0.05radian RMS (≈<#comment/>λ<#comment/>/130) and an extended exoplanet scene. The joint regression of the bias-corrected estimator simultaneously achieved an NCPA estimate with an accuracy of∼<#comment/>5×<#comment/>10−<#comment/>3radian RMS and an estimate of the exoplanet scene that was free of the self-subtraction artifacts typically associated with differential imaging. The5σ<#comment/>contrast achieved by imaging of the exoplanet scene was∼<#comment/>1.7×<#comment/>10−<#comment/>4at a distance of3λ<#comment/>/Dfrom the star and∼<#comment/>2.1×<#comment/>10−<#comment/>5at10λ<#comment/>/D. These contrast values are comparable to the very best on-sky results obtained from multi-wavelength observations that employ both angular differential imaging (ADI) and spectral differential imaging (SDI). This comparable performance is despite the fact that our simulations are quasi-monochromatic, which makes SDI impossible, nor do they have diurnal field rotation, which makes ADI impossible. The error covariance matrix of the joint regression shows substantial correlations in the exoplanet and NCPA estimation errors, indicating that exoplanet intensity and NCPA need to be estimated self-consistently to achieve high contrast.

    « less
  3. The success of nonlinear optics relies largely on pulse-to-pulse consistency. In contrast, covariance-based techniques used in photoionization electron spectroscopy and mass spectrometry have shown that a wealth of information can be extracted from noise that is lost when averaging multiple measurements. Here, we apply covariance-based detection to nonlinear optical spectroscopy, and show that noise in a femtosecond laser is not necessarily a liability to be mitigated, but can act as a unique and powerful asset. As a proof of principle we apply this approach to the process of stimulated Raman scattering in α-quartz. Our results demonstrate how nonlinear processes in the sample can encode correlations between the spectral components of ultrashort pulses with uncorrelated stochastic fluctuations. This in turn provides richer information compared with the standard nonlinear optics techniques that are based on averages over many repetitions with well-behaved laser pulses. These proof-of-principle results suggest that covariance-based nonlinear spectroscopy will improve the applicability of fs nonlinear spectroscopy in wavelength ranges where stable, transform-limited pulses are not available, such as X-ray free-electron lasers which naturally have spectrally noisy pulses ideally suited for this approach.

  4. A spatial heterodyne Raman spectrometer (SHRS), constructed using a modular optical cage and lens tube system, is described for use with a commercial silica and a custom single-crystal (SC) sapphire fiber Raman probe. The utility of these fiber-coupled SHRS chemical sensors is demonstrated using 532 nm laser excitation for acquiring Raman measurements of solid (sulfur) and liquid (cyclohexane) Raman standards as well as real-world, plastic-bonded explosives (PBX) comprising 1,3,5- triamino- 2,4,6- trinitrobenzene (TATB) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) energetic materials. The SHRS is a fixed grating-based dispersive interferometer equipped with an array detector. Each Raman spectrum was extracted from its corresponding fringe image (i.e., interferogram) using a Fourier transform method. Raman measurements were acquired with the SHRS Littrow wavelength set at the laser excitation wavelength over a spectral range of ∼1750 cm−1with a spectral resolution of ∼8 cm−1for sapphire and ∼10 cm−1for silica fiber probes. The large aperture of the SHRS allows much larger fiber diameters to be used without degrading spectral resolution as demonstrated with the larger sapphire collection fiber diameter (330 μm) compared to the silica fiber (100 μm). Unlike the dual silica fiber Raman probe, the dual sapphire fiber Raman probe did not include filtering at the fiber probe tip nearest the sample. Even so,more »SC sapphire fiber probe measurements produced less background than silica fibers allowing Raman measurements as close as ∼85 cm−1to the excitation laser. Despite the short lengths of sapphire fiber used to construct the sapphire probe, well-defined, sharp sapphire Raman bands at 420, 580, and 750 cm−1were observed in the SHRS spectra of cyclohexane and the highly fluorescent HMX-based PBX. SHRS measurements of the latter produced low background interference in the extracted Raman spectrum because the broad band fluorescence (i.e., a direct current, or DC, component) does not contribute to the interferogram intensity (i.e., the alternating current, or AC, component). SHRS spectral resolution, throughput, and signal-to-noise ratio are also discussed along with the merits of using sapphire Raman bands as internal performance references and as internal wavelength calibration standards in Raman measurements.

    « less
  5. Abstract: 100 words Jurors are increasingly exposed to scientific information in the courtroom. To determine whether providing jurors with gist information would assist in their ability to make well-informed decisions, the present experiment utilized a Fuzzy Trace Theory-inspired intervention and tested it against traditional legal safeguards (i.e., judge instructions) by varying the scientific quality of the evidence. The results indicate that jurors who viewed high quality evidence rated the scientific evidence significantly higher than those who viewed low quality evidence, but were unable to moderate the credibility of the expert witness and apply damages appropriately resulting in poor calibration. Summary: <1000 words Jurors and juries are increasingly exposed to scientific information in the courtroom and it remains unclear when they will base their decisions on a reasonable understanding of the relevant scientific information. Without such knowledge, the ability of jurors and juries to make well-informed decisions may be at risk, increasing chances of unjust outcomes (e.g., false convictions in criminal cases). Therefore, there is a critical need to understand conditions that affect jurors’ and juries’ sensitivity to the qualities of scientific information and to identify safeguards that can assist with scientific calibration in the courtroom. The current project addresses thesemore »issues with an ecologically valid experimental paradigm, making it possible to assess causal effects of evidence quality and safeguards as well as the role of a host of individual difference variables that may affect perceptions of testimony by scientific experts as well as liability in a civil case. Our main goal was to develop a simple, theoretically grounded tool to enable triers of fact (individual jurors) with a range of scientific reasoning abilities to appropriately weigh scientific evidence in court. We did so by testing a Fuzzy Trace Theory-inspired intervention in court, and testing it against traditional legal safeguards. Appropriate use of scientific evidence reflects good calibration – which we define as being influenced more by strong scientific information than by weak scientific information. Inappropriate use reflects poor calibration – defined as relative insensitivity to the strength of scientific information. Fuzzy Trace Theory (Reyna & Brainerd, 1995) predicts that techniques for improving calibration can come from presentation of easy-to-interpret, bottom-line “gist” of the information. Our central hypothesis was that laypeople’s appropriate use of scientific information would be moderated both by external situational conditions (e.g., quality of the scientific information itself, a decision aid designed to convey clearly the “gist” of the information) and individual differences among people (e.g., scientific reasoning skills, cognitive reflection tendencies, numeracy, need for cognition, attitudes toward and trust in science). Identifying factors that promote jurors’ appropriate understanding of and reliance on scientific information will contribute to general theories of reasoning based on scientific evidence, while also providing an evidence-based framework for improving the courts’ use of scientific information. All hypotheses were preregistered on the Open Science Framework. Method Participants completed six questionnaires (counterbalanced): Need for Cognition Scale (NCS; 18 items), Cognitive Reflection Test (CRT; 7 items), Abbreviated Numeracy Scale (ABS; 6 items), Scientific Reasoning Scale (SRS; 11 items), Trust in Science (TIS; 29 items), and Attitudes towards Science (ATS; 7 items). Participants then viewed a video depicting a civil trial in which the defendant sought damages from the plaintiff for injuries caused by a fall. The defendant (bar patron) alleged that the plaintiff (bartender) pushed him, causing him to fall and hit his head on the hard floor. Participants were informed at the outset that the defendant was liable; therefore, their task was to determine if the plaintiff should be compensated. Participants were randomly assigned to 1 of 6 experimental conditions: 2 (quality of scientific evidence: high vs. low) x 3 (safeguard to improve calibration: gist information, no-gist information [control], jury instructions). An expert witness (neuroscientist) hired by the court testified regarding the scientific strength of fMRI data (high [90 to 10 signal-to-noise ratio] vs. low [50 to 50 signal-to-noise ratio]) and gist or no-gist information both verbally (i.e., fairly high/about average) and visually (i.e., a graph). After viewing the video, participants were asked if they would like to award damages. If they indicated yes, they were asked to enter a dollar amount. Participants then completed the Positive and Negative Affect Schedule-Modified Short Form (PANAS-MSF; 16 items), expert Witness Credibility Scale (WCS; 20 items), Witness Credibility and Influence on damages for each witness, manipulation check questions, Understanding Scientific Testimony (UST; 10 items), and 3 additional measures were collected, but are beyond the scope of the current investigation. Finally, participants completed demographic questions, including questions about their scientific background and experience. The study was completed via Qualtrics, with participation from students (online vs. in-lab), MTurkers, and non-student community members. After removing those who failed attention check questions, 469 participants remained (243 men, 224 women, 2 did not specify gender) from a variety of racial and ethnic backgrounds (70.2% White, non-Hispanic). Results and Discussion There were three primary outcomes: quality of the scientific evidence, expert credibility (WCS), and damages. During initial analyses, each dependent variable was submitted to a separate 3 Gist Safeguard (safeguard, no safeguard, judge instructions) x 2 Scientific Quality (high, low) Analysis of Variance (ANOVA). Consistent with hypotheses, there was a significant main effect of scientific quality on strength of evidence, F(1, 463)=5.099, p=.024; participants who viewed the high quality evidence rated the scientific evidence significantly higher (M= 7.44) than those who viewed the low quality evidence (M=7.06). There were no significant main effects or interactions for witness credibility, indicating that the expert that provided scientific testimony was seen as equally credible regardless of scientific quality or gist safeguard. Finally, for damages, consistent with hypotheses, there was a marginally significant interaction between Gist Safeguard and Scientific Quality, F(2, 273)=2.916, p=.056. However, post hoc t-tests revealed significantly higher damages were awarded for low (M=11.50) versus high (M=10.51) scientific quality evidence F(1, 273)=3.955, p=.048 in the no gist with judge instructions safeguard condition, which was contrary to hypotheses. The data suggest that the judge instructions alone are reversing the pattern, though nonsignificant, those who received the no gist without judge instructions safeguard awarded higher damages in the high (M=11.34) versus low (M=10.84) scientific quality evidence conditions F(1, 273)=1.059, p=.30. Together, these provide promising initial results indicating that participants were able to effectively differentiate between high and low scientific quality of evidence, though inappropriately utilized the scientific evidence through their inability to discern expert credibility and apply damages, resulting in poor calibration. These results will provide the basis for more sophisticated analyses including higher order interactions with individual differences (e.g., need for cognition) as well as tests of mediation using path analyses. [References omitted but available by request] Learning Objective: Participants will be able to determine whether providing jurors with gist information would assist in their ability to award damages in a civil trial.« less