Life Detection on Icy Moons Using Flow Cytometry and Intrinsically Fluorescent Biomolecules.

In a previous experiment, we demonstrated the capability of flow cytometry as a potential life detection technology for icy moons using exogenous fluorescent stains (Wallace et al., 2023). In this companion experiment, we demonstrated the capability of flow cytometry to detect life using intrinsically fluorescent biomolecules in addition to exogenous stains. We used a method similar to our previous work to positively identify six classes of intrinsically fluorescent biomolecules: flavins, carotenoids, chlorophyll, tryptophan, NAD+, and NAD(P)H. We demonstrated the effectiveness of this method with six known organisms and known abiotic material and showed that the cytometer is easily able to distinguish the known organisms and the known abiotic material by using the intrinsic fluorescence of these six biomolecules. To simulate a life detection experiment on an icy moon lander, we used six natural samples with unknown biotic and abiotic content. We showed that flow cytometry can identify all six intrinsically fluorescent biomolecules and can separate the biotic material from the known abiotic material on scatter plots. The use of intrinsically fluorescent biomolecules in addition to exogenous stains will potentially cast a wider net for life detection on icy moons using flow cytometry.

Life Detection on Icy Moons Using Flow Cytometry and Exogenous Fluorescent Stains.

Flow cytometry is a potential technology for in situ life detection on icy moons (such as Enceladus and Europa) and on the polar ice caps of Mars. We developed a method for using flow cytometry to positively identify four classes of biomarkers using exogenous fluorescent stains: nucleic acids, proteins, carbohydrates, and lipids. We demonstrated the effectiveness of exogenous stains with six known organisms and known abiotic material and showed that the cytometer is easily able to distinguish between the known organisms and the known abiotic material using the exogenous stains. To simulate a life-detection experiment on an icy world lander, we used six natural samples with unknown biotic and abiotic content. We showed that flow cytometry can identify all four biomarkers using the exogenous stains and can separate the biotic material from the known abiotic material on scatter plots. Exogenous staining techniques would likely be used in conjunction with intrinsic fluorescence, clustering, and sorting for a more complete and capable life-detection instrument on an icy moon lander.

The Raman Spectrum of a Single Molecule on an Electrochemically Etched Silver Tip.

We recorded the Raman spectrum of a single azobenzene thiol molecule upon picking it up from an atomically flat gold surface, using an electrochemically etched silver tip, in an ultrahigh vacuum cryogenic scanning tunneling microscope. While suppressed at the junction, the stationary spectrum appeared once the molecule was transferred to the tip, with line intensities that increased by a factor of ∼5 as the tip was retracted from 1 nm to 161 nm. The effect, and the enhanced tensorial Raman spectrum was reproduced using an explicit treatment of the electromagnetic fields to identify a cis-azobenzene thiol molecule, adsorbed on a nanometric asperity removed from the tip apex, lying in the plane normal to the tip z-axis, with enhanced incident and radiative local fields polarized in the same plane. Tips decorated with asperities break the rules and give unique insights on Raman driven by cavity modes of a plasmonic junction.

Visualizing vibrational normal modes of a single molecule with atomically confined light.

The internal vibrations of molecules drive the structural transformations that underpin chemistry and cellular function. While vibrational frequencies are measured by spectroscopy, the normal modes of motion are inferred through theory because their visualization would require microscopy with ångström-scale spatial resolution-nearly three orders of magnitude smaller than the diffraction limit in optics1. Using a metallic tip to focus light and taking advantage of the surface-enhanced Raman effect2 to amplify the signal from individual molecules, tip-enhanced Raman spectromicroscopy (TER-SM)3,4 reaches the requisite sub-molecular spatial resolution5, confirming that light can be confined in picocavities6-10 and anticipating the direct visualization of molecular vibrations11-13. Here, by using TER-SM at the precisely controllable junction of a cryogenic ultrahigh-vacuum scanning tunnelling microscope14-16, we show that ångström-scale resolution is attained at subatomic separation between the tip atom and a molecule in the quantum tunnelling regime of plasmons6,8,9,17. We record vibrational spectra within a single molecule, obtain images of normal modes and atomically parse the intramolecular charges and currents driven by vibrations. Our analysis provides a paradigm for optics in the atomistic near-field.

Microscopy with a single-molecule scanning electrometer.

The vibrational spectrum of a single carbon monoxide molecule, adsorbed on the tip apex of a scanning tunneling microscope, is used to image electrostatic fields with submolecular spatial resolution. The method takes advantage of the vibrational Stark effect to image local electrostatic fields and the single-molecule sensitivity of tip-enhanced Raman scattering (TERS) to optically relay the signal. We apply the method to single metalloporphyrins adsorbed on Au(111) to image molecular charges, intramolecular polarization, local photoconductivity, atomically resolved hydrogen bonds, and surface electron density waves.

Bias-Dependent Chemical Enhancement and Nonclassical Stark Effect in Tip-Enhanced Raman Spectromicroscopy of CO-Terminated Ag Tips.

Tip-enhanced Raman spectromicroscopy (TERS) with CO-terminated plasmonic tips can probe angstrom-scale features of molecules on surfaces. The development of this technique requires understanding of how chemical environments affect the CO vibrational frequency and TERS intensity. At the scanning tunneling microscope junction of a CO-terminated Ag tip, we show that rather than the classical vibrational Stark effect, the large bias dependence of the CO frequency shift is due to ground-state charge transfer from the Ag tip into the CO π* orbital softening the C-O bond at more positive biases. The associated increase in Raman intensity is attributed to a bias-dependent chemical enhancement effect, where a positive bias tunes a charge-transfer excited state close to resonance with the Ag plasmon. This change in Raman intensity is contrary to what would be expected based on changes in the tilt angle of the CO molecule with bias, demonstrating that the Raman intensity is dominated by electronic rather than geometric effects.

Tip-Enhanced Raman Spectromicroscopy on the Angstrom Scale: Bare and CO-Terminated Ag Tips.

The tip is key to the successful execution of tip-enhanced Raman scattering (TERS) measurements in the single molecule limit. We show that nanoscopically smooth silver tips, batch produced through field-directed sputter sharpening, reliably attain TERS with enhancement factors that reach 1013, as measured by the Raman spectra of single CO molecules attached to the tip apex. We validate the bare tips by demonstrating spectromicroscopy with submolecular spatial resolution and underscore that TERS is a near-field effect that does not obey simple selection rules. As a more gainful analytical approach, we introduce TERS-relayed molecular force microscopy using CO-terminated tips. By taking advantage of the large Stark tuning rate of the CO stretch, molecular structure and charges can be imaged with atomic resolution. As illustration, we image a single Ag atom adsorbed on Au(111) and show that the adatom carries +0.2e charge.

Tip-Enhanced Raman Spectromicroscopy of Co(II)-Tetraphenylporphyrin on Au(111): Toward the Chemists' Microscope.

Atomically terminated and nanoscopically smooth silver tips effectively focus light on the angstrom scale, allowing tip-enhanced Raman spectromicroscopy (TER-sm) with single molecule sensitivity and submolecular spatial resolution. Through measurements carried out on cobalt-tetraphenylporphyrin (CoTPP) adsorbed on Au(111), we highlight peculiarities of vibrational spectromicroscopy with light confined on the angstrom scale. Field-gradient-driven spectra, orientational fingerprinting, and sculpting of local fields by atomic morphology of the junction are elucidated through measurements that range from 2D arrays at room temperature to single molecule manipulations at 5 K. Notably, vibrational Stark tuning within molecules, reflecting intramolecular charge distributions, becomes accessible when light is more localized than the interrogated normal modes. The Stark images of CoTPP reveal that it is saddled, and the distortion is accompanied by charge transfer to gold through the two anchoring pyrroles.

Hovering and Twirling of Tethered Molecules by Confinement between Surfaces.

Through STM images, we show that azobenzene-terminated alkanethiols hover and twirl when confined between the Ag tip and Au(111) substrate of an STM junction. In contrast with mechanisms of activation used to drive molecular rotors, twirling is induced by the effective elimination of lateral corrugation in the energy landscape when molecules hover by their van der Waals attraction to the approaching tip. While in the stationary state the benzenes of the head group lie flat with an inter-ring separation of 7.5 Å, they stand on-edge as the molecule twirls and their separation contracts to 5.2 Å, close to the value of the free molecule. The captured images of motion allow the characterization of physisorption potentials.

Isomerization of One Molecule Observed through Tip-Enhanced Raman Spectroscopy.

While exploring photoisomerization of azobenzyl thiols (ABT) adsorbed on Au(111), through joint scanning tunneling microscopy (STM) and tip-enhanced Raman scattering (TERS) studies, the reversible photoisomerization of one molecule is captured in TERS trajectories. The unique signature of single molecule isomerization is observed in the form of anticorrelated flip-flops between two distinct spectra with two discrete, on- and off-levels. The apparently heterogeneously photocatalyzed reaction is assigned to cis-trans isomerization of an outlier, which is chemisorbed on the silver tip of the STM. Otherwise, the ensemble of ABT molecules that lie flat on Au(111) remain strongly coupled to the surface, excluding the possibility of photoisomerization or detection through TERS.

Single electron bipolar conductance switch driven by the molecular Aharonov-Bohm effect.

We demonstrate a conductance switch controlled by the spin-vibronic density of an odd electron on a single molecule. The junction current is modulated by the spin-flip bistability of the electron. Functional images are provided as wiring diagrams for control of the switch's frequency, amplitude, polarity, and duty-cycle. The principle of operation relies on the quantum mechanical phase associated with the adiabatic circulation of a spin-aligned electron around a conical intersection. The functional images quantify the governing vibronic Hamiltonian.

Note: Automated electrochemical etching and polishing of silver scanning tunneling microscope tips.

Fabrication of sharp and smooth Ag tips is crucial in optical scanning probe microscope experiments. To ensure reproducible tip profiles, the polishing process is fully automated using a closed-loop laminar flow system to deliver the electrolytic solution to moving electrodes mounted on a motorized translational stage. The repetitive translational motion is controlled precisely on the µm scale with a stepper motor and screw-thread mechanism. The automated setup allows reproducible control over the tip profile and improves smoothness and sharpness of tips (radius 27 ± 18 nm), as measured by ultrafast field emission.