Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy

We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.     

Kinetic multichannel spectroscopy of biological molecules: decomposition of the spectral matrix

The time-resolved difference spectra after flash excitation of various biological molecules are measured on a gated optical multichannel analyzer. Electron transfer between the photoactive covalent label thiouredopyrene-trisulfonate and the heme of cytochrome c and the photocycle of the E204Q mutant bacteriorhodopsin are studied. The spectral matrices containing consecutive difference spectra are analyzed to reveal the reaction kinetics and individual component spectra. Singular value decomposition combined with stoichiometric analysis and self-modeling is demonstrated as a tool for successful dissection of the matrix, despite the difficulties arising from broad, featureless, overlapping spectra and the overlapping kinetics of the components.     

Collective vibrational modes in biological molecules investigated by terahertz time-domain spectroscopy

We present well-resolved absorption spectra of biological molecules in the far-IR (FIR) spectral region recorded by terahertz time-domain spectroscopy (THz-TDS). As an illustrative example we discuss the absorption spectra of benzoic acid, its monosubstitutes salicylic acid (2-hydroxy-benzoic acid), 3- and 4-hydroxybenzoic acid, and aspirin (acetylsalicylic acid) in the spectral region between 18 and 150 cm(-1). The spectra exhibit distinct features originating from low-frequency vibrational modes caused by intra- or intermolecular collective motion and lattice modes. Due to the collective origin of the observed modes the absorption spectra are highly sensitive to the overall structure and configuration of the molecules, as well as their environment. The THz-TDS procedure can provide a direct fingerprint of the molecular structure or conformational state of a compound.     

High-resolution magnetic resonance spectroscopy of the mouse vomeronasal organ

The chemical composition of the vomeronasal organ (VNO) was investigated by means of in vitro proton magnetic resonance spectroscopy (MRS) in prepubertal and adult mice of both sexes. Results demonstrate that MRS detects several chemical constituents in the VNO, showing their age- and sex-associated changes in concentration. Preliminary experiments also suggest the ability of MRS to show compositional changes in the VNO after pheromonal stimulation. MRS can serve as a useful technique to investigate vomeronasal chemoreception.     

Nonlinear optical spectroscopy of chiral molecules

We review nonlinear optical processes that are specific to chiral molecules in solution and on surfaces. In contrast to conventional natural optical activity phenomena, which depend linearly on the electric field strength of the optical field, we discuss how optical processes that are nonlinear (quadratic, cubic, and quartic) functions of the electromagnetic field strength may probe optically active centers and chiral vibrations. We show that nonlinear techniques open entirely new ways of exploring chirality in chemical and biological systems: The cubic processes give rise to nonlinear circular dichroism and nonlinear optical rotation and make it possible to observe dynamic chiral processes at ultrafast time scales. The quadratic second-harmonic and sum-frequency-generation phenomena and the quartic processes may arise entirely in the electric-dipole approximation and do not require the use of circularly polarized light to detect chirality. They provide surface selectivity and their observables can be relatively much larger than in linear optical activity. These processes also give rise to the generation of light at a new color, and in liquids this frequency conversion only occurs if the solution is optically active. We survey recent chiral nonlinear optical experiments and give examples of their application to problems of biophysical interest.     

Resonant interactions between biological molecules

Forces between localized, oscillating vibrations on molecules show strongly resonant dependence on the relative frequencies of the two oscillators; i.e. there is a force between the vibrating parts of two molecules if the frequencies are the same but none if the frequencies are different. A vibrating site on a large molecule can attract another specific type of molecule out of a medium, one which has a vibrational mode at the same frequency, without influencing other types of molecules in the medium. These resonant forces are thus highly selective. Other pairs of molecules could similarly interact using vibrations at a different frequency. In a complex medium such as a cell, many different types of interactions could be assisted by these resonant forces wtihout interfering with each other. These forces could be found to be useful in assisting some metabolic reactions. It is interesting that they are vulnerable to selective suppression or enhancement by externally applied electromagnetic radiation of correspondingly specific frequencies. Supported by NIH Grant #GM 24443-01 and Amer. Cancer Society Grant #IN17Q.     

Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans-cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.     

Workshop--Predicting the structure of biological molecules

This April, in Cambridge (UK), principal investigators from the Mathematical Biology Group of the Medical Research Council's National Institute of Medical Research organized a workshop in structural bioinformatics at the Centre for Mathematical Sciences. Bioinformatics researchers of several nationalities from labs around the country presented and discussed their computational work in biomolecular structure prediction and analysis, and in protein evolution. The meeting was intensive and lively and gave attendees an overview of the healthy state of protein bioinformatics in the UK.     

Optical waveguide detection of small molecules with two immobilized reagents

A benzyl viologen containing Si(OMe)₃ groups was covalently attached to the surface of glass waveguides. The viologen-coated waveguides were then silanized and formate dehydrogenase was bound to pendant amino groups on the silane via glutaraldehyde. Successful linking of both the redox-sensitive viologen dye and the enzyme was demonstrated by reaction of the bound enzyme with formate ion in the presence of NAD. Production of NADH by the enzyme-catalyzed reaction resulted in a reduction of the positive charge on the bound viologen and a reversible color change that was detectable with the optical waveguide.     

Periodic acceptor excitation spectroscopy of single molecules

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.     

From molecules to dynamic biological communities

Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease.     

On a possible biological spectroscopy

A new type of physical transition, denotedS→S ^*, has been detected in irradiated organic molecules (λ=546 nm) through their interaction with specific biological macromolecules. In a specific enzyme-substrate interaction, a clear enhancement of the reaction rate is observed, when the substrate is irradiated with sharply well defined times. These “efficient irradiation times” are always of the 5k sec type (k=1, 2, 3, …). They have been consistently revealed in a great number of specific biological interactions. The present note demonstrates an important property, i.e. that forevery irradiation time aS→S ^* transition is induced in organic molecules. It is shown that for any irradiation times different from the 5k sec type (k=1, 2, 3, …) states of theS ^* type may occur, but the biological macromolecules may “detect” only theS ^* states induced by irradiations of the 5k sec type.     

Laser Doppler spectroscopy of biological objects

Laser Doppler spectroscopy permits to investigate the velocity distribution of the biological objects either single cells or ensembles. The main principles of laser Doppler spectroscopy and its practical application connected with the spectral correlation dependence of biological objects have been studied.     

Synthesis of biological molecules on molecular sieves

Catalytic properties of aluminosilicates may play a role in the synthesis of biological molecules from simple gaseous molecules commonly found in planetary atmospheres. Urea, amino acids and UV absorbing substances have been obtained by heating CO and NH₃ with Linde molecular sieves saturated with Ca⁺², NH₄ ⁺ or Fe⁺³. The yields of amino acids produced have been determined by an amino acid analyzer. The quantity of urea produced largely depends on the nature of the saturating cation. Experiments using¹⁴CO confirm that the amino acids are not due to contaminants adsorbed on the surface of the molecular sieves.