Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of (...) fluorescence fluctuation spectroscopy. (shrink)

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution‐based single molecule technique, fluorescence correlation spectroscopy (FCS). This time‐averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has (...) proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual‐color cross‐correlation and two‐photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research. BioEssays 24:758–764, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

A recent article argued that signals from conventional Raman spectroscopy of organic materials are overwhelmed by edge filter and fluorescence artefacts. The article targeted a subset of Raman spectroscopic investigations of fossil and modern organisms and has implications for the utility of conventional Raman spectroscopy in comparative tissue analytics. The inferences were based on circular reasoning centered around the unconventional analysis of spectra from just two samples, one modern, and one fossil. We validated the disputed signals with (...) in situ Fourier‐Transform Infrared (FT‐IR) Spectroscopy and through replication with different lasers, filters, and operators in independent laboratories. Our Raman system employs a holographic notch filter which is not affected by edge filter or other artefacts. Multiple lines of evidence confirm that conventional Raman spectra of fossils contain biologically and geologically meaningful information. Statistical analyses of large Raman and FT‐IR spectral data sets reveal patterns in fossil composition and yield valuable insights into the history of life. (shrink)

Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio‐temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of (...) fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross‐correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio‐temporal map of molecular interactions. (shrink)

Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence microscopy (...) offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review. The functional mechanism of proteins is often driven by underlying conformational changes. These intramolecular changes and dynamics can be investigated using single pair Förster resonance energy transfer. The kinetic timescales of the dynamics in the range of nanoseconds to seconds is then quantified using different experimental and analytical implementations. (shrink)

Actin plays several essential roles in cellular processes and is a vital component in the contractile apparatus. To accomplish its many cellular tasks, actin must interact with a wide range of other proteins in addition to self‐assembling into filaments. Characterization of these functional domains and localized binding regions on the actin monomer is therefore an important undertaking. Strategies for elucidating the many interaction sites include X‐ray diffraction, NMR and fluorescence spectroscopy, chemical modification, chemical cross‐linking, protein cleavage, and the (...) study of sequence homologies between the many isotypes of actin. Based on these varied data, we discuss the possible spatial relationships between the interaction sites. (shrink)

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the (...) impact of modern technologies is influenced not only by further hard‐ and software developments, but also by the availability and suitability of protein‐engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. (shrink)

Luminescence‐based sensing schemes for oxygen have experienced a fast growth and are in the process of replacing the Clark electrode in many fields. Unlike electrodes, sensing is not limited to point measurements via fiber optic microsensors, but includes additional features such as planar sensing, imaging, and intracellular assays using nanosized sensor particles. In this essay, I review and discuss the essentials of (i) common solid‐state sensor approaches based on the use of luminescent indicator dyes and host polymers; (ii) fiber optic (...) and planar sensing schemes; (iii) nanoparticle‐based intracellular sensing; and (iv) common spectroscopies. Optical sensors are also capable of multiple simultaneous sensing (such as O2 and temperature). Sensors for O2 are produced nowadays in large quantities in industry. Fields of application include sensing of O2 in plant and animal physiology, in clinical chemistry, in marine sciences, in the chemical industry and in process biotechnology. (shrink)

We consider the possibility that the relative phase in quantum mechanics plays a role in determining measurement outcome and could therefore serve as a “hidden” variable. The Born rule for measurement equates the probability for a given outcome with the absolute square of the coefficient of the basis state, which by design removes the relative phase from the formulation. The value of this phase at the moment of measurement naturally averages out in an ensemble, which would prevent any dependence from (...) being observed, and we show that conventional frequency-spectroscopy measurements on discrete quantum systems cannot be imposed at a specific phase due to a straightforward uncertainty relation. We lay out general conditions for imposing measurements at a specific value of the relative phase so that the possibility of its role as a hidden variable can be tested, and we discuss implementation for the specific case of an atomic two-state system with laser-induced fluorescence for measurement. (shrink)

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live‐cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high (...) class='Hi'>fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. (shrink)

Spectroscopy is a scientific topic at the interface between Chemistry and Physics, which is taught at high school level in relation with its fundamental applications in Analytical Chemistry. In the first part of the paper, the topic of spectroscopy is analyzed having in mind the well-known Johnstone’s triangle of chemistry education, putting in evidence the way spectroscopy is usually taught at the three levels of chemical knowledge: macroscopic/phenomenological, sub-microscopic/molecular and symbolic ones. Among these three levels, following Johnstone’s (...) recommendations the macroscopic one is the most useful for high school students who learn spectroscopy for the first time. Starting from these premises, in the second part of the paper, we propose a didactic sequence which is inspired by the historical evolution of spectroscopic instruments from the first spectroscopes invented by Gustav Kirchhoff and Robert Bunsen in 1860 to the UV–vis spectrophotometers which became common since the 1960s. The idea behind our research is to analyze the conceptual advancements through the history of spectroscopy and to identify the key episodes/experiments and spectroscopic instruments. For each of them, a didactic activity, typically an experiment, is then proposed underlining the relevant aspects from the chemistry education point of view. The present paper is the occasion to reflect on the potentialities of an historical approach combined with a laboratorial one, and to discuss the role of historical instruments and related technological improvements to teach spectroscopy. (shrink)

Phototoxicity frequently occurs during live fluorescence microscopy, and its consequences are often underestimated. Damage to cellular macromolecules upon excitation light illumination can impair sample physiology, and even lead to sample death. In this review, we explain how phototoxicity influences live samples, and we highlight that, besides the obvious effects of phototoxicity, there are often subtler consequences of illumination that are imperceptible when only the morphology of samples is examined. Such less apparent manifestations of phototoxicity are equally problematic, and can (...) change the conclusions drawn from an experiment. Thus, limiting phototoxicity is a prerequisite for obtaining reproducible quantitative data on biological processes. We present strategies to reduce phototoxicity, e.g. limiting the illumination to the focal plane and suggest controls for phototoxicity effects. Overall, we argue that phototoxicity needs increased attention from researchers when designing experiments, and when evaluating research findings. (shrink)

Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of (...) the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem‐specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment. (shrink)

A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. (...) Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180–187, 2000. ©2000 John Wiley & Sons, Inc. (shrink)

We show that linear Regge trajectories for mesons and glueballs, and the cubic mass spectrum associated with them, determine a relation between the masses of the ρ meson and the scalar glueball, $M(0^{ + + } ) = 3/\sqrt 2 M(\rho )$ , which implies $M(0^{ + + } ) = 1650_ \pm 10$ MeV. We also discuss relations between the masses of the scalar, tensor and 3-- glueballs, $M(2^{ + + } ) = \sqrt 2 M(0^{ + + } (...) ),{\text{ }}M(3^{ - - } ) = 2M(0^{ + + } )$ , which imply $M(2^{ + + } ) = 2290_ \pm {\text{ }}15{\text{ MeV, }}M(3^{ - - } ) = 3240_ \pm {\text{ }}20$ MeV. (shrink)

A hundred years ago the science of spectroscopy, though not yet christened, may be said to have attained its majority and to be just entering on its period of full adult development. It was born, of course, with Newton's explanation of the formation of the spectrum, and for many years thereafter little of importance was added to what he had discovered. It was not, in fact, until the nineteenth century that anything of outstanding importance occurred, and then, in 1802, (...) Wollaston substituted a slit for the round hole through which Newton's sunlight passed into his prism, and thereby not only saw for the first time the dark lines in the solar spectrum but also took the first step towards the perfection of the spectroscope on which all later progress depended. The next step was taken by Fraunhofer who, in 1814, examined the spectrum through a telescope instead of letting it fall on a screen. The last essential improvement—the introduction of the collimator to make the light from the slit parallel before it entered the prism—was introduced in 1839 independently by Simms and Swan, so that before our period begins, the complete spectroscope existed, though it was not to be converted into a spectrograph, for photographing spectra, until much later. (shrink)

Principles of Laser Spectroscopy and Quantum Optics is an essential textbook for graduate students studying the interaction of optical fields with atoms. It also serves as an ideal reference text for researchers working in the fields of laser spectroscopy and quantum optics. The book provides a rigorous introduction to the prototypical problems of radiation fields interacting with two- and three-level atomic systems. It examines the interaction of radiation with both atomic vapors and condensed matter systems, the density matrix (...) and the Bloch vector, and applications involving linear absorption and saturation spectroscopy. Other topics include hole burning, dark states, slow light, and coherent transient spectroscopy, as well as atom optics and atom interferometry. In the second half of the text, the authors consider applications in which the radiation field is quantized. Topics include spontaneous decay, optical pumping, sub-Doppler laser cooling, the Heisenberg equations of motion for atomic and field operators, and light scattering by atoms in both weak and strong external fields. The concluding chapter offers methods for creating entangled and spin-squeezed states of matter. Instructors can create a one-semester course based on this book by combining the introductory chapters with a selection of the more advanced material. A solutions manual is available to teachers. Rigorous introduction to the interaction of optical fields with atoms Applications include linear and nonlinear spectroscopy, dark states, and slow light Extensive chapter on atom optics and atom interferometry Conclusion explores entangled and spin-squeezed states of matter Solutions manual. (shrink)

Brain-computer interfaces (BCIs) are being investigated as an access pathway to communication for individuals with physical disabilities, as the technology obviates the need for voluntary motor control. However, to date, minimal research has investigated the use of BCIs for children. Traditional BCI communication paradigms may be suboptimal given that children with physical disabilities may face delays in cognitive development and acquisition of literacy skills. Instead, in this study we explored emotional state as an alternative access pathway to communication. We developed (...) a pediatric BCI to identify positive and negative emotional states from changes in hemodynamic activity of the prefrontal cortex (PFC). To train and test the BCI, 10 neurotypical children aged 8–14 underwent a series of emotion-induction trials over four experimental sessions (one offline, three online) while their brain activity was measured with functional near-infrared spectroscopy (fNIRS). Visual neurofeedback was used to assist participants in regulating their emotional states and modulating their hemodynamic activity in response to the affective stimuli. Child-specific linear discriminant classifiers were trained on cumulatively available data from previous sessions and adaptively updated throughout each session. Average online valence classification exceeded chance across participants by the last two online sessions (with 7 and 8 of the 10 participants performing better than chance, respectively, in Sessions 3 and 4). There was a small significant positive correlation with online BCI performance and age, suggesting older participants were more successful at regulating their emotional state and/or brain activity. Variability was seen across participants in regards to BCI performance, hemodynamic response, and discriminatory features and channels. Retrospective offline analyses yielded accuracies comparable to those reported in adult affective BCI studies using fNIRS. Affective fNIRS-BCIs appear to be feasible for school-aged children, but to further gauge the practical potential of this type of BCI, replication with more training sessions, larger sample sizes, and end-users with disabilities is necessary. (shrink)

Carbon monoxide intoxication leads to acute and chronic neurological deficits, but little is known about the specific noxious mechanisms. 1 H magnetic resonance spectroscopy may allow insight into the pathophysiology of CO poisoning by monitoring neurochemical disturbances, yet only limited information is available to date on the use of this protocol in determining the neurological effects of CO poisoning. To further examine the short-term and long-term effects of CO on the central nervous system, we have studied seven patients with (...) CO poisoning assessed by gray and white matter MRS, magnetic resonance imaging and neuropsychological testing. Five patients suffered from acute high-dose CO intoxication and were in coma for 1–6 days. In these patients, MRI revealed hyperintensities of the white matter and globus pallidus and also showed increased choline and decreased N -acetyl aspartate ratios to creatine, predominantly in the white matter. Lactate peaks were detected in two patients during the early phase of high-dose CO poisoning. Two patients with chronic low-dose CO exposure and without loss of consciousness had normal MRI and MRS scans. On follow-up. five of our seven patients had long-lasting intellectual impairment, including one individual with low-dose CO exposure. The MRS results showed persisting biochemical alterations despite the MRI scan showing normalization of morphological changes. In conclusion, the MRS was normal in patients suffering from chronic low-dose CO exposure; in contrast, patients with high-dose exposure showed abnormal gray and white matter levels of NAA/Cr, Cho/Cr and lactate, as detected by 1 H MRS, suggesting disturbances of neuronal function, membrane metabolism and anaerobic energy metabolism, respectively. Early increases in Cho/Cr and decreases of NAA/Cr may be related to a poor long-term outcome, but confirmation by future studies is needed. (shrink)

Members of the family of the Green Fluorescent Protein (GFP) are the only known type of natural pigments that are essentially encoded by a single gene, since both the substrate for pigment biosynthesis and the necessary catalytic moieties are provided within a single polypeptide chain. In sharp contrast to the state of knowledge just three years ago when GFP was the only known protein of its kind, a whole family of related proteins, exhibiting striking diversity of features have now been (...) identified. This provides new possibilities for a variety of studies ranging from applied biotechnology to evolutionary ecology. BioEssays 24:953–959, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

The present study aimed to examine the neural mechanisms underlying the ability to process the mental rotation with mirrored stimuli for different depressive tendencies with psychomotor retardation. Using functional near-infrared spectroscopy, we measured brain cortex activation of participants with higher and lower depressive tendencies while performing a left-right paradigm of object mental rotation or a same-different paradigm of subject mental rotation. Behavioral data revealed no differences in reaction time and rotation speed. The fNIRS data revealed a higher deactivation of (...) oxyhemoglobin change for the higher depression group in the perceptual stage of object mental rotation with mirrored stimuli in the superior external frontal cortex, inferior frontal gyrus, premotor cortex, and primary motor cortex. In addition, there existed a significant difference between the two groups in premotor cortex in subject mental rotation with mirrored stimuli. These results suggest that the neural mechanism of higher depression individuals connected with psychomotor retardation exists in the frontal and motor areas when processing object mental rotation with mirrored stimuli, and the motor cortex when processing subject mental rotation. (shrink)

This article looks at Vesto Slipher’s work on nebular spectroscopy between 1912 and 1922as well as related research by other astronomers of the period, and it examines the dissem-ination of their results more widely. Slipher’s observations are viewed as marking the di-viding line between speculation about the universe in traditional astronomy and theadvent of modern cosmology and the theory of an expanding universe. The intent is todocument the dissemination of Slipher’s results in the period leading up to the publicationof (...) studies of relativistic cosmology by Willem de Sitter in 1917 and Alexander Friedmannin 1922. Themes touched on in the article include the unprecedented character ofSlipher’sfindings and the interaction of observation and theory in modern cosmology.A prominent concern is the role of technology in astronomical science over the past cen-tury and a half. Here reference is made to the writings of Paul Forman on historical shiftsthat have taken place in our understanding of the relationship of science and technology. (shrink)

Widespread preservation of fossilized biomolecules in many fossil animals has recently been reported in six studies, based on Raman microspectroscopy. Here, we show that the putative Raman signatures of organic compounds in these fossils are actually instrumental artefacts resulting from intense background luminescence. Raman spectroscopy is based on the detection of photons scattered inelastically by matter upon its interaction with a laser beam. For many natural materials, this interaction also generates a luminescence signal that is often orders of magnitude (...) more intense than the light produced by Raman scattering. Such luminescence, coupled with the transmission properties of the spectrometer, induced quasi‐periodic ripples in the measured spectra that have been incorrectly interpreted as Raman signatures of organic molecules. Although several analytical strategies have been developed to overcome this common issue, Raman microspectroscopy as used in the studies questioned here cannot be used to identify fossil biomolecules. (shrink)

The shape of the spectral lines of an optically active system interacting with one or more strong radiation fields in the presence of a perturbing bath is studied. A method based on the statistics of the fluctuation of the interaction between the radiator and the perturbing environment (the model Markov microfield theory) is used. This method permits the foundations of line shape theory in modern statistical mechanics to be seen clearly. Multiphoton processes and homogeneous, inhomogeneous, and power broadening mechanisms are (...) included in the analysis. The correlations between radiative and collisional processes which arise in nonlinear spectroscopy are included explicitly. A discussion of the new information that is obtained from these correlations in nonlinear spectroscopy is also presented. Several model systems are presented as illustrative examples of the theory. (shrink)

This study aims to examine the neural correlates of cognitive shifting during the Dimensional Change Card Sort Task task with functional near-infrared spectroscopy. Altogether 49 children completed the DCCS tasks, and 25 children passing all items were classified into the Switch group. Twenty children committing more than one perseverative errors were grouped into the Perseverate group. The Switch group had Brodmann Area 9 and 10 activated in the pre-switch period and BA 6, 9, 10, 40, and 44 in the (...) post-switch period. In contrast, the Perseverate group had BA 9 and 10 activated in the pre-switch period and BA 8, 9, 10 in the post-switch period. The general linear model results afford strong support to the “V-shape curve” hypothesis by identifying a significant decrease–increase cycle in BA 9 and 44, the neural correlations of cognitive shifting. (shrink)

The so-called Ramsey–Carnap approach, or Ramseyfication, has gone out of fashion in the philosophy of science. Advocates have tried to argue for a revival by writing methodological and metatheoretical studies of Ramseyfication. For this paper I have chosen a different approach; I will apply Ramseyfication to infrared spectroscopy—a method used in analytical chemistry—in order to logically analyse the relation between measurements and mathematical structures. My aim in doing so is to contribute to the debate about the _Application Problem of (...) Mathematics_, thereby making a case for Ramseyfication as a method of study for scientific theories. (shrink)

Recent magnetic resonance imaging and pathological studies have indicated that axonal loss is a major contributor to disease progression in multiple sclerosis. 1 H magnetic resonance spectroscopy, through measurement of N -acetyl aspartate, a neuronal marker, provides a unique tool to investigate this. Patients with primary progressive multiple sclerosis have few lesions on conventional MRI, suggesting that changes in normal appearing white matter, such as axonal loss, may be particularly relevant to disease progression in this group. To test this (...) hypothesis NAWM was studied with MRS, measuring the concentration of N -acetyl derived groups. Single-voxel MRS using a water-suppressed PRESS sequence was carried out in 24 patients with primary progressive multiple sclerosis and in 16 age-matched controls. Ratios of metabolite to creatine concentration were calculated in all subjects, and absolute concentrations were measured in 18 patients and all controls. NA/Cr was significantly lower in NAWM in patients than in controls, as was the absolute concentration of NA. There was no significant difference in the absolute concentration of creatine between the groups. This study supports the hypothesis that axonal loss occurs in NAWM in primary progressive multiple sclerosis and may well be a mechanism for disease progression in this group. (shrink)

Behaviorally, children’s explicit theory of mind (ToM) proceeds in a progression of mental-state understandings: developmentally, children demonstrate accurate explicit desire-reasoning before accurate explicit belief-reasoning. Given its robust and cross-cultural nature, we hypothesize this progression may be paced in part by maturation/specialization of the brain. Neuroimaging research demonstrates that the right temporoparietal junction (TPJ) becomes increasingly selective for ToM reasoning as children age, and as their ToM improves. But this research has narrowly focused on beliefs or on undifferentiated mental-states. A recent (...) ERP study in children included a critical contrast to desire-reasoning, and demonstrated that right posterior potentials differentiated belief-reasoning from desire-reasoning. Taken together, the literature suggests that children’s desire-belief progression may be paced by specialization of the right TPJ for belief-reasoning specifically, beyond desire-reasoning. In the present study, we tested this hypothesis directly by examining children’s belief- and desire-reasoning using functional near-infrared spectroscopy in conjunction with structural magnetic resonance imaging to pinpoint brain activation in the right TPJ. Results showed greatest activation in the right TPJ for belief-reasoning, beyond desire-reasoning, and beyond non-mental reasoning (control). Findings replicate and critically extend prior ERP results, and provide clear evidence for a specific neural mechanism underlying children’s progression from understanding desires to understanding beliefs. (shrink)