The development of optical nanosensors for biological measurements

This article discusses and documents the basic concepts of, and developments in, the field of optical nanosensors and nanobiosensors. It describes the progression of this field of research from its birth up to the present, with emphasis on the techniques of sensor construction and their application to biological systems. After a brief overview of the techniques for fabricating nanometer-sized optical fibers, we describe the various types of transducer and bioreceptor molecule presently used for nanosensor and nanobiosensor fabrication.     

Improved differentiation between luminescence decay components by use of time-resolved optical activity measurements and selective lifetime modulation.

The analysis of luminescence decay experiments from proteins is typically modeled as a combination of independent first-order decay functions. However, Poisson noise in the photon counting experiment limits the ability of this approach to resolve decay components from separate lumiphores with similar lifetimes. To provide further differentiation, we incorporate time-resolved circular polarization of luminescence, an additional independent observable, into the analysis. In the simplest case, for example, each lumiphore's chirality is assumed to be time independent and is determined by the position of the lumiphore with respect to the surrounding chiral environment within the protein. In this paper, we describe the analysis of simultaneously recorded time-resolved luminescence and circularly polarized luminescence data to obtain improved temporal resolution. When combined with selective dynamic luminescence quenching, in a model system comprising a mixture of Tb/transferrin and Tb/conalbumin, we demonstrate resolution between two decay components with a lifetime difference of 7% and a difference in emission anisotropy of 5 X 10(-2). Evidence for the improved discrimination is further demonstrated by the increase in curvature of the chi 2 surface that results from the additional information.     

The optical activity of polypeptides

The present status of the theory of the optical properties of polypeptides is discussed in a general way. Arguments are presented that indicate that the interaction of exciton bands in helices will occur in a tensorial way with the parallel polarized bands normally having stronger interactions than the perpendicular polarized bands, with only small cross-interactions. The net result is an anisotropy of the hyperchromism of the ππ* band and an imbalance of the rotatory strengths of the parallel and perpendicular components. According to the evidence of Mandel and Holzwarth [(1973) Biopolymers 13 , 655–674], this is, in fact, the origin of the strongly nonconservative CD bands of polyproline II and related structures. The implications of anisotropic hyperchromicity on the interpretation of the linear dichroism of crystals is discussed.     

The use of image analysis for morphological measurements on filamentous microorganisms

Characterization of mycelial morphology is important for the design and operation of filamentous fermentations. Initial investigations have been made of semiautomated image analysis as a replacement for a digitizing-table method. It was shown that the image analysis method was more precise than the digitizing-table method used, although this extra precision is unimportant in this application. On average, image analysis gave mean hyphal lengths 6% greater than digitizing, because the latter used chord lengths to represent are lengths. For short branches image analysis was less accurate. In time and convenience the image analysis method had an overwhelming advantage, and this advantage might be enhanced by full automation. The resulting ability to characterize mycelial morphology rapidly would permit such characterization to be used routinely in studies of filamentous fermentations.     

The use of Lanthanum for intracellular calcium measurements in cultured cells

The Lanthanum method, used extensively to measure the intracellular Ca content ([Ca]i) of muscle, was adapted for cell culture using the carcinoma line C-4II. Reproducible values of [Ca]i were obtained by atomic absorption spectrophotometry after 30 min of rinsing of monolayer fragments with cold 10 mM LaCl₃ in 160 mM Tris-HCl buffer, pH 7.4. [Ca]i values were not influenced by the extracellular Ca concentration, the size of the monolayer fragments or by several culture variables examined. [Ca]i levels were inversely proportional to cell density. The modified La method provides a relatively simple means for [Ca]i measurements and the estimation of net Ca fluxes in and out of cultured cells.     

Interlaced optical force-fluorescence measurements for single molecule biophysics

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence, does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-basepair DNA segment labeled with Cy3.     

Local presynaptic activity gates homeostatic changes in presynaptic function driven by dendritic BDNF synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.     

The use of FPLC for microdetermination of enzyme activity

Summary The FPLC system was used for the microdetermination of enzyme activities via the micromeasurement of absorbance of the reaction mixture. The sample loop was directly connected with the monitor. The solution to be measured was injected into the flow of water and the monitor response was measured as a peak height with the built-in integrator. Only 40 l of the solution are needed for one absorbance measurement.     

Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.     

Advances in optical detection strategies for reporter signal measurements

Many recent advances in bioreporter technology focus on challenges related to bioengineering, yet in many applications implementation of optical signal measurement is equally susceptible to improvement. For bioluminescent bioreporters, one area of effort lies in the development of semiconductor chip-based detector modules; this holds great promise for ultra-compact and field-deployable instrumentation, but has not yet had a palpable impact on improved detection limits. Regarding lower detection limits, single-molecule detection techniques have seen their first application to bioreporters, and preliminary results serve as an indication of future promise. Another technique applicable to fluorescent bioreporters is fluorescence flow cytometry, which is rapid, suitable for high-throughput screening, and lends itself to increased analytical specificity through simple algorithmic approaches to data treatment.     

The use of optical spectroscopy in combinatorial chemistry

Infrared and Raman spectroscopy allow direct spectral analysis of the solid‐phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as “pins” or “crowns.” Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on‐bead monitoring of solid‐phase reactions. Providing identification based on molecular structure, HPLC‐FTIR is, therefore, complementary to LC‐MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and—using a Raman microscope—to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:179–187, 1998/1999.     

Single-step stereolithography of complex anatomical models for optical flow measurements

Transparent stereolithographic rapid prototyping (RP) technology has already demonstrated in literature to be a practical model construction tool for optical flow measurements such as digital particle image velocimetry (DPIV), laser doppler velocimetry (LDV), and flow visualization. Here, we employ recently available transparent RP resins and eliminate time-consuming casting and chemical curing steps from the traditional approach. This note details our methodology with relevant material properties and highlights its advantages. Stereolithographic model printing with our procedure is now a direct single-step process, enabling faster geometric replication of complex computational fluid dynamics (CFD) models for exact experimental validation studies. This methodology is specifically applied to the in vitro flow modeling of patient-specific total cavopulmonary connection (TCPC) morphologies. The effect of RP machining grooves, surface quality, and hydrodynamic performance measurements as compared with the smooth glass models are also quantified.     

The presynaptic active zone

Neurotransmitters are released by synaptic vesicle exocytosis at the active zone of a presynaptic nerve terminal. In this review, I discuss the molecular composition and function of the active zone. Active zones are composed of an evolutionarily conserved protein complex containing as core constituents RIM, Munc13, RIM-BP, α-liprin, and ELKS proteins. This complex docks and primes synaptic vesicles for exocytosis, recruits Ca(2+) channels to the site of exocytosis, and positions the active zone exactly opposite to postsynaptic specializations via transsynaptic cell-adhesion molecules. Moreover, this complex mediates short- and long-term plasticity in response to bursts of action potentials, thus critically contributing to the computational power of a synapse.