This PDF file contains the editorial “Special Section Guest Editorial: Tools for Biomolecular and Cellular Analysis” for JBO Vol. 7 Issue 04

Semiconductor nanoparticles in the size range of 2–6 nm are of great current interest, not only because of their size-tunable properties but also because of their dimensional similarity with biological macromolecules (e.g., nucleic acids and proteins). This similarity could allow an integration of nanomaterials with biological molecules, which would have applications in medical diagnostics, targeted therapeutics, and high-throughput drug screening. Here we report new developments in preparing highly luminescent and biocompatible CdSe quantum dots (QDs), and in synthesizing QD-encoded micro- and nano-beads in the size range of 100 nm–10 μm. We show that the optical properties of ZnS-capped CdSe quantum dots are sensitive to environmental factors such as pH and divalent cations, leading to the potential use of quantum dots in molecular sensing. We also show that chemically modified proteins can be used to coat the surface of water-soluble QDs, to restore their fluorescence, and to provide functional groups for bioconjugation. For multiplexed optical encoding, we have prepared large microbeads with sizes similar to that of mammalian cells, and small nanobeads with sizes similar to that of viruses.

We have developed a new series of glucose sensitive fluorophores that display shifts in emission wavelengths and/or intensity change upon the binding of monosaccharides. Complexation of glucose with the boronic acid moiety changes both its orbital hybridization and its ability to accept and donate electrons. This change results in distinct emission spectra for the fluorophores when free in solution or complexed with monosaccharide. The spectral changes upon saccharide binding can be modified by substitution of electron donor or acceptor group on the fluorophore allowing rational design of the spectral response.

We investigate optoelectronic properties of integrated structures comprising semiconductor light-emitting materials for optical probes of microscopic biological systems. Compound semiconductors are nearly ideal light emitters for probing cells and other microorganisms because of their spectral match to the transparency wavelengths of biomolecules. Unfortunately, the chemical composition of these materials is incompatible with the biochemistry of cells and related biofluids. To overcome these limitations, we investigate functionalized semiconductor surfaces and structures to simultaneously enhance light emission and the flow of biological fluids in semiconductor microcavities. We have identified several important materials problems associated with the semiconductor/biosystem interface. One is the biofluid degradation of electroluminescence by ionic diffusion into compound semiconductors. Ions that diffuse into the active region of a semiconductor light emitter can create point defects that degrade the quantum efficiency of the radiative recombination process. In this paper we discuss ways of mitigating these problems using materials design and surface chemistry, and suggest future applications for these materials.

Zinc is a metal ion of increasing significance in several biomedical fields, including neuroscience, immunology, reproductive biology, and cancer. Fluorescent indicators have added greatly to our understanding of the biology of several metal ions, most notably calcium. Despite substantial efforts, only recently have zinc indicators been developed which are sufficiently selective for use in the complex intra- and extracellular milieus, and which are capable of quantifying the free zinc levels with some degree of reliability. However, these indicators (such as FuraZin-1 and Newport Green DCF) have only modest sensitivity, and there is growing evidence that significantly lower levels of free zinc may be biologically relevant in some instances. We have adapted the peerless selectivity and sensitivity of a carbonic anhydrase-based indicator system to an excitation ratiometric format based on resonance energy transfer: i.e., where the zinc ion level is transduced as the ratio of fluorescence intensities excited at two different excitation wavelengths, which is preferred for fluorescence microscopy. The system exhibits more than a 60% increase in the ratio of intensity excited at 365 nm to that excited at 546 nm (emission observed at 617 nm). The detection limit is about 10 pM in zinc buffered systems, a 10–1000-fold improvement on the Fura indicators (which respond to Ca and Mg as well), and a 10 000-fold improvement on the recently described FuraZin-1.

Pharmaceutical oral dosage forms are used in this paper to test the sensitivity and spatial resolution of hyperspectral imaging instruments. The first experiment tested the hypothesis that a near-infrared (IR) tunable diode-based remote sensing system is capable of monitoring degradation of hard gelatin capsules at a relatively long distance (0.5 km). Spectra from the capsules were used to differentiate among capsules exposed to an atmosphere containing 150 ppb formaldehyde for 0, 2, 4, and 8 h. Robust median-based principal component regression with Bayesian inference was employed for outlier detection. The second experiment tested the hypothesis that near-IR imaging spectrometry of tablets permits the identification and composition of multiple individual tablets to be determined simultaneously. A near-IR camera was used to collect thousands of spectra simultaneously from a field of blister-packaged tablets. The number of tablets that a typical near-IR camera can currently analyze simultaneously was estimated to be approximately 1300. The bootstrap error-adjusted single-sample technique chemometric-imaging algorithm was used to draw probability-density contour plots that revealed tablet composition. The single-capsule analysis provides an indication of how far apart the sample and instrumentation can be and still maintain adequate signal-to-noise ratio (S/N), while the multiple-tablet imaging experiment gives an indication of how many samples can be analyzed simultaneously while maintaining an adequate S/N and pixel coverage on each sample.

The near-infrared region of the spectrum (650–1100 nm) offers distinct advantages over the traditional UV/vis region for spectroscopic measurements. In the past, the lack of commercially available equipment capable of working in the near infrared limited the utility of near-infrared techniques. However, since the advent of photodiodes and semiconductor lasers, much progress has been made in the development of near-infrared techniques. This paper discusses the use of near-infrared dyes used in DNA and protein applications.

We have developed and applied single live cell imaging for real-time monitoring of resistance kinetics of Pseudomonas aeruginosa. Real-time images of live cells in the presence of a particular substrate (EtBr) provided the first direct insights of resistance mechanism with both spatial and temporal information and showed that the substrate appeared to be accumulated in cytoplasmic space, but not periplasmic space. Three mutants of P. aeruginosa, PAO4290 (a wild-type expression level of MexAB-OprM), TNP030#1 (nalB-1, MexAB-OprM over expression mutant), and TNP076 (ΔABM, MexAB-OprM deficient mutant), were used to investigate the roles of these three membrane proteins (MexAB-OprM) in the resistance mechanism. Ethidium bromide (EtBr) was chosen as a fluorescence probe for spectroscopic measurement of bulk cell solution and single cell imaging of bulk cells. Bulk measurement indicated, among three mutants, that nalB-1 accumulated the least EtBr and showed the highest resistance to EtBr, whereas ΔABM accumulated the most EtBr and showed the lowest resistance to EtBr. This result demonstrated the MexAB-OprM proteins played the roles in resistance mechanism by extruding EtBr out of cells. Unlike the bulk measurement, imaging and analysis of bulk cells at single cell resolution demonstrated individual cell had its distinguished resistance kinetics and offered the direct observation of the regulation of influx and efflux of EtBr with both spatial and temporal resolution. Unlike fluorescent staining assays, live cell imaging provided the real-time kinetic information of transformation of membrane permeability and efflux pump machinery of three mutants. This research constitutes the first direct imaging of resistance mechanism of live bacterial cells at single cell resolution and opens up the new possibility of advancing the understanding of bacteria resistance mechanism.

Optical technologies, in particular fluorescence spectroscopy, have shown the potential to provide improved detection methods for cervical neoplasia that are sensitive and cost effective through accurate, objective, instantaneous point-of-care diagnostic tools. The specific goals of this study were to analyze reflectance spectra of normal and neoplastic cervical tissue in vivo and to evaluate the data for use in diagnostic algorithm development. Spectroscopic measurements were obtained at four distinct source–detector separations from 324 sites in 161 patients. As the source–detector separation increases, greater tissue depth is probed. The average spectra of each diagnostic class differed at all source–detector separations, with the greatest differences occurring at the smallest source–detector separations. Algorithms, based on principal-component analysis and Mahalanobis distance classification, were developed and evaluated for all combinations of source–detector separations relative to the gold standard of colposcopically directed biopsy. The diagnostic combination of squamous normal versus high-grade squamous intraepithelial lesions gave good discrimination with a sensitivity of 72% and a specificity of 81%; discrimination of columnar normal versus high-grade squamous intraepithelial lesions also was good, with sensitivity of 72% and specificity of 83%. Thus, reflectance spectroscopy appears promising for in vivo detection of cervical precancer. Strategies that combine fluorescence and reflectance spectroscopy may enhance the discrimination capabilities.

This study assesses one possible cause of inter-patient variation in fluorescence spectroscopy of the cervix: the menstrual cycle. Ten patients with no history of an abnormal Pap smear were seen daily throughout 30 consecutive days of their cycle. Fluorescence excitation-emission matrices were measured from three cervical sites on each patient. Principal component analysis was used to determine which spectral regions varied with the day of the cycle. Classification was performed to assess the influence of menstrual cycle on precancer diagnosis. Variations in the principal component scores and the redox ratio values show that the fluorescence emission spectra at 340–380 nm excitation appear to correlate with the cell metabolism of the cervical epithelium throughout the menstrual cycle; these changes do not affect diagnostic classification. The menstrual cycle affects intra-patient variation but does not appear to cause a significant level of inter-patient variation. It does not need to be controlled for in optical detection strategies based on fluorescence spectroscopy.

A GaN based ultraviolet (UV) laser diode (LD) was used to study the autofluorescence (AF) spectrum of the normal and tumor human bronchial tissues under ex vivo conditions. The UV LD generates a coherent short wavelength (around 400 nm) light beam with an intensity of about a few watts. AF spectrum data can be obtained without interference by excitation light. A clear blue peak located at around 483 nm was observed along with a green peak at around 560 nm in the normal tissue. The peak intensities observed were very weak for the tumor tissues. The AF imaging and spectrum analysis were performed along with a histopathological study. The spatial distribution of the elastin in the bronchial tissue affected the intensity of the AF whereas the spectrum shape was not affected. Strong AF was observed from regions that include a high density of the elastin. Biopsy measurements were performed for ex vivo samples, and depth profiling of the elastin was studied along with variations of the AF spectrum. AF spectra excited by the UV LD for fluorescence materials including FAD, NADH, and elastin were measured. The spectrum shape of the elastin as well as of NADH was similar to that of normal bronchial tissues.

This study was undertaken to compare the effect of glucose injection on the pharmacokinetic behavior of a soluble dye in normal and tumoral tissues. The measurements were done using a noninvasive fluorescent spectroscopy in situ and in real time. The experiments were performed on three groups of animals with calcein as a soluble pH-insensitive fluorescent dye combined or not with glucose. Glucose solution was injected 5 or 30 min before calcein. Fluorescence emission intensity was recorded on normal and tumor tissues with an optical multichannel analyzer. Calcein concentration was also measured in blood using repetitive blood sampling. In the control group (without glucose injection), calcein is rapidly cleared from the blood, with a slow tissue clearance. Fluorescence of normal tissue was higher than fluorescence measured in tumor tissue. When glucose is injected 5 min before calcein, there was a rapid increase of tissue fluorescence followed by a plateau remaining during the whole experiment. No difference between tumor and normal tissue fluorescence intensity was observed. When glucose was injected 30 min before calcein, the plateau phase was reduced to 50 min in normal tissue. Tumor tissue fluorescence displays no distinct plateau phase. These results clearly showed the effect of glucose injection in situ and in real time, by a noninvasive method, on the pharmacokinetic of a soluble dye in a tumor tissue compared to a normal tissue. Differences between blood compartment and tissues kinetic profiles were also clearly demonstrated.

Tight glucose monitoring is essential for the reduction of diabetic complications. This research investigated the changes of absorption spectra observed in serum at three prominent glucose absorption peaks in the middle infrared using a demountable liquid, transmission cell. Two frequencies of light were used to determine the glucose absorption: one at 1193 cm^–1 to determine the background water absorption and the other at one of the characteristic peaks (1035, 1080, and 1109 cm^–1). The peak at 1035 cm^–1 was best for quantitative determination with a standard of error of 20.6 mg/dl (1.1 mmol/L). While interference from other serum constituents could cause problems, urea and albumin—two constituents known to have close absorption peaks—were determined to have no effect on the ability to determine the glucose levels at 1035 cm^–1.

New diagnostic tools are needed for the characterization of dental caries in the early stages of development. If carious lesions are detected early enough, they can be arrested without the need for surgical intervention. The objective of this study was to demonstrate that polarization sensitive optical coherence tomography (PS-OCT) can be used for the imaging of early caries lesions and for the monitoring of lesion progression over time. High-resolution polarization resolved images were acquired of natural caries lesions and simulated caries lesions of varying severity created over time periods of 1 to 14 days. Linearly polarized light was incident on the tooth samples and the reflected intensity in both orthogonal polarizations was measured. PS-OCT was invaluable for removing the confounding influence of surface reflections and native birefringence necessary for the enhanced resolution of the surface structure of caries lesions. This study demonstrated that PS-OCT is well suited for the imaging of interproximal and occlusal caries, early root caries, and for imaging decay under composite fillings. Longitudinal measurements of the reflected light intensity in the orthogonal polarization state from the area of simulated caries lesions linearly correlated with the square root of time of demineralization indicating that PS-OCT is well suited for monitoring changes in enamel mineralization over time.

This paper presents two techniques based on optical coherence tomography, the "focus tracking method" and the "optical path shifting method," for determining refractive index and thickness simultaneously, which are especially useful for bio tissues. From comparison of these two methods, it was found that the focus tracking method is suitable for in vivo measurement, but does not have high precision. The optical path shifting method is limited to in vitro measurement, but has high precision. Using the optical path shifting method, the refractive indices of cucumber were measured at the wavelength of 850 and 1300 nm.

Objective. To improve the precision of refractive surgery, a new approach for determination of the removed corneal thickness profile in situ with laser ablation by optical coherence tomography (OCT) is developed. Study Design/Materials and Methods. The traditional method for precision (less than 10 µm) measurements of intraocular distances is based on the use of the reflected component of probing radiation. This component is characterized by a small range of operating angles between a probing beam and a normal to the surface under study. To enhance this range of operating angles we suggest using a light component backscattered from a biological object. This will enable precision measurements over the entire surface of the cornea without any changes in the orientation between a probing beam and the eye, a necessary condition for in situ monitoring of laser refraction correction in the eye. We suggest a specially developed algorithm of OCT signal processing to measure the corneal thickness by the backscattered light component for a single longitudinal scan (A scan). The corneal thickness profile is obtained by a series of such A scans acquired by successively scanning a probing beam along the corneal surface. The thickness profile of removed layer is determined by changes in the corneal thickness profile in the process of ablation. When the cornea is ablated by a beam with a fixed transverse profile, we propose using integral characteristics of the ablated layer profile, for example, the maximum ablation depth, as criteria of changes in refractive power of the eye. The measurement precision by these characteristics is considerably higher than by a single A scan. Since the cornea is a poorly scattering medium, the Fourier filtering is employed to increase reliability and precision of the method. Model experiments on monitoring the ablation process in a lavsan film and ex vivo human cornea are described. Preliminary experiments on in vivo measurements of human corneal thickness are performed. Results. In model experiments the precision of measurement of laser ablation depth by one A scan was 5–20 μm, depending on the signal-to-noise ratio (SNR), whereas the precision of measurement of laser ablation depth as the integral characteristic of the ablated layer profile was 0.3–5 μm. The experimental results showed that at small SNR Fourier filtering might considerably increase reliability and precision of measurements. When SNR is high, the measurement precision does not change. The precision of measurements of the corneal thickness in preliminary in vivo experiments was higher than in ex vivo experiments. This factor is very promising for application of the method suggested herein in refractive surgery.

Frequency-domain near-infrared techniques have been widely used to detect the optical properties of biological tissues noninvasively. In this paper we propose an analytical model to evaluate the performance of frequency-domain instruments. Based on the diffusion equation and the transfer properties of optoelectronic components, we treat all parts, including the medium, as two-port networks and apply systematic methods to answer questions concerning frequency-domain instruments. Experiments show that this method can reasonably reflect the properties of the instrument within an accuracy of 7%. This kind of method can be used to design suitable instruments for various applications. We also analyze the selection of the instrument parameters to achieve optimal performance at an efficient cost using this analytical model.

Chen, Dougherty, and Bittner [Y. Chen, E. R. Dougherty, and M. L. Bittner, J. Biomed. Opt. 2(4), 364–374 (1997)] provided the derivation of a probability density function (PDF) for a signal ratio from a DNA microarray. This PDF is potentially useful for testing whether a pair of signals from the same gene has a common mean. The derivation of the PDF assumes the normality of all signal distributions and a common coefficient of variation (CV) for all signals within a microarray. The testing procedure requires the calculation of a common confidence interval for a microarray, based on a maximum likelihood estimator of the "common" CV, and the determination of whether or not a ratio for a particular gene falls within this interval. This study used Monte Carlo techniques and demonstrated that the procedure is robust to violations of normality and also to constancy in the coefficients of variation. A closer examination of the dynamics of the procedure found that the robustness was the result of offsetting effects. The size of the confidence interval was increased as a result of higher estimates of the common CV, as the actual CV pattern became heterogeneous. This effect mitigated the inflation in the size of the ratio as a result of increasing CV heterogeneity. These findings suggest that the Chen–Dougherty–Bittner procedure may be used even if underlying assumptions do not hold.