Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Optical Absorption Microspectroscopy (µ-OAS) Based on Schwarzschild-Type Cassegrain Optics.

A new experimental setup, combining a custom-designed Schwarzschild-type Cassegrain-based microscope and an ultraviolet-visible-near infrared (UV-Vis-NIR) spectrophotometer, has been developed, focusing the light beam down to 20 µm diameter. Optical absorption spectra (in the 300-2500 nm range) have been measured on micrometer-sized natural glass inclusions providing information on iron speciation in magmatic melts. The absence of contribution from the host crystal matrix provides a test of the efficiency of micro-focusing. A microthermometric stage has been adapted on the microscope for measuring optical absorption spectra up to 900 K with application to the thermochromism of minute natural spinel crystals (MgAl2O4:Fe(2+),Cr(3+)). This experimental setup provides an easy and fast way to follow the evolution of spectral properties and color of glasses or crystals with temperature as well as the possibility of measuring spatially resolved optical absorption spectra.

Real-time luminescence microspectroscopy monitoring of singlet oxygen in individual cells.

A new setup for direct microspectroscopic monitoring of singlet oxygen ((1)O2) has been developed in our laboratory using a novel near-infrared sensitive InGaAs 2D-array detector. An imaging spectrograph has been inserted in front of the 2D-array detector, which allows us to acquire spectral images where one dimension is spatial and the other is spectral. The work presents a detailed examination of sensitivity and noise characteristics of the setup and its ability to detect (1)O2. The (1)O2 phosphorescence-based images and near-infrared luminescence spectral images recorded from single TMPyP-containing fibroblast cells reflecting spectral changes during irradiation are demonstrated. The introduction of spectral images addresses the issue of a potential spectral overlap of (1)O2 phosphorescence with near-infrared-extended luminescence of the photosensitizer and provides a powerful tool for distinguishing and separating them, which can be applied to any photosensitizer manifesting near-infrared luminescence.

A Cell Phone-Based Microphotometric System for Rapid Antimicrobial Susceptibility Testing.

This study demonstrates a low-cost, portable diagnostic system for rapid antimicrobial susceptibility testing in resource-limited settings. To determine the antimicrobial resistance phenotypically, the growth of pathogens in microwell arrays is detected under different antibiotic conditions. The use of a colorimetric cell viability reagent is shown to significantly improve the sensitivity of the assay compared with standard absorbance spectroscopy. Gas-permeable microwell arrays are incorporated for facilitating rapid bacterial growth and eliminating the requirement of bulky supporting equipment. Antibiotics can also be precoated in the microwell array to simplify the assay protocol toward point-of-care applications. Furthermore, a low-cost cell phone-based microphotometric system is developed for detecting the bacterial growth in the microwell array. By optimizing the operating conditions, the system allows antimicrobial susceptibility testing for samples with initial concentrations from 10(1) to 10(6) cfu/mL. Using urinary tract infection as the model system, we demonstrate rapid antimicrobial resistance profiling for uropathogens in both culture media and urine. With its simplicity and cost-effectiveness, the cell phone-based microphotometric system is anticipated to have broad applicability in resource-limited settings toward the management of infectious diseases caused by multidrug-resistant pathogens.

Simple and robust method for lithium traces determination in drinking water by atomic emission using low-power capacitively coupled plasma microtorch and microspectrometer.

A method for Li determination in drinking water using atomic emission spectrometry in a new low-power Ar capacitively coupled plasma microtorch (15 W, 0.6 L min(-1)) with a detection limit of 0.013 µg L(-1) was developed. The method is based on external calibration in the presence of a buffering solution containing 5 mg L(-1) Na, K, Ca, Mg added both to calibration standards and water samples. The statistical validation on 31 bottled drinking water samples (0.4-2140 µg L(-1) Li) using the Bland and Altman test and regression analysis has shown results similar to those obtained by the standard additions method. The buffering solution approach is simpler than the standard additions and has demonstrated good intra- and interday precision, accuracy and robustness. It was successfully applied over a wide concentration range of Li and multimineral matrix with a pooled precision of 2.5-3.5% and 99±9% accuracy.

Validation of a new 96-well plate spectrophotometric method for the quantification of compound 48/80 associated with particles.

A new, simple, inexpensive, and rapid 96-well plate UV spectrophotometric method was developed and validated for the quantification of compound 48/80 (C48/80) associated with particles. C48/80 was quantified at 570 nm after reaction with acetaldehyde and sodium nitroprusside in an alkaline solution (pH 9.6). The method was validated according to the recommendations of the ICH Guidelines for specificity, linearity, range, accuracy, precision, and detection and quantification limits (DL and QL). All the validation parameters were assessed in three different solvents, i.e., deionized water, blank matrix of chitosan nanoparticles, and blank matrix of chitosan/alginate nanoparticles. The method was found to be linear in the concentration range of 5 to 160 µg/ml (R(2)>0.9994). Intraday and interday precision was adequate, with relative standard deviation lower than those given by the Horwitz equation. The mean recoveries of C48/80 from spiked samples ranged between 98.1% and 105.9% for calibration curves done with the blank matrices and between 89.3% and 103.3% for calibration curves done with water, respectively. The DL were lower than 1.01 µg/ml and the QL were lower than 3.30 µg/ml. The results showed that the developed method is sensitive, linear, precise, and accurate for its intended use, with the additional advantages of being cost-effective and time-effective, allowing the use of small-volume samples, and the simultaneous analysis of a large number of samples. The proposed method was already successfully applied to evaluate the loading efficacy of C48/80 chitosan-based nanoparticles and can be easily applied during the development of other C48/80-based formulations.

Linear variable optical filter-based ultraviolet microspectrometer.

An IC-compatible linear variable optical filter (LVOF) for application in the UV spectral range between 310 and 400 nm has been fabricated using resist reflow and an optimized dry-etching. The LVOF is mounted on the top of a commercially available CMOS camera to result in a UV microspectrometer. A special calibration technique has been employed that is based on an initial spectral measurement on a xenon lamp. The image recorded on the camera during calibration is used in a signal processing algorithm to reconstruct the spectrum of the mercury lamp and the calibration data is subsequently used in UV spectral measurements. Experiments on a fabricated LVOF-based microspectrometer with this calibration approach implemented reveal a spectral resolution of 0.5 nm.

Micro-lightguide spectrophotometry for tissue perfusion in ischemic limbs.

OBJECTIVE: To validate micro-lightguide spectrophotometry (O2C) in patients with lower limb ischemia and to compare results with those obtained from toe blood pressure. METHODS: We prospectively examined 59 patients, 24 of whom complained of claudication, 31 had critical ischemia, and four were asymptomatic. Diabetes was present in 19 (32%) patients. Saturation (SO(2)) and flow measured with O2C were determined with the limb in the horizontal position followed by a 55-cm elevation. Toe pressures were determined in the horizontal position only. In addition, 13 patients were examined before and, on average, 3 days after revascularization. RESULTS: Median SO(2) was 62% (25%-75% percentile: 37%-75%) with the limb in the horizontal position and 16% (3%-41%) with the limb elevated. Comparing the individual toe pressures with SO(2) values measured in the horizontal position and elevated position revealed a significant correlation (r(s) = 0.40; P < .01 and r(s) = 0.56; P < .01, respectively). A low SO(2) (ie, <40% in the horizontal position and <20% in the elevated position) was highly predictive of a toe pressure of 40 mm Hg or less. In the horizontal position, the positive predictive value was 100%, whereas the negative predictive value was 47%. The similar figures in the elevated position were a positive predictive value of 97% and a negative predictive value of 68%. Postoperatively, SO(2) increased significantly from 27% (P25%-75%: 11%-75%) to 79% (68%-87%) in the horizontal position (P = .008) and from 14% (P25%-75%: 2%-39%) to 55% (30%-73%) in the elevated position (P = .011), respectively. Looking at the individual 13 cases in which revascularization was performed, three patients had a partial reconstruction (ie, superficial femoral artery occlusion distal to a central reconstruction or reconstruction to a popliteal blind segment). These patients had significantly lower postoperative SO(2) as well as toe pressure compared with the 10 patients with unobstructed flow to the foot. CONCLUSIONS: O2C was easy to use, fast, and painless. The most useful finding was the high predictive value of a low saturation and the rise in O2C values after successful revascularization.

Fiber confocal back-scattering micro-spectral analysis for single cell.

A fiber confocal back scattering micro-spectrometer (FCBS) was established, which combined fiber confocal microscopy with light scattering spectroscopy (LSS) for early diagnosis of the cancer cell at cellular level. An adherent monolayer human normal gastric epithelium line GES-1 and a carcinoma cell line NCI-N87 as well as a normal liver cell line L02 and a high-metastatic-potential hepatocellular carcinoma cell line HCC-LM3 were measured respectively. The spectral results showed that micro-back-scattering intensity from GES-1 cell and L02 cell possessed interesting oscillations in contrast to NCI-N87 and HCC-LM3 cells. There was significant difference between the spectra of the normal and the cancer cells (p<0.001). This demonstrates that the FCBS system here is able to distinguish dysplastic cells from normal cells at cellular level.

High resolution on-chip spectroscopy based on miniaturized microdonut resonators.

We experimentally demonstrate a high resolution integrated spectrometer on silicon on insulator (SOI) substrate using a large-scale array of microdonut resonators. Through top-view imaging and processing, the measured spectral response of the spectrometer shows a linewidth of ~0.6 nm with an operating bandwidth of ~50 nm. This high resolution and bandwidth is achieved in a compact size using miniaturized microdonut resonators (radius ~2 µm) with a high quality factor, single-mode operation, and a large free spectral range. The microspectrometer is realized using silicon process compatible fabrication and has a great potential as a high-resolution, large dynamic range, light-weight, compact, high-speed, and versatile microspectrometer.

Rejection of fluorescence background in resonance and spontaneous Raman microspectroscopy.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

Infrared monitoring of dinitrotoluenes in sunflower and maize roots.

Infrared microspectroscopy (IMS) is emerging as an important analytical tool for the structural analysis of biological tissue. This report describes the use of IMS coupled to a synchrotron source combined with principal components analysis (PCA) to monitor the fate and effect of dinitrotoluenes in the roots of maize and sunflower plants. Infrared imaging revealed that maize roots metabolized 2,4-dinitrotoluene (DNT) and 2,6-DNT. The DNTs and their derivative aromatic amines were predominantly associated with epidermis and xylem. Both isomers of DNT altered the structure and production of pectin and pectic polysaccharides in maize and sunflower plant roots. Infrared peaks diagnostic for aromatic amines were seen at the 5 mg L concentrations for both DNTs in maize and sunflower treated tissue. However, only infrared peaks for nitro groups, not aromatic amines, were present in the maize treated at 10 mg L For sunflower, the 10 mg L level was toxic and also produced very dark root systems making spectra difficult to obtain. Maize and sunflower seem unable to metabolize effectively at concentrations higher than about 5 mg L DNT in hydroponic solution. Based on the results of this study, IMS combined with PCA can be an effective means of determining the fate and metabolism of organic contaminants in plant tissue when isotopically labeled compounds are not available.

The investigation of a relative contrast index model for fingerprint quantification.

The quantification of fingerprint contrast is a relatively new concept in fingerprint enhancement research. It has emerged as a mode of fingerprint assessment to reduce the potential biased of visual qualitative assessment. Subjective qualitative methods that are currently reported in the literature include; side-by-side assessment, assigning a score to a treatment based on visible criteria and stating observed results without presenting supporting validation. These qualitative methods often do not state clearly the visual assessment parameters and produce a degree of ambiguity when defining the enhancement results. The relative contrast index model was constructed to empirically quantify the difference in contrast between fingerprint ridges and valleys, using measurements gained from a microspectrophotometer. This paper seeks to further investigate this recent research and test the model using three different microspectrophotometers. Data from these separate sources will determine whether the theoretical aspects of the model would pragmatically produce reliable and repeatable results across a range of microspectrophotometers found in forensic laboratories.

Detecting reactive oxygen species in primary hepatocytes treated with nanoparticles.

This chapter describes a protocol for testing nanoparticle formulations for reactive oxygen species generation in male Sprague-Dawley (SD) primary hepatocytes. The protocol utilizes the fluorescent redox active probe, dichlorofluorescein diacetate (DCFH-DA). Primary hepatocytes were chosen for this assay since they have greater metabolic activity than hepatocyte cell lines. This method extends previous standardized cytotoxicity methods for particulates by evaluating mechanisms of toxicity in potential target organ cells. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered an important component of biocompatibility screens.

Raman-assisted crystallography of biomolecules at the synchrotron: instrumentation, methods and applications.

Raman spectroscopy is a powerful technique that, in recent years, has been successfully coupled to X-ray crystallography for the analysis of biological macromolecular systems. The complementarity between both techniques is illustrated at multiple stages, including sample preparation, data collection and structural interpretation with a mechanistic perspective. The current state of instrumentation is described, focusing on synchrotron based setups. Present and future applications of Raman microspectrophotometry are reviewed with reference to recent examples dealing with metallo-, photosensitive-, and redox-proteins. The added value of Raman microspectrophotometry to assess X-radiation damage is discussed, and its applicability to investigate crystalline DNA molecules is also emphasized. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Protein crystal microspectrophotometry.

Single crystal microspectrophotometry has emerged as a valuable technique for monitoring molecular events that take place within protein crystals, thus tightly coupling structure to function. Absorption and fluorescence spectra, ligand binding affinities and kinetic constants can be determined, allowing i) the definition of the experimental conditions for X-ray crystallography experiments and their interpretation, ii) the assessment of whether crystal lattice forces have altered conformational equilibria, iii) the comparison with data obtained in solution. Microspectrophotometric measurements using oriented crystals and linearly polarized light are carried out usually off-line with respect to X-ray data collection and are aimed at an in- depth characterization of protein function in the crystal, leading to robust structure-function relationships. The power of this approach is highlighted by reporting a few case studies, including hemoglobins, pyridoxal 5'-phosphate-dependent enzymes and acetylcholinesterases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Demountable liquid/flow cell for in vivo infrared microspectroscopy of biological specimens.

We have developed a liquid/flow cell/chamber allowing infrared measurements of living biological specimens with high spatial resolution under a controlled aqueous environment. This flow chamber features sub-micrometer thick diamond windows exhibiting low spherical and chromatic aberrations. Diamond has excellent transmission properties and minimal dispersion over the entire mid-infrared and visible spectral ranges. In contrast to current commercially available infrared liquid chambers, the flow chamber has a slim profile, which accommodates high resolution/magnification microscope objectives with small working distances, down to 0.6 mm above the chamber and 6 mm below the flow chamber. We have coupled a pump to the flow chamber to provide medium exchange. As an example, we present microspectroscopic infrared maps and spectra of the freshwater green alga Micrasterias sp. in the new flow chamber and compare them to maps obtained with a conventional liquid chamber. Pulse-amplitude-modulated fluorescence measurements on Micrasterias sp. cells inside the new flow chamber have been evaluated to demonstrate the viability of the algal cells.

Near-infrared microspectroscopic analysis of rat skin tissue heterogeneity in relation to noninvasive glucose sensing.

BACKGROUND: Noninvasive glucose measurements are possible by analysis of transmitted near-infrared light over the 4000- to 5000-cm(-1) spectral range. Such measurements are highly sensitive to the exact position of the fiber-optic interface on the surface of the skin sample. A critical question is the degree of heterogeneity of the major chemical components of the skin matrix in relation to the size of the fiber-optic probed used to collect noninvasive spectra. Microscopic spectral mapping is used to map the chemical distribution for a set of excised sections of rat skin. METHOD: A Fourier transform near-infrared microspectrometer was used to collect transmission spectra from 16 tissue samples harvested from a set of four healthy Harlan-Sprague male rats. A reference point in the center of the tissue sample was probed regularly to track dehydration, changes in tissue composition, and changes in instrument performance. Amounts of the major skin constituents were determined by fitting microspectra to a set of six pure component absorbance spectra corresponding to water, type I collagen protein, keratin protein, fat, an offset term, and a slope term. RESULTS: Microspectroscopy provides spectra with root mean square noise levels on 100% lines between 418 and 1475 microabsorbance units, which is sufficient for measuring the main chemical components of skin. The estimated spatial resolution of the microscope is 220 microm. The amounts of each tissue matrix component were determined for each 480 x 360-microm(2) location of a 4.8 x 3.6-mm(2) rectangular block of skin tissue. These spectra were used to generate two-dimensional distribution maps for each of the principal skin components. CONCLUSIONS: Distribution of the chemical components of rat skin is significant relative to the dimensions of noninvasive glucose sensing. Chemical distribution maps reveal that variations in the chemical composition of the skin samples are on the same length scale as the fiber-optic probe used to collect noninvasive near-infrared spectra. Analysis of variance between tissue slices collected for one animal and analysis of variations between animals indicate that animal-to-animal variation for all four chemical components is significantly higher than variations between samples for a given animal. These findings justify the collection and interpretation of near-infrared microspectroscopic maps of human skin to establish chemical heterogeneity and its impact on noninvasive glucose sensing for the management of diabetes.

Sub-100 nm IR spectromicroscopy of living cells.

We have performed IR spectromicroscopy of cells immersed in liquid water, with a lateral resolution better than 100 nm. Here, we use the motion of an atomic force microscope tip, probing the local transient deformation induced by an IR pulsed laser tuned at a sample absorbing wavelength. By Fourier analysis of the vibration of the cantilever tip, we can discriminate frequencies that are characteristic of the object, thus eliminating the influence of the water absorption. This opens the door of chemical imaging of living species in vivo, with spatial resolution of the order of the size of cell components.

Fourier transform-infrared microspectroscopy and microscopic imaging.

For age- and sex-matched subjects, osteoporotic bone is more fragile than healthy bone. Vibrational infrared spectroscopy and in particular infrared microspectroscopic imaging is a useful tool for investigating and characterizing changes associated with metabolic bone diseases including osteoporosis in biopsied tissues. Strength-related measures such as bone mineral content/composition as well as spectroscopically determined bone quality-related measures such as mineral crystallinity, carbonate substitution, and collagen cross-linking consequently differ between osteoporotic patients and normal subjects. Validated IR parameters specific to the mineral and matrix components of bone have been defined and can now be used to quantify anatomical/spatial variations and the effect of new therapies on osteoporotic bone.