Quantification of biofilm accumulation by an optical approach

Methods for non-invasive, in situ, measurements of biofilm optical density and biofilm optical thickness were evaluated based on Pseudomonas aeruginosa experiments. Biofilm optical density, measured as intensity reduction of a light beam transmitted through the biofilm, correlates with biofilm mass, measured as total carbon and as cell mass. The method is more sensitive and less labor intensive than other commonly used methods for determining extent of biofilm mass accumulation. Biofilm optical thickness, measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements. Biofilm refractive index was found to be close to the refractive index of water. The P. aeruginosa biofilms studied reached a pseudo steady state in less than a week, with stable liquid phase substrate, cell and TOC concentrations and average biofilm thickness. True steady state was, however, not reached as both biofilm density and roughness were still increasing after 3 weeks.     

The Phycomyces lens: measurement of the sporangiophore intensity profile using a fiber optic microprobe

A fiber optic microprobe, 5.5 m in diameter, was used as a detector to measure the light intensity profile at the distal cell surface of Phycomyces blakesleeanus (Burgeff) sporangiophores that were irradiated unilaterally by a collimated xenon source. The light intensity at a fixed location of the cell surface showed large random variations over time which were probably the result of optical effects of particles being carried past the probe by cytoplasmic streaming. The intensity profile, formed around the distal periphery of the cell by the lens action of the sporangiophore, was determined from intensity measurements made while the probe was held fixed and the incident beam direction was varied in angle of azimuth. The resulting profile consisted of two steeply rising sides enclosing a central plateau or shallow well which ranged in fluence rate from 1.6 to 2.2 times that of the incident beam. These experimental findings differ from theoretical modeling where much greater contrast between the sides and central portion of the lens profile was predicted. These results also indicate that the mechanism of phototropic sensory perception in Phycomyces may filter out cytoplasmic light flicker and may not require strong contrasting regions within the lens profile to detect light direction.     

Cell monitoring with optical coherence tomography

Background aimsWe evaluated a commercially available instrument, OCTiCell (chromologic.com/octicell), for monitoring cell growth in suspended agitated bioreactors based on optical coherence tomography. OCTiCell is an in-line, completely non-invasive instrument that can operate on any suspended-cell bioreactor with a window or transparent wall. In traditional optical coherence tomography, the imaging beam is rastered over the sample to form a three-dimensional image. OCTiCell, instead uses a fixed imaging beam and takes advantage of the motion of the media to move the cells across the interrogating optical beam.ResultsWe found strong correlations between the non-invasive, non-contact, reagent-free OCTiCell measurements of cell concentration and viability and those obtained from the automated cell counter, and the XTT viability assay, which is a colorimetric assay for quantifying metabolic activity.ConclusionsThis novel cell monitoring method can adapt to different bioreactor form factors and could reduce the labor cost and contamination risks associated with cell growth monitoring.     

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.     

Measurement of the Growth of a Floc Forming Bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1

Pseudomonas putida CP1 formed clumps of cells when grown on mono-chlorophenols but not on phenol or glucose. An increase in cell numbers for the organism grown on mono-chlorophenols was accompanied by a decrease in the dry weight. The change in shape of the bacterium from rod shape to coccus shape coupled with a reduction in cell size when the organism was grown under nutritional stress was found. This result together with cell aggregation affected the measurement of growth parameters in the system by conventional methods (optical density measurements, dry weight measurements and the plate count technique). Monitoring growth of Pseudomonas putida CP1 by a direct microscopic count technique was found to be more representative than conventional methods including optical density measurements, dry weight measurements and the plate count technique when grown on phenolics.     

Irradiance-induced variability in light scatter from marine phytoplankton in culture

A series of laboratory experiments are reported that illustratethe response of beam attenuation ( = 660 nm) and single-celllight scatter ( = 488 nm) properties of several species of marinephytoplankton to light intensity. When unialgal cultures weresubjected to an increase in light intensity, the particle-scatteringcomponent of beam attenuation and near-forward single-cell lightscatter were found to increase rapidly in response. Cell abundanceincreased only slightly over the course of the experiments,leading to the conclusion that the response in beam attenuationwas due to irradiance-induced changes in the single-cell opticalproperties. The percent hourly increase in beam attenuation,normalized to cell abundance, and single-cell light scatterrangedfrom 5% for a culture of the coccolithophore Emiliania huxleyito 25% for a culture of Thalassiosira pseudonana. In a separateset of experiments, carbon-specific beam attenuation (c^*_c; thepaniculate material component of beam attenuation normalizedto the concentration of paniculate organic carbon) was foundto be species specific and, to some extent, sensitive to irradiance.The positive response in phytoplankton light scatter, both atthe population and at the single-cell level, to an increasein light intensity is similar to diet patterns in beam attenuationreported for the near-surface ocean. If a component of the observeddiel pattern in beam attenuation is due to irradiance-induced,carbon-independent optical variability in the phytoplanktonassemblage, as the results of the high-light experiments suggest,neglecting such variability can result in either an overestimationor an underestimation of primary production, depending on theresponse in cc^*_c. Natural variability in cc^*_c is poorly understoodand responses to environmental factors, such as irTadiance,have yet to be addressed outside of the laboratory.     

Single cell viability and impact of heating by laser absorption

Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s.     

Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran

A capsular-polysaccharide-producing strain, LM-17, was isolated from kefir grains and was identified as a slime-forming, rod-shaped Lactobacillus. According to ¹H- and ¹³C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from kefir grains and therefore known as kefiran. The kefiran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l purified kefiran from the culture supernatant of the LM-17 strain. Received: 6 April 1999 / Received revision: 30 July 1999 / Accepted: 13 August 1999     

Leaf orientation and distribution in a Phaseolus vulgaris L. crop and their relation to light microclimate

Changes in canopy structure parameters (leaflet orientation, leaflet inclination and leaf area index) were measured in crops of beans (Phaseolus vulgaris L.) in the field as the canopy developed between July and October. These changes were compared with the corresponding changes in seasonal light transmission. The beans showed clear heliotropic behaviour, with preferential orientation of leaflets towards the sun’s beam, especially on sunny days. Nevertheless a significant proportion of the leaves pointed in other directions, with as much as 20% oriented towards the north. The highest proportion of leaf inclinations was in the range 30–40° on cloudy days and between 40° and 50° on sunny days. Two methods were compared for assessing changes in light transmission: (a) the use of a Sunfleck Ceptometer and (b) the use of continuous records obtained with sensors installed in the canopy. Over the growth period studied, the total of the leaf plus stem area indices (L _S) increased from 0.26 to 5.2 with the transmission coefficient (τ) for photosynthetic photon flux density (PPFD), obtained using the Ceptometer, correspondingly decreasing from 0.72 to 0.05, and the canopy extinction coefficient decreasing from 1.4 to 0.62. The continuous records of light transmission gave generally similar estimates of τ. Some contrasting leaf angle distribution functions were compared for estimation of L _S from the light measurements. The best leaf angle function to predict L _S from the observed light transmission was a conical function corrected by the degree of heliotropism. Received: 27 January 1999 / Accepted: 11 June 1999     

Measurements of Light Transmission Through Single Melanophores

A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.). The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 μm². Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor-mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.     

An optical monitor of tension for small cardiac preparations.

When a light beam is focused on a muscle preparation which is allowed to contract, large changes in the intensity of the emerging light accompany the contraction. These movement-related optical signals were studied and compared to simultaneous measurements of force in isolated cardiac Purkinje fibers. The two signals were compared in response to action potentials and to graded changes in membrane potential controlled under voltage clamp. These experiments indicate that the optical signal is a sensitive monitor of tension development under these conditions. This technique is particularly well-suited to force measurements in smaller preparations in which direct mechanical techniques are not feasible.     

Application of optical classifications to North European lakes

Two classifications (rough and fine) based on the optical properties of water were used to classify 42 Estonian, Finnish, and Swedish lakes. The rough classification was based on the amount of optically active substances (OAS: chlorophyll a, suspended matter, and colored dissolved organic matter) in the water. The basic variables of the fine classification were chlorophyll a concentration, beam attenuation coefficient of light in the photosynthetically active radiation (PAR) region in the water (or corresponding Secchi depth), and the beam attenuation coefficient for filtered water at 380 nm. All optical classes were represented in the studied lakes by both classifications, when they changed their class depending on the seasonal and biological conditions. In a large lake, different parts of the lake belonged to different optical classes. The results obtained by both classifications were in good agreement. There was a concurrence between optical classes and water transparency by Secchi depth. Often the bio-optical variations of water properties are described from the measurements of all three OAS and Secchi depth because these parameters belong to routine monitoring datasets and have been carefully recorded. The water classes provide a method to summarize the influences of the different factors.     

Cell cycle studies of murine leukemia cell L5l78Y by polarization effects of light scattering

The structural changes during the life cycle of a synchronized population of mouse leukemia cell line L5l78Y have been described by polarized light scattering measurements. Exponentially growing cells were synchronized by an automatic excess thymidine-colcemid treatment technique. Samples were removed from the suspension culture and fixed with glutaraldehyde at hourly intervals throughout the life cycle. The effect these cell samples had in changing right-hand circularly polarized light to 45° linearly polarized light during the scattering process was measured at angles 6–l60° to the incident beam. The reproducibility of the light scattering signals for each time interval was statistically evaluated and found to have good intertrial correlation for each time period in the angular range 6–60° to the incident beam. Statistically significant changes were seen between cell samples during the synchronous life cycle. Therefore, the system developed has applications as an extremely sensitive measure of cell structure, and of structural changes caused by low-level chemical, physical or biological agents.     

THE KINETICS OF THE PHOTOACCLIMATION RESPONSE OF NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE): A STUDY OF CHANGES IN ULTRASTRUCTURE AND PSU DENSITY

In this study we report the kinetics of photoacclimation of the unicellular alga Nannochloropsis sp. grown under high light (HL), and subsequently transferred to low light (LL). We examined the changes in ultrastructural features, pigmentation, and photosynthetic parameters over short intervals until the LL steady state was reached. The ultrastructural changes were followed by quantitative morphometric measurements of transmission electron micrographs. We found that the increase in the relative volume of the chloroplast during acclimation to LL (twofold) was accompanied by an increase in number of stacks (twofold) and in the surface area of thylakoids per cell (2.5-fold). The increase in photosynthetic unit (PSU) density was about 2.15-fold. Maximal density was about 84 PSU·μm⁻² in LL cells, and minimal density was 39 PSU·μm⁻² in HL cells. The HL/LL ratio of the in vivo optical absorption cross-section of PSU (σ_PSU) was 2.8, whereas in the in vivo optical absorption cross-section of the cell (σ_cell), the trend of change was in the opposite direction: 1.7-fold higher in LL-acclimated cells than in HL-acclimated cells. We propose a partial sequence of the photoacclimation processes based on our data and the derived rate constants.     

Acousto-optic laser-scanning cytometer

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.     

Investigation of luminescent banding in solid coral: the contribution of phosphorescence

This study investigates the nature and components of annual luminescent banding in massive Porites coral skeletons, with a view to refining the technique for using this banding to reconstruct past environmental conditions. Three-dimensional excitation-emission-matrix spectroscopy and optical fibre beam delivery have been used to investigate the luminescence properties of the bright and dull bands of solid coral. Six characteristic excitation/emission peaks have been identified: 280/450–600, 340/450, 370/470, 390/485, 420/505 and 450/530 nm. The first peak corresponds to protein-type fluorescence. The others are characteristic of humic acid luminescence. The difference in luminescence intensity between bright and dull bands has been quantified and characterised spectroscopically. The luminescence of the bright bands is up to 25% more intense than their neighbouring dull bands with the greatest increase in relative intensity in the long wavelength emission region, between 500 and 600 nm. The contribution of long-lived phosphorescence to the total luminescence intensity has been determined by time-resolved measurements on the 100 ms timescale. Both bright and dull bands show long-lived phosphorescence with decay times up to 1.5 s. This phosphorescence accounts for about 10% of the total luminescence intensity of bright bands. The difference in phosphorescence intensity between bright and dull bands is substantially greater than the difference in total luminescence intensity: the phosphorescence of bright bands is up to twice as intense as that of dull bands. This suggests that phosphorescence plays an important role in defining luminescent banding in coral. Furthermore, the large observable difference in phosphorescence between bright and dull bands indicates that measurement of phosphorescence profiles across growth bands in corals may prove to be a more sensitive indicator of past environmental conditions than measurements of total luminescence. Received: 18 March 1999 / Accepted: 20 December 1999     

Photic Volume in Photobioreactors Supporting Ultrahigh Population Densities of the Photoautotroph Spirulina platensis

Characterization of the photic zone and light penetration depth in cultures with ultrahigh cell densities represents a major issue in mass cultures of phytoautotrophic microorganisms grown in enclosed photobioreactors. In a study of the effect of underwater optical properties on the penetration depth of photosynthetically active radiation, the inherent optical properties of algal suspensions, i.e., absorption and scattering coefficients, as well as their apparent optical properties, i.e., the reflectance and the vertical attenuation coefficient of downwelling irradiance, were determined by using high-spectral-resolution radiometric measurements. The vertical attenuation coefficient was used to estimate quantitatively the depth of light penetration into a reactor containing an ultrahigh cell density (chlorophyll concentration, up to 300,000 mg m(sup-3)). For such a high cell density, the photic volume in the reactor was found to be extremely small; nevertheless, it differed between the blue and red light (less than 0.06 mm) and the green light (about 0.5 mm). This suggests a singular role for green light under the unique circumstances existing in ultrahigh-cell-density cultures of photoautotrophs.     

A simple light scattering method to assay magnetism in Magnetospirillum gryphiswaldense

Abstract A simple optical method was developed for assaying cellular magnetism in culture samples of magnetic spirilla. Cells are aligned parallel to the field lines in a magnetic field, resulting in a change in light scattering. The ratio of scattering intensities at different angles of magnetic field relative to the light beam ( C _mag) is used to characterize the average magnetic orientation of the cells. C _mag was found to be well correlated with the average number of particles in different magnetic cell populations. Thus, estimations of magnetosome content can be made using magnetically induced differential light scattering. The method provides a fast and sensitive tool for monitoring the magnetite formation in growing cultures of Magnetospirillum gryphiswaldense .     

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam-Bruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.     

Low-angle light-scattering instrument for DNA solutions

A light-scattering instrument capable of measurements on native DNA at angles as low as 10° is described. The major features of the instrument, which make it capable of low-angle measurements, are the use of an intense light source in which the incident beam is monitored directly and the use of a long, rectangular sample cell in which the scattered light can be measured at low angles with no interference from the incident beam. Methods for calibrating the instrument and for determining scattering correction factors are described. Procedures for the preparation and use of various calibration standards are given.