Microphotometric Method Research Articles

Large scale micro-photometry for high resolution pH-characterization during electro-osmotic pumping and modular micro-swimming

Micro-fluidic pumps as well as artificial micro-swimmers are conveniently realized exploiting phoretic solvent flows based on local gradients of temperature, electrolyte concentration or pH. We here present a facile micro-photometric method for monitoring pH gradients and demonstrate its performance and scope on different experimental situations including an electro-osmotic pump and modular micro-swimmers assembled from ion exchange resin beads and polystyrene colloids. In combination with the present microscope and DSLR camera our method offers a 2 μm spatial resolution at video frame rate over a field of view of 3920 × 2602 μm2. Under optimal conditions we achieve a pH-resolution of 0.05 with about equal contributions from statistical and systematical uncertainties. Our quantitative micro-photometric characterization of pH gradients which develop in time and reach out several mm is anticipated to provide valuable input for reliable modeling and simulations of a large variety of complex flow situations involving pH-gradients including artificial micro-swimmers, microfluidic pumping or even electro-convection.

A simple micro-photometric method for urinary iodine determination

Urinary iodide concentration (UIC) is useful to evaluate nutritional iodine status. In clinical settings UIC helps to exclude blocking of the thyroid gland by excessive endogenous iodine, if diagnostic or therapeutic administration of radio-iodine is indicated. Therefore, this study established a simple test for the measurement of UIC. UIC was analyzed in urine samples of 200 patients. Samples were pre-treated at 95°C for 45 min with ammonium persulfate in a thermal cycler, followed by a photometric Sandell-Kolthoff reaction (SK) carried out in microtiter plates. For method comparison, UIC was analyzed in 30 samples by inductivity coupled plasma mass spectro-metry (ICP-MS) as a reference method. Incubation conditions were optimized concerning recovery. The photometric test correlated well to the reference method (SK=0.91*ICP-MS+1, r=0.962) and presented with a functional sensitivity of 20 μg/L. UIC of patient samples ranged from <20 to 750 μg/L (median 110 μg/L); 90% of the urine samples had iodide concentrations below 210 μg/L. The modified SK-test takes approximately 90 min for analyses of 20 urine samples compared with 27 h for ICP-MS. The photometric test provides satisfactory results and can be performed with the basic equipment of a clinical laboratory.

Identification of kelp zoospores from in situ plankton samples

Studies of the link between planktonic and benthic stages of marine macroalgae have been hampered by the inability to identify planktonic stages from field samples. Here I describe a microphotometric method for identifying kelp zoospores from in situ plankton samples based on light-absorbance characteristics of zoospore plastids. Absorption spectra were obtained from plastids within Macrocystis pyrifera (L.) C. Agardh, Pterygophora californica Ruprecht, Eisenia arborea Areschoug and Laminaria farlowii Setchell zoospores released in the laboratory from adult specimens collected in the field; these are the dominant subtidal kelps in most southern California kelp forests. Absorption spectra were highly specific to each kelp species. Average absorbance decreased from P. californica to E. arborea to L. farlowii to M. pyrifera at most wavelengths between 400 and 750 nm. Discriminant-function analyses provided a significant discriminant rule, based on zoospore-plastid absorbance values at 25 different wavelengths, for identifying unknown zoospores among the four kelp species. Cross-validation techniques indicated that the discriminant rule could successfully identify 97.8% M. pyrifera, 96.4% P. californica, 86.2% E. arborea and 69.7% L. farlowii zoospores. Most misidentified E. arborea zoospores were identified as L. farlowii, and vice-versa; when the relatively rare E. arborea was pooled with L. farlowii and treated as a single complex, the discriminant rule could successfully identify 98.2% M. pyrifera, 97.4% P. californica and 89.5% E. arborea/L. farlowii zoospores. Coupled with a protocol for isolating kelp zoospores from non-kelp cells, this discriminant rule was used to determine kelp zoospore abundances and species-compositions of in situ plankton samples. This is the first description of a method for identifying macroalgal zoospores or spores from field samples.

Microphotometric determination of structure-bound oxidoreductase activities avoiding nothing-dehydrogenase artefacts: succinate dehydrogenase.

A tetrazolium-based microphotometric method has been devised for the determination of structure-bound dehydrogenase activities with correction for nothing-dehydrogenase artefacts. The method is based on the microphotometric recording of maximum reaction rates in a simple incubation chamber and consists of two successive measurements on the same section, the first in the absence and the second in the presence of the substrate. Following the first measurement, the substrate-free medium is quickly exchanged with the substrate-containing medium and a second measurement is taken. Subtraction of the first from the second reaction rate yields the enzyme activity corrected for nothing-dehydrogenase. Measurements of succinate dehydrogenase (SDH) in skeletal muscle fibres, liver, cardiac atrium and ventricle demonstrate the feasibility of the method. Measurements on the extensor digitorum longus muscle of rat reveal a range of up to fivefold differences in SDH activity within the fibre population of this muscle.

Relationship between immunostaining intensity and antigen content in sections.

We studied the relationship between staining intensity of immunohistochemical reaction and antigen content in sections. Alpha-fetoprotein (AFP) and albumin in sections cut from livers of newborn, 5-, 10-, 20-, and 60-day-old rats were examined as examples. First, we compared average immunostaining intensity (sum of specific absorbance in pixel/number of pixels) measured by image processing (IP), with antigen content measured by immunochemical assay to determine whether the intensity is proportional to antigen content. The intensity of AFP was proportional to the antigen content, whereas that of albumin was not. Subsequently, the antigen preservation test was carried out to determine whether the intensity was decreased by fixation and, if so, which type of decrease (proportional or disproportionate) occurred. Thereafter, antigen content in the same portion in the same immunostained section was measured by the microphotometric (MP) method followed by the IP method, because the MP method gives a low average antigen content when a decrease in antibody binding occurs in sections, whereas the average antigen content measured by the IP method is unchanged. The intensity of AFP decreased primarily by a proportional decrease in antigenicity during fixation. However, the intensity of albumin decreased not only by a proportional decrease during fixation but also by a disproportionate reduction in antibody binding during immunostaining or before fixation. The results indicate that AFP content in sections is measurable by quantitative immunohistochemical methods, whereas albumin content is not.

Acetylcholinesterase regeneration in mouse CNS studied by a microphotometric method.

Acetylcholinesterase (AChE) regeneration after single and repeated doses of diisopropylfluorophosphate (DFP) was investigated in neostriatum of central nervous system of Balb mice, by means of a microphotometric method. In the present study, three experimental groups received the following DFP treatments: group I was administered a single dose of 3 mg/kg; group II received weekly doses of 3 mg/kg for 9 weeks, and group III was exposed to 28 weekly treatments of 1 mg/kg, plus a last treatment of 3 mg/kg, for a total of 29 doses. The results of microphotometric analysis for all the experimental groups have shown a distinct reversibility pattern of DFP inhibition, consistent with the process of AChE regeneration. Nevertheless, one week following the last DFP treatment, a persisting difference (approximately 22%) in the AChE concentration between control and the experimental groups was observed. Based on our results and on the correlation reported in the literature between AChE reduction and muscarinic receptor density, the hazardous implications of organophosphate environmental contamination on human and animal health are pointed out.

Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processor.

We have a quantitative light microscopic immunohistochemical method using video image processing. First, an antigen (NADPH-cytochrome P-450 reductase) content in homogenates of livers of rats was measured by enzyme immunoassay. Then frozen sections from rat livers were incubated with the anti-NADPH-cytochrome P-450 reductase antibody under saturation conditions by the indirect immunoperoxidase method. Subsequently, relative staining intensities in small portions and those in wide areas in the sections were measured with a video image processor. Finally, the resulting relative values obtained from the small portions were converted into absolute NADPH-cytochrome P-450 reductase contents using the results of enzyme immunoassay and the average relative staining intensity obtained from the wide areas in the sections. The reductase content in sections from rat livers measured by the image processing method coincided with the content measured by the microphotometric method using a nitrocellulose model system. The present image processing method is applicable to measurement of contents of antigens that can not be immobilized in model systems.

Kinetic Modelling of Chloroplast Phototranslocations in Lemna trisulca L.: Two Rate Limiting Components?

An experimental model is derived from analytical action spectroscopy, in which total chloroplast translocation response to blue light is a linear combination of independent positive (low-intensity response) and negative (high-intensity response) components. This model explains transmission changes in Lemna trisulca L. leaves under steady-state light conditions and transient displacements after single pulses of strong blue light. The initial rate of chloroplast phototranslocation as measured by the microphotometric method is dependent on the fluence rate of the actinic light in both positive and negative response ranges. In both cases the absolute level of response is proportional to the initial rate of chloroplast translocation. Model considerations and kinetic experiments indicate that positive and negative chloroplast responses to light are controlled by two different rate-limiting processes and consequently may have separate control mechanisms.

Quantitative succinate dehydrogenase analysis in normal and ragged-red muscle fibers

Single fiber analyses were performed in normal and diseased muscle by means of a high-resolution microphotometric method. We investigated the activity distribution of a mitochondrial marker enzyme, succinate dehydrogenase, within single muscle fibers. We differentiated between the central and the subsarcolemmal region. Both normal muscle fibers, and ragged-red fibers from patients with a mitochondrial myopathy showed significantly higher succinate dehydrogenase activities in the subsarcolemmal region. Since the fibers' supply of oxygen is accomplished by diffusion from capillaries located close to the sarcoplasmic membrane our results are of functional importance.

An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm.

To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.

Is There a Third Photoreceptor Involved in the Control of Chloroplast Movements in Mougeotia?

The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor.

Coalification and metamorphism in Torlesse Supergroup rocks, North Island, New Zealand

Abstract Microphotometric methods of coal rank assessment have been applied to dispersed coal fragments (phytoclasts) in metasedimentary rocks of the central North Island Torlesse sequence. To the east (Kaweka Range), zeolite facies rocks have phytoclast mean maximum reflectances of <1%, increasing through the prehnite-pumpellyite and stilpnomelane zones in the west (Kaimanawa Mountains) to a maximum of 10% reflectance. Graphite has been found in rocks approaching greenschist grade, but faulting has complicated the pattern of mineral and reflectance isograds. Reaction kinetic models show that the duration of heating has a major influence on the rank attained by the coal and this must be taken into account when comparing data from different metamorphic terranes.

A new microphotometric method for measurement of cytochrome P-450 in sections of liver.

We developed a new microphotometric method for measuring the amounts of cytochrome P-450 (P-450) in fresh frozen sections of liver. Four serial frozen sections cut from the liver were separately incubated in 50 mM Tris-HCl buffer (pH 8.0) alone, in buffer containing sodium dithionite, in buffer saturated with carbon monoxide (CO), and in buffer saturated with CO and containing sodium dithionite. The difference between absorbance at 450 nm and that at 490 nm was measured in these sections with a simple microphotometer system. This method yielded precise amounts of P-450 in sections by measuring the true extinction of P-450 and by minimizing the effect of contaminating hemoproteins. Livers of adult rats contained large amounts of P-450, which was greater in perivenular hepatocytes than in periportal hepatocytes. In livers of newborn rats, however, small amounts of the enzyme were distributed evenly throughout the lobule.

Dispersion Behavior of Solutes in an Ideal Laminar Flow with Small-Bore Glass Tubes

Abstract The dispersion behavior of solute molecules injected as a sample plug into an ideal laminar capillary flow was experimentally analyzed by means of microscopic and microphotometric methods. The flow pattern in small-bore glass tubes was directly observed by the microscopic method using a camera. The peak responses obtained for the colored solutes by means of the microphotometric method consist of double-humped peak features which resulted from both laminar flow convection and the molecular diffusion of solute molecules at the parabolic interface between the sample plug and the carrier fluid. The influences of the diffusion coefficient of the solute, the flow rate, the tube length, the tube diameter, etc. were mainly investigated in order to guide the design and optimization of the flow systems of capillary liquid chromatography (CLC), capillary electrophoresis (CEP), flow-injection analysis (FIA), etc. The experimental results obtained here are in good agreement with the theoretical results obtained by the numerical solution for the diffusion-convection equation.

Effect of anticancer drugs and hyperthermia on nasopharyngeal carcinoma cell line in vitro

The therapeutic effects of 10 anticancer drugs on a cultured nasopharyngeal carcinoma (NPC) cell line were examined by tritiated thymidine incorporation-inhibition assay and growth inhibition assay by a microphotometric method. The NPC cell line was found to be heterogeneous in terms of sensitivity to these anticancer drugs. Therapeutic efficacies of selected drugs could be increased by either increasing the drug concentration or the time of exposure of cells to drugs. Short duration of hyperthermia alone was also effective in inhibiting cell growth. Combined hyperthermia and actinomycin D or vincristine sulfate are markedly cytotoxic for the NPC cells in vitro.

Microphotometric investigation of fixed holograms

Holographic gratings are written in copper-doped LiNbO3 crystals at temperatures of about 180 degrees C. At room temperature the gratings are fixed. The subsequent development process under homogeneous illumination is investigated by the microphotometric method. Measurements of the light intensity pattern at the exit face of the crystal reveal not only the change of the amplitude but also a phase shift of nearly pi . The dynamics of the development process can be fully understood by the assumption of a quasi-permanent modulated charge distribution and a second modulated charge distribution of optically excitable ions.

Enzyme activity measured in single muscle fibers in partial cytochrome c oxidase deficiency.

Single-fiber analyses using a kinetic microphotometric method were performed on three patients with chronic progressive external ophthalmoplegia and proximal myopathy accompanied by a partial deficiency of cytochrome c oxidase. Two patients had subsarcolemmal accumulation of mitochondria (ragged-red fibers). Qualitative histochemical demonstration of cytochrome c oxidase showed a mosaic of fibers without detectable cytochrome c oxidase activity. Quantitative single fiber measurements in the patients' biopsies showed that the majority of the muscle fibers had decreased cytochrome c oxidase activity without selective involvement of a specific fiber type. Succinate dehydrogenase was measured and the ratio of the activities (succinate dehydrogenase/cytochrome c oxidase) was calculated. Normal muscle showed a ratio of about 2, whereas diseased muscle showed values between 10 and 20, due to a decrease in cytochrome c oxidase activity. Ragged-red fibers showed very low or undetectable cytochrome c oxidase activity.

Estimation of the transfer coefficients of oxygen and carbon monoxide in the boundary of human and chicken red blood cells by a microphotometric method.

The reaction rates of O2 and CO with the human and chicken red blood cell (RBC) were measured by using a microphotometric apparatus. In the experiments on the human RBC, a small amount of RBCs were put in an air-tight reaction cuvette. Gas mixtures containing various concentrations of O2 and CO were sequentially injected into the cuvette and the change in O2 and CO saturation of hemoglobin was measured from the change in transmission of the RBCs at 402 and 416.5 nm. The reaction rate of CO with RBCs was significantly influenced by photodissociation of carboxyhemoglobin (COHb). To eliminate this, a short-pass filter (400 to 435 nm) and a sector (100 Hz) were used. By comparing the measured reaction rates of O2 and CO with the theoretical rates obtained from the numerical solutions of the partial differential equations of the diffusions of O2 and CO, the transfer coefficients of O2 and CO (eta O2 and eta CO) in the RBC boundary, including the RBC membrane and water layer around the RBC, were estimated. Both the values showed good agreement, ranging from 0.3 to 2.5 x 10(-6) cm.sec-1.Torr-1. Furthermore, the chorioallantoic capillary of chicken embryo was used for the measurements of the reaction rates of O2 and CO with RBC through the capillary membrane. The reaction rates of O2 and CO in the chorioallantoic capillary were slower than those obtained in the human RBC. By comparing the measured reaction rates and the numerical solutions, the eta O2 and eta CO in the boundary, including the capillary membrane, plasma, and RBC membrane, were estimated. These two values ranged from 0.1 to 0.4 x 10(-6) cm.sec-1.Torr-1 and showed good agreement. These results suggest that the diffusion rates for O2 and CO across the capillary and RBC membrane are similar.

Studies of Thermal Diffusion of Copper in Amorphous Germanium Chalcogenides

Step-like doping profiles are observed by means of the microphotometric edge tracing method in thermal copper-doping of amorphous Ge30Se70. The doping profiles are described theoretically by supposing that the diffusion coefficient switches between a low and a high value after the doping concentration reaches a percolation threshold. The diffusivity within the undoped chalcogenide increases with duration of a pre-exposure.

Investigation of the dynamic recording process of thick refractive index gratings in photorefractive crystals by the microphotometric method

Diffraction-efficiency measurements are compared with the microphotometric determination of the intensity pattern at the exit face of a LiNbO3-sample during the holographic recording process. The special advantage of the microphotometric method is the possibility to determine phase shifts of the refractive index grating and of the light intensity pattern simultaneously and independently. Both quantities are very important for an understanding of dynamical effects during the holographic writing process. Furthermore, direct experimental evidence for the change of the modulation degree, i.e. the change of the contrast of the light pattern writing the grating, has been found. This change is caused by a Moire-like effect which is closely related to the energy transfer between writing beams.