The fluorescence of elastic fibres stained with Eosin and excited by visible light

Synopsis Fluorescent light of any wavelength, emitted by a microscopical specimen excited with visible light, can be observed using crossed polarizers and a dark-field condenser. The absorbance of Eosin is high in the green portion of the spectrum, so that visible light from a tungsten lamp is highly effective in exciting the fluorescence of this dye.Elastic fibres in routine sections stained with Haemalum and Eosin have been found to fluoresce rather strongly, while most other Eosin-stained structures fluoresce less or not at all. The reason for this relatively selective fluorescence of Eosin-stained elastic fibres is obscure, although the phenomenon may prove useful in routine histology. It is possible, however, that in most stained substrates the fluorescence of Eosin is largely quenched by aggregation of the dye molecules, but that this is inhibited inside the relatively dense (i.e. impermeable) elastic fibres.     

An Inexpensive Microscope Lamp for Critical Illumination

A microscope lamp with light of adjustable intensity, which will afford critical illumination of a quality satisfactory for the most exacting cytological work, can be constructed for about $7.50. The essential parts needed are: auto headlight bulb, radio power transformer, radio “potentiometer”, and simple plano-convex lens. A ground glass is not used with 4 and 2 mm. objectives, a solid source of light being obtained by placing the bulb so that one filament coil is directly behind the other. This light source is brought to a focus by the condensing lens at a field diaphragm and this image is projected by the microscope condenser into the plane of focus of the microscope.     

Action spectra of the light-growth response of Phycomyces

The light-growth response of Phycomyces blakesleeanus (Burgeff) is a transient change in elongation rate of the sporangiophore caused by a change in light intensity. Previous investigators have found that the light-growth response has many features in common with phototropism; the major difference is that only the light-growth response is adaptive. In order to better understand the light-growth response and its relationship to phototropism, we have developed a novel experimental protocol for determining light-growth-response action spectra and have examined the effect of the reference wavelength and intensity on the shape of the action spectrum. The null-point action spectrum obtained with broadband-blue reference light has a small peak near 400 nm, a flat region from 430 nm to 470 nm, and an approximately linear decline in the logarithm of relative effectiveness above 490 nm. The shape of the action spectrum is different when 450-nm reference light is used, as has been shown previously for the phototropic-balance action spectrum. However, the action spectrum of the light-growth response differs from that for phototropic balance, even when the same reference light (450 nm) is used. Moreover, for the light-growth response, the relative effectiveness of 383-nm light decreases as the intensity of the 450-nm reference light increases; this trend is the opposite of that previously found for phototropic balance. The dependence of the lightgrowth-response action spectrum on the reference wavelength, its difference from the phototropic-balance action spectrum, and the reference-intensity dependence of the relative effectiveness at 383 nm may be attributable to dichroic effects of the oriented photoreceptor(s), and to transduction processes that are unique to the light-growth response.I dedicated to Masaki Furuya on the occasion of his 65th birthdayThis work was supported by a grant from the National Institutes of Health (GM29707) to E.D. Lipson. Anuradha Palit, Promod Pratap, and Benjamin Horwitz participated in the early phases of this work. We thank Leonid Fukshansky and Benjamin Horwitz for helpful discussions, David Durant for computer programming, and Steven Block for providing us with a C-language program of Reinsch's procedure for cubic spline interpolation. One of us (R.S.) gratefully acknowledges a junior faculty fellowship leave from the Department of Physics at Yale University.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.     

Analysis of fluorescence decay curves by means of the Laplace transformation.

A computational procedure is described for the analysis of fluorescence decay data convolved with a lamp flash of finite width. The computer program calculates the ratio of the Laplace transforms of the decay and the lamp flash for different values of s to give the transforms of the impulse response for each value of s. These are set equal to the analytical Laplace transforms of the decay law involved. Solution of the nonlinear simultaneous equations yields the desired decay parameters. The method can be modified to analyze data that contains a component due to scattered light and can also provide essential information regarding transit time changes of the photomultiplier with changes in emission wavelength. The method was tested by the analysis of real and simulated data. The accuracy of the analysis depends on the degree of correlation among the parameters.     

Laser surgery: using the carbon dioxide laser.

In 1917 Einstein theorized tha through an atomic process a unique kind of electromagnetic radiation could be produced by stimulated emission. When such radiation is in the optical or infrared spectrum it is termed laser (light amplification by stimulated emission of radiation) light. A laser, a high-intensity light source, emits a nearly parallel electromagnetic beam of energy at a given wavelength that can be captured by a lens and concentrated in the focal spot. The wavelength determines how the laser will be used. The carbon dioxide laser is now successfully employed for some surgical procedures in gynecology, otorhinolaryngology, neurosurgery, and plastic and general surgery. The CO2 laser beam is directed through the viewing system of an operating microscope or through a hand-held laser component. Its basic action in tissue is thermal vaporization; it causes minimal damage to adjacent tissues. Surgeons require special training in the basic methods and techniques of laser surgery, as well as in the safety standards that must be observed.     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

Red region excitation spectra of protochlorophyllide in dark-grown leaves from plant species with different proportions of its spectral forms

Etiolated leaves of three different species, maize, wheat, and pea, as well as a pea mutant (lip1) were used to compare the excitation spectra of protochlorophyllide (Pchlide) in the red region. The species used have different composition of short-wavelength and long-wavelength Pchlide forms. The relation between different forms was furthermore changed through incubating the leaves in 5-aminolevulinic acid (ALA), which caused an accumulation of short-wavelength Pchlide forms, as shown by changes in absorption and fluorescence spectra. This is the first time a comprehensive comparison is made between excitation spectra from different species covering an emission wavelength range of 675–750 nm using fluorescence equipment with electronic compensation for the variations in excitation irradiance. The different forms of Pchlide having excitations peaks at 628, 632, 637, 650, and 672 nm could be best measured at 675, 700, 710, 725, and 750 nm, respectively. Measuring emission at wavelengths between 675– 710 nm gave an exaggeration of the short-wavelength forms and measuring at longer wavelengths gave for the pea leaves an exaggeration of the 672 nm peak. In general, an energy transfer from short-wavelength Pchlide forms to long-wavelength Pchlide forms occurred, but such an energy transfer sometimes seemed to be limited as a result of a discrete location of the Pchlide spectral forms. The excitation spectra resembling the absorption spectrum most were measured at an emission wavelength of 740 nm. Measuring the excitation at 710 nm gave higher intensity of the spectra but the short-wavelength forms were accentuated.     

Quinacrine and acridine-R banding without a fluorescence microscope

Summary A technique is described in which a standard fluorescence microscope equipped with a high-pressure mercury lamp is replaced with an ordinary laboratory microscope fitted with a quartz-iodine lamp. A dark field condenser and a set of three filters, including an FITC interference filter, complete a fluorescence microscope.The microscope has proved itself satisfactory in the study of Y-chromatin, chromosome Q-bands including Q-polymorphism, and acridine-R band. It is very easy to operate and does not emit ultraviolet light, which might harm operators. Total cost of the quartz-iodine lamp's outfit, filters, and a dark-field condenser is much less than that of a standard fluorescence microscope. The cost is especially low when a laboratory microscope with a quartz-iodine lamp is already at hand. Spectrofluorometric studies of QM and Q indicate that the present system will show even better performance if an interference filter with a transmission range of about 400 to 440–450 nm is designed and used in combination with a 455–475 nm barrier filter.     

Spectral Sensitivity of Melanophores in the Primary Color Response of the Rose Bitterling, <Emphasis Type="Italic">Rhodeus ocellatus ocellatus</Emphasis>

Melanophores of young Rhodeus ocellatus ocellatus have the ability to respond by melanosome dispersion to the direct action of visible light. The effective wavelength within visible light region for inducing melanosome dispersion was investigated using melanophores located around the base of the dorsal fin of young fish set on the stage of a light microscope. The melano- -’ phores were exposed to light of various wavelengths (420–680 nm) but of the same intensity by placing interference filters under the condenser diaphragm. The most effective wavelength was about 420 nm. Longer wavelengths were less effective for the induction of melanosome dispersion.     

Wavelength dependence of suppression of potato sprout growth by light

Abstract Light is used commercially to prevent the sprouts on seed potatoes from growing so long that they are knocked off at planting. We investigated the relationship between wavelength and growth inhibition, using a very long irradiation time of 23 d with a 12-h photoperiod. The inhibition showed a narrow peak of far-red activity centered on 707 nm, with a shoulder in the red whose size depended on the degree of inhibition chosen as standard, because the log photon-fluence rate/response lines were not parallel. There was also inhibitory activity in the blue (< 500 nm). In many respects the wavelength relationship resembled the action spectra for growth inhibition in dark-grown seedlings, but in the latter the red peak is, or quickly becomes, more pronounced than the far-red peak: this did not happen with potato sprouts. Blue light caused a positive phototropic response at similar or lower fluence rates. Greening became visible only at the highest fluence rates, between the two spectral regions inhibitory to growth. Broadband sources had much less inhibitory activity in the 650–750-nm region.     

On the use of eosin as a fluorescent dye to demonstrate mucous cells and other structures in tissue sections

Summary The fluorescence of Eosin taken up by mucins and other structures in sections of tissue stained with Haematoxylin and Eosin is readily observable with an ordinary microscope using tungsten light and a dark-field condenser. The demonstration of mucin in this manner in histopathologic diagnosis may obviate the need for a special stain for mucin with the attendant delay.The intensity and colour of the fluorescence emitted by Eosin-stained structures depends on the amount of dye taken up by them. This in turn appears to be related to the number of amine groups in the protein component of the mucin complex that are available for ionic combination with the anionic groups of the dye. The saccharide component of the mucin does not appear to be responsible for its eosinophilia.Fluorescein and Fluorescein isothiocyanate, utilised as simple acid dyes, induced slight to moderate fluorescence in various proteins and mucins. However, sections of tissues containing antigens that had been incubated with Fluorescein-labelled antibodies did not show significant dark-field fluorescence when illuminated with a tungsten lamp.Wellcome Trust Fellow.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.     

Action spectra for killing and mutation of Chinese hamster cells exposed to mid- and near-ultraviolet monochromatic light

We have examined the response of Chinese hamster V79 cells to monochromatic light of selected wavelengths in the mid- to near-UV region, using cell survival and induction of mutants resistant to 6-thioguanine (6-TG) or ouabain (OUA) as end points. As the wavelength increased from 313 to 405 nm, the induction of mutants resistant to 6-TG and to OUA decreased to a greater degree than did cell survival. Cells resistant to OUA were induced with considerably lesser efficiency at wavelengths of 313 and 334 nm than cells resistant to 6-TG. No mutants resistant to either 6-TG or OUA were induced by 405-nm light, and no mutants resistant to OUA were induced by 365-nm light. Thus, cell killing and mutation induction have different action spectra, and furthermore, action spectra for mutation induction at the HGPRT and Na+/K+-ATPase loci are different from each other. These observations imply important differences in the cellular mechanisms, and/or lesions, for cell inactivation, induction of 6-TG and OUA resistance for V79 cells exposed to near-UV monochromatic light.     

Plasmonic Lens with Multiple-Turn Spiral Nano-Structures

In this paper, we investigate the focusing properties of a plasmonic lens with multiple-turn spiral nano-structures, and analyze its field enhancement effect based on the phase matching theory and finite-difference time-domain simulation. The simulation result demonstrates that a left-hand spiral plasmonic lens can concentrate an incident right-hand circular polarization light into a focal spot with a high focal depth. The intensity of the focal spot could be controlled by altering the number of turns, the radius and the width of the spiral slot. And the focal spot is smaller and has a higher intensity compared to the incident linearly polarized light. This design can also eliminate the requirement of centering the incident beam to the plasmonic lens, making it possible to be used in plasmonic lens array, optical data storage, detection, and other applications.     

Inhibitory effect of p-nitrothiophenol in the light on the photosystem II activity of spinach chloroplasts.

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH'S caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiopehnol to causing the specific inhibition of Photosystem II.     

A circular dichroism microspectrophotometer

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.     

Light dependent oxygen uptake by the blue green alga Anacystis nidulans.

Cells of Anacystis nidulans consume oxygen when illuminated with 750 nm light. The same process occurs with 675 nm light when the photosynthetic production of oxygen has been halted by gentle heating of the cells. These reactions do not require the addition of artificial redox compounds. There seem to be two separate systems, one activated by 750 nm light, the other by 675 nm light. Polarographic action action spectra reveal that the 675 nm system utilises pigments of the photosynthetic apparatus, excluding phycocyanin. Fluorescence excitation spectra suggest that only the pigment P750 is involved in the 750 nm system. Purified P750 recombines spontaneously with extracted pigment-free cell fragments. After recombination the P750 has the same spectroscopic properties as the pigment in vivo.     

207-nm UV Light - A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. I: In Vitro Studies

Background

0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.

Aims

The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.

Methods

A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.

Results

We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.

Conclusions

As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.