Microscope Eyepiece Research Articles

Conjunctivitis in Clean Room Workers

Conjunctivitis, in the industrial setting, can be attributed to eye contact with contaminated microscope oculars. Establishing a simple, convenient process for cleaning microscope oculars as well as educating employees and their management is important to reduce risk of contamination. Early identification and treatment of an isolated infection by the occupational health nurse and temporary exclusion from microscope work is essential in controlling the spread of infection and preventing a potential epidemic. The occupational health nurse plays a pivotal role in the recognition of and response to workplace health concerns and development of targeted strategies to promote and maintain health in the workplace.

A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi.

Previously described methods to quantify the proportion of root length colonized by vesicular-arbuscular (VA) mycorrhizal fungi are reviewed. It is argued that these methods give observer-dependent measures of colonization which cannot be used to compare, quantitatively, roots examined by different researchers. A modified method is described here to estimate VA mycorrhizal colonization on an objective scale of measurement, involving inspection of intersections between the microscope eyepiece crosshair and roots at magnification × 200; it is referred to as the magnified intersections method. Whether the vertical eyepiece crosshair crosses one or more arbuscules is noted at each intersection. The estimate of colonization is the proportion of root length containing arbuscules, called the arbuscular colonization (AC). The magnified intersections method also determines the proportion of root length containing vesicles, the vesicular colonization (VC), and the proportion of root length containing hyphae, the hyphal colonization (HC). However, VC and HC should be interpreted with caution because vesicles and hyphae, unlike arbuscules, can be produced in roots by non-mycorrhizal fungi.

Ocular infections and the industrial use of microscopes.

A cluster of ocular infections occurred in one area of a computer fabrication facility that relied on the use of industrial microscopes. A questionnaire was administered to all employees in this area. Microscope oculars were cultured and compared with control microscopes from a nonindustrial setting. Risk of infection was correlated with the number of hours of microscope use per day and subjective indicators of cleanliness. Bacterial cultures confirmed increased colony counts in industrial oculars compared with control oculars. Hygienic practices were instituted similar to those employed in medical settings. No further outbreaks of conjunctivitis have been reported in a 1-year follow-up.

Quantitative assessment of atherosclerotic lesions in mice

The well-defined genetic systems of the mouse are proving useful in experimental studies of atherosclerosis. Inbred mouse strains differ in atherosclerosis susceptibility, and several variants of apolipoproteins have been identified and mapped. This report explores the location and timing of lesion formation in the mouse in an effort to provide a basis for quantitatively comparing groups of mice. After 14 weeks on an atherogenic diet containing 1.25% cholesterol, 15% fat, and 0.5% cholic acid, C57BL/6J female mice had aortic lesions at each of the intercostal arteries, at the junction of the aorta to the heart, and in scattered areas covering 1.1% ± 0.5 (SD) of the aortic surface. After 9 months on the atherogenic diet, those lesions near the heart and intercostal arteries were extensive, 8% ± 3 (SD) of the remainder of the aorta was involved in lesions, and lesions were found in the coronary arteries. Results indicated that one suitable location for scoring lesions was in a 300 [tin area of the aorta just beyond the aortic sinus. The mean number of lesions/mouse in the selected area after 14 weeks on the atherogenic diet was 1.1 ± 0.3 (SD). The results were reproducible over 10 separate experiments. The number of lesions per mouse fit a Poisson distribution indicating that the presence of one lesion did not predispose the mouse to acquiring a second lesion. Lesion formation and cholesterol levels did not vary with the season of the year as demonstrated by 9 separate experiments over more than 12 months. Methods of evaluating the number and size of lesions were compared including sizing with a microscope eyepiece grid and computer-assisted planimetry. The resulting data provide reproducible methods of quantitatively comparing lesion formation in various strains or groups of mice, thereby increasing the usefulness of the mouse as an experimental system for atherosclerosis research.

Microscopes and ocular infections.

Environmental microbial assays of industrial microscope eyepieces were conducted following reports of multiple intershift ocular infections. Pathogenic Staphylococcus aureus was identified among the microorganisms cultured. This paper suggests that direct contact with industrial microscope eyepieces provides a potentially significant route of transmission of both bacterial and viral ocular infections. An industrial hygiene ocular health questionnaire was distributed to a first and second shift manufacturing operation to assess the incidence of ocular infections. These data were compared to the questionnaire responses of 122 control manufacturing workers who did not use microscopes. Based on self-reporting by employees, those who used microscopes were found to have statistically significant incidence of sites and conjunctivitis that was 8.3 times that of the control group. Sterilization of eyepieces by ethylene oxide, formaldehyde and isopropyl alcohol were considered, but ultimately rejected. These biocides were found respectively to damage ocular lens coatings, contribute to volatile organic emissions, or be ineffective against spore-forming bacteria. This article presents a detailed evaluation of a commercially available ultraviolet sanitization unit (manufactured by the King Bactostat Corp., 7115 Armistad Street, El Paso, TX 79912). This ultraviolet disinfection process proved to be rapid and emission free; it also yielded eyepieces free of residual chemical biocides that have the potential for ocular irritation. Field tests involving 60 eyepieces demonstrated effective disinfection by a Chi-Square statistical comparison, at values greater than 95% confidence level, as compared to unirradiated eyepieces.

Microscopes and Ocular Infections

Environmental microbial assays of industrial microscope eyepieces were conducted following reports of multiple intershift ocular infections. Pathogenic Staphylococcus aureus was identified among the microorganisms cultured. This paper suggests that direct contact with industrial microscope eyepieces provides a potentially significant route of transmission of both bacterial and viral ocular infections. An industrial hygiene ocular health questionnaire was distributed to a first and second shift manufacturing operation to assess the incidence of ocular infections. These data were compared to the questionnaire responses of 122 control manufacturing workers who did not use microscopes. Based on self-reporting by employees, those who used microscopes were found to have statistically significant incidence of sties and conjunctivitis that was 8.3 times that of the control group. Sterilization of eyepieces by ethylene oxide, formaldehyde and isopropyl alcohol were considered, but ultimately rejected. These biocides were found respectively to damage ocular lens coatings, contribute to volatile organic emissions, or be ineffective against spore-forming bacteria. This article presents a detailed evaluation of a commercially available ultraviolet sanitization unit (manufactured by the King Bactostat Corp., 7115 Armistad Street, El Paso, TX 79912). This ultraviolet disinfection process proved to be rapid and emission free; it also yielded eyepieces free of residual chemical biocides that have the potential for ocular irritation. Field tests involving 60 eyepieces demonstrated effective disinfection by a Chi-Square statistical comparison, at values greater than 95% confidence level, as compared to unirradiated eyepieces.

Specific tension measurements in single soleus and medial gastrocnemius muscle fibers of the cat

Direct measurements of the sizes of and forces produced by single fibers of the cat soleus and medial gastrocnemius muscles were made to determine whether or not different fiber types have characteristically distinct specific tensions. Single fibers (5-mm lengths), whose sarcolemmas had been chemically removed using a 5-mM EGTA “skinning” solution, were attached to a photodiode force transducer. Each single fiber was first placed in “relaxing” solution (22 ± 1°C, pH 7.0, pCa 8), its sarcomere length set at 2.7 μm using its laser diffraction pattern, and its diameter measured using the calibrated graticule of a microscope eyepiece (±2 μm). Subsequently, each fiber was transferred to an activating bathing solution ( pCa 3.6) in which the fiber produced its maximum tension. The specific tension values for single soleus muscle fibers displayed a threefold range (1.19 to 3.53 kg/cm 2) with a mean value of 2.30 ± 0.61 (SD) kg/cm 2 ( N = 42). The medial gastrocnemius fibers studied had a fourfold range in specific tensions (1.05 to 4.47 kg/cm 2) and a mean value of 2.42 ± 0.61 kg/cm 2 ( N = 104). Many medial gastrocnemius fibers ( N = 64) were type-identified using a standard actomyosin ATPase histochemical assay. Type I medial gastrocnemius fibers had mean specific tension values of 2.45 ± 0.47 kg/cm 2 ( N = 18), whereas, type II single fibers had mean specific tension values of 2.43 ± 0.67 kg/cm 2 ( N = 46). Our results suggest that there is no significant difference between the specific tensions of the different muscle fiber types within the cat medial gastrocnemius muscle.

Three-dimensional reconstruction from serial sections III. Autoscan, a software package in FORTRAN for semiautomated photomicrography

A FORTRAN program, called AUTOSCAN, is described. This program permits the collection of photomicrographic data from serial sections to be semiautomated. In essence the user defines a box around a microscopic field of interest. Then the program drives the stage incrementally in the x and y directions, taking photographs of contiguous subfields. The box is defined by the use of a joystick and the “return” key. That is, movements of a joystick cause the stage to translate in the x and y directions. When a corner of the object is reached, as defined by cross-hairs in the microscope eyepiece, the user hits the return key. Repetition of this process at each corner defines a “box” within which photographs are to be taken. AUTOSCAN then calculates the step size and the number of frames to be taken from the user-defined values for the magnification. The actual movements of the stage in the x and y directions and the photography are fully automated. Each frame of film has the x and y coordinates of the center of the subfield being photographed imprinted in one corner, along with other relevant data. The x and y coordinates permit the resultant information to be assembled correctly into two-dimensional montage.

Left ventricular cellular hypertrophy in pressure- and volume-overload valvular heart disease

To determine the dependence of myocyte hypertrophy in chronic valvular heart disease on the site and type of lesion, the myocardium was studied from 11 patients with either pressure-overload hypertrophy (PO; four patients with aortic stenosis and two with mixed aortic stenosis/insufficiency) or pure volume-overload hypertrophy (VO; two patients with mitral regurgitation and three with aortic insufficiency). These patients, all without coronary artery disease, died zero to 34 days after valve replacement surgery. Diameters of 25 longitudinally oriented myocytes in the circular midwall myocardium were measured with a calibrated light microscope eyepiece reticle on each of five transmural, transverse, histologic sections from the apical, anterolateral, posterolateral, anteroseptal, and posteroseptal left ventricle. Statistical analysis by modified two-way analysis of variance (ANOVA) demonstrated that mean myocyte size (based on 125 measurements) varied widely among cases but was not statistically different among sites. The myocyte diameter for PO lesions (25.9 +/- 1.1 micron, mean +/- SEM) was significantly greater (P less than 0.05) than that for pure VO lesions (20.4 +/- 0.7 micron), despite equal relative heart weights (measured/predicted from body weight: 2.5 +/- 0.2 [mean +/- SD] versus 2.5 +/- 0.5). This study suggests that 1) cellular hypertrophy in valvular heart disease occurs uniformly throughout the left ventricular myocardium; and 2) mean myocyte diameters are greater in PO than in VO hypertrophy for equivalent cardiac enlargement.

Cellular basis of morphogenesis

ABSTRACT The relationship between cell density distributions and differentiation is of interest to workers in many different areas of developmental biology. Traditionally, such studies have required tedious counting of individual cells through a microscope eyepiece grid or on photographs. We have developed a rapid, reliable, and inexpensive microcomputer based system for counting cells in histological sections. The system employs an Apple He computer, Digisector video board (Micro Works, Del Mar, CA), Super Scan program (Magna Soft, La Canada, CA), Gibson Light Pen System (Gibson Laboratories, Laguna Hills, CA), and Telestar video camera (Bell and Howell). We have applied this to identify specific patterns of cell density in feathers. Our system is significantly different from other image analysis systems available because its low price (about $2500) makes it accessible to average laboratories, as opposed to the expensive image analysis systems which are available commercially or in a few special research laboratories. Normal feather germs, serial sectioned at 6 μ, and stained using the cold hydrolysis Feulgen technique were photographed at 400× using Kodak Tech Pan film and printed on Kodabromide paper (no. 5). One inch squares of the 4×6 inch print were exposed to the video camera. The correlation coefficient for the number of black pixels counted by the computer and the number of cells counted by hand from the photograph was >·98. Dramatic local increases in mesenchymal cell density in the area of the germ which protrudes distally were observed in the saggital sections of all the specimens examined. (Supported by NIH Training Grant 5T32AM07376.)

The image-combining computer microscope — an interactive instrument for morphometry of the nervous system

The image-combining computer microscope is a new configuration of light microscope and computer graphics instrumentation which provides remarkable morphometric capabilities over a wide range of applications. The key component is the well known camera lucida, an image-combining device that is used to superimpose the computer graphics image upon the microscope's image of the object under study. Illumination need not be limited to the bright-field variety. The image superposition permits the investigator to study the object and acquire data from it while always looking through the microscope oculars. A computer controlled stepping motor driven stage is integrated with translations of the graphics image to permit image superposition and data acquisition at maximum visual magnification over the full expanse of a 25 × 25 mm tissue section. The investigator controls the system by means of a graphics tablet and a “Menu” area visible to him in the microscope's field of view. Spatial data in all 3 dimensions can be acquired easily since the z-axis is also stepping motor controlled. EM micrograph transparencies can be studied similarly. Stereological applications are possible by superimposing test grids upon the image. The system is self-contained and carries out data analysis programs written in C or Basic. Simplicity of operation has been emphasized throughout.

Operating microscope eyepiece heaters.

Precipitation of water vapour from expired air on to exposed eyepiece lenses of operating microscopes can seriously interfere with the surgeon's view. By warming these optical surfaces to temperatures well above the dew point of expired air this problem is eliminated. A pair of small heaters were designed to clamp over the eyepiece tubes of a standard operating microscope and their effectivity assessed.

Small vessel constriction in the rat cremaster during the early phase of moderate hemorrhagic hypotension

Diameters of small vessels (89 ± 3 (SEM) μm arteries and 135 ± 8 μm veins) in the cremaster muscle of six experimental and seven control Wistar male rats (weight, 119 ± 5 g) were measured with a microscope eyepiece micrometer. After induction of anesthesia with sodium pentobarbital (50 mg/kg, ip), the femoral artery was cannulated to allow pressure measurement and to control hemorrhage with a servo-syringe. The experimental protocol was a 30-min control period, a 30-min hemorrhage period during which the arterial pressure was lowered to and maintained at 30 mmHg, and a 30-min recovery period which followed reinfusion of the hemorrhaged blood. The average hemorrhage volume was 25 ± 2.6 ml/kg of body wt. The principal responses were small artery and vein constriction to 78 ± 3 and 88 ± 3% of their control values respectively, and a pronounced bradycardia. All variables returned to their prehemorrhage control values during the recovery period and all of the animals survived a 48-hr postexperimental period. Observations of initial venoconstriction in the rat cremaster were considerably different than our previous observations of initial venodilatation in the wing of unanesthetized bats. The arterial responses to hemorrhage were very similar in both preparations. Presently, we do not have an adequate explanation for the lack of venoconstriction in similar experiments by Hutchins et al. ( Microvas. Res. 5, 131–140, 1973) .

Nonrandom distribution of goblet cells around the circumference of colonic crypt.

The ascending colon of the guinea pig was cut serially and stained with the periodic-acid-Schiff-hematoxylin method. Maps of transversely sectioned crypts were prepared using a microscopic eye-piece projector. The number and angular positions of goblet cells were mapped around the circumference of transverse sections of the crypt. A method of the "statistics of the circumference" was introduced in order to assess the pattern of distribution of goblet cells. This distribution was found to be significantly nonrandom. The nonrandomness is such that the goblet cells tend to occupy positions of the crypt circumference in maximal distances from each other.

An instrument for measuring radial immunodiffusion precipitates.

A standard stereozoom microscope was instrumented to measure directly areas of small circles. The zoom axis knob was replaced with a calibrated dial and a circular reticle was placed in the microscope eyepiece. This allowed selection of the “best fit” of the circular reticle to a slightly irregular precipitate and permitted measurement of area directly from the dial.

Journal article

A critical issue in quantitative neuromorphology is the accuracy and subsequent reliability of the tracing techniques employed to characterize neuronal components. Historically, the camera lucida was the only option for such investigations. In 1987, MBF Bioscience, Inc. (Williston, VT) developed the integrative Neurolucida computer-microscope system, replacing the camera lucida drawing tube with a Lucivid cathode ray tube, thereby allowing computer overlays directly on the view through microscope oculars. Subsequent advances in digital cameras have allowed the Lucivid system to be replaced so that microscope images can be traced by viewing the digital image on a computer monitor. Indeed, the camera systems now outsell Lucivid systems 9 to 1 (J. Glaser, personal communication, 08/2008). Nevertheless, researchers seldom note which of these configurations are being used (which may confound the accuracy of data sharing), and there have been no published comparisons of the Lucivid and camera configurations. The present study thus assesses the relative accuracy of these two hardware configurations by examining reconstructions of human pyramidal neurons. We report significant differences with respect to dendritic spines, with the camera estimates of spine counts being greater than those obtained with the Lucivid system. Potential underlying reasons (e.g., magnification, illumination, and resolution, as well as observer ergonomic differences between the two systems) for these quantitative findings are explored here, along with qualitative observations on the relative strengths of each configuration.

The Fiber-Optics Hypodermic Microscope

This paper presents the prototype of a hypodermic fiber-optics microscope. The instrument permits the researcher to observe tissues beneath the skin of the living animal or human, microscopically, through a hypodermic needle. The device utilizes fiber optics for image transmission, making it possible to observe through needles with inside diameters of 0.81 mm. Special features of this device include a self-contained illumination system within the same needle as the imaging bundle, and a unique microscope eyepiece which corrects for distortion caused by the necessary bevel at the far end of the needle. The resolving power of the present system is about 10 μ. A major problem is the definition of structures from each other within living tissue. Experiments are under way with various vital stains to improve this definition. Successful observations to date have been made of blood vessels and muscle fibers. Possible applications of the instrument are discussed.

Dimensions of microscope eyepiece graticules

Dimensions of microscope eyepiece graticules

Modifications to a travelling microscope used for measuring X-ray powder photographs

A travelling microscope has been adapted for measuring powder photographs. The cross-hairs in the microscope eyepiece were removed and the reference mark was obtained from a light spot thrown on the photograph from a projector clamped to the barrel of the microscope. Cross-hairs seen in silhouette tend to obscure faint lines on the photograph and prevent their measurement. The light spot eliminates this effect, allows change of magnification for measuring different lines without moving the reference mark, eliminates parallax and allows the use of a binocular eyepiece. These features reduce eyestrain while measuring a photograph and improve the accuracy of measurement of faint or diffuse lines. The photograph is illuminated by a short fluorescent tube, the light of which is cooler and harder in quality than that of filament lamps. Means are described of adjusting the light intensity of the tube over a wide range.

The Size of Living Bacteria

ABSTRACT The difficulties involved in the direct measurement, by eyepiece-micrometer or from photomicrographs, of small microscopic objects such as living bacteria are discussed. The accuracy with which this can be done is limited by the numerical aperture of the optical system and the wavelength of light used. With visible light it is scarcely possible to determine the dimensions of an object more accurately than to the nearest O’4 μ. It also seems probable, from the nature of the diffraction pattern at the edges of images of objects of circular cross-section such as bacteria, that direct measurement of the width of the image will tend to give an underestimate of the true width of the object. An interference microscope enables thickness measurements to be made that are not subject to these particular limitations, because with it, the phase-change in the light passing through the middle of a bacterium can be measured very accurately. This phase-change is proportional to the product of the refractive index of the bacterium minus that of the mounting medium, and its true thickness. Two methods were used to determine the mean thickness of the living bacilli in a number of different cultures of Lactobacillus bulgaricus. With the first, the mean refractive index of the bacilli was measured directly by the method of immersion refractometry first used by Barer and Ross (1952), and phase-change measurements were made on the bacilli mounted in dilute saline. Their mean thickness was calculated from these measurements. With the second method, phase-change measurements were made on the bacilli mounted in saline and also mounted in protein solutions with refractive indices ranging from 1·365 to 1·376; and, from these, both their mean thickness and their mean refractive index were calculated. The phase-change measurements were made with a Smith interference microscope and half-shade eyepiece (manufactured by Messrs. Charles Baker). The values for the mean thickness of the living L. bulgaricus from 14 different cultures obtained by the first method ranged from 1-13 μ to i·23 μ·, and those from 9 different cultures obtained by the second method ranged from 1 ·02 μ to 1 · 14 μ. The mean refractive indices of the latter calculated by the second method agreed very closely with that obtained by immersion refractometry, and differed by a maximum of o·oo9 in all the cultures measured. It therefore seems unlikely that the mean thickness measurements obtained by either method are wrong by more than about ±o·1 μ.