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PubMed1957

The structure and concentration of solids in photoreceptor cells studied by refractometry and interference microscopy.

Fragments of freshly obtained retinas of several vertebrate species were studied by refractometry, with reference to the structure of the rods and cones. The findings allowed a reassessment of previous descriptions based mainly on fixed material. The refractometric method was used also to measure the refractice indices and to calculate the concentrations of solids and water in the various cell segments. The main quantitative data were confirmed by interference microscopy. When examined by the method of refractometry the outer segments of freshly prepared retinal rods appear homogeneous. Within a few minutes a single eccentric longitudinal fiber appears, and transverse striations may develop. These changes are attributed to imbibition of water and swelling in structures normally too small for detection by light microscopy. The central "core" of outer segments and the chromophobic disc between outer and inner segments appear to be artifacts resulting from shrinkage during dehydration. The fresh outer segments of cones, and the inner segments of rods and cones also are described and illustrated. The volumes, refractive indices, concentrations of solids, and wet and dry weights of various segments of the photoreceptor cells were tabulated. Rod outer segments of the different species vary more than 100-fold in volume and mass but all have concentrations of solids of 40 to 43 per cent. Cone outer segments contain only about 30 per cent solids. The myoids, paraboloids, and ellipsoids of the inner segments likewise have characteristic refractive indices and concentrations of solids. Some of the limitations and particular virtues of refractometry as a method for quantitative analysis of living cells are discussed in comparison with more conventional biochemical techniques. Also the shapes and refractive indices of the various segments of photoreceptor cells are considered in relation to the absorption and transmission of light. The Stiles-Crawford effect can be accounted for on the basis of the structure of cone cells.

Histological Techniques↗

PubMed1960

Studies in histochemistry. LVII. Determination of the total dry mass of human erythrocytes by interference microscopy and x-ray microradiography.

The total dry mass of human erythrocytes was determined by both interference microscopy and x-ray microradiography. The determination of mass per unit area, and calculation of total dry mass per cell were simplified by changing the shape of the cells to spheres which were then flattened to discs of constant thickness when smeared on glass slides for measurement of fixed cells by interferometry, and to oblate spheroids when smeared on parlodion-coated slides for measurement of fixed cells by x-ray absorption. From x-ray measurements of 100 smeared and alcohol-fixed cells a mean dry mass per cell of 33.7 x 10(-12) g was obtained. Interference measurements of 100 fresh cells suspended in isotonic saline gave a mean value of 32.4 x 10(-12)g while interference measurement of 100 smeared and alcohol-fixed cells gave a mean value of 30.8 x 10(-12)g. The first two values compare well with a mean corpuscular hemoglobin of 31.2 x 10(-12)g, obtained from determinations of erythrocyte count and hemoglobin, since 95 per cent of the dry mass of the cell is hemoglobin. The difference in interference values between the fixed and fresh cells is possibly due to a difference between the specific refractive increment of alcohol-denatured hemoglobin and that of the unmodified substance. The value for the latter was used since that of the former is unknown.

Erythrocyte Indices↗

PubMed2005

Image of a straight edge in confocal self-interference microscopy.

An image of a straight edge in confocal self-interference microscopy (CSIM) is analyzed. Simulations of edge images based on a two-dimensional imaging equation are presented that show a 103% increase in edge gradient and a 43.1% decrease in the 10-90% width. The first experimental results, to our knowledge, for CSIM are presented and show good agreement with the simulation results and a 23% decrease in the 10-90% width.

Algorithms↗

PubMed2005

Two-dimensional imaging theory of confocal self-interference microscopy.

A two-dimensional coherent imaging equation is derived for confocal self-interference microscopy (CSIM), which uses a birefringent material to generate an interference pattern in the detection optics. This interference pattern, called a self-interference pattern, sharpens the point-spread function (PSF) along the lateral direction. To derive the imaging equation, an equation for the self-interference pattern is derived. Numerical simulation results based on the imaging equation are presented. One-point response results show a 42.8% reduction in the FWHM of the lateral PSF. Two-point response results show a nearly twofold improvement in two-point resolution.

Algorithms↗

PubMed1969

Spatial patterns of threadlike elements in the axoplasm of the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interference microscopy and by electron microscopy.

The giant nerve fiber of the squid (Loligo pealii L.) has been investigated in situ, and in fresh and fixed preparations, by differential interference microscopy and electron microscopy. A continuous, three-dimensional network, composed of threadlike elements, was disclosed in the axoplasm. The threadlike elements in the axoplasm are twisted as a whole into a steep, right-handed helix. In a peripheral ectoplasmic region, the elements are more parallel to one another and more densely packed than in a central endoplasmic core. The threadlike elements can be resolved into a hierarchy of decreasing order of size. Successive levels of the hierarchy are formed by the association of smaller elements into larger ones. The following levels in the hierarchy of network elements have been distinguished: 1-3-micro-wide threads, 0.1-0.35-micro-wide strands, and 70-250-A-wide unit-filament strands. The differential interference microscope selects, from the network, threads oriented at a specific angle to the long axis of the axon. The specific angle depends upon the orientation of the long axis of the axon relative to the direction of shear. It is postulated that the network configuration is expressed in the solid-state properties of the axoplasm essential for the normal functioning of the nerve fiber.

Animals↗

PubMed2002

Determination of fringe order in white-light interference microscopy.

Combining phase and coherence information for improved precision in white-light interference microscopy requires a robust strategy for dealing with the inconsistencies between these two types of information. We correct for these inconsistencies on every measurement by direct analysis of the difference map between the coherence and the phase profiles. The algorithm adapts to surface texture and noise level and dynamically compensates for optical aberrations, distortions, diffraction, and dispersion that would otherwise lead to incorrect fringe order. The same analysis also provides the absolute height data that are essential to relational measurements between disconnected surfaces.

Journal Article↗

PubMed2004

Effects of phase change on reflection in phase-measuring interference microscopy.

We show by analytical and numerical calculations that the phase change on reflection that occurs in interference microscopy is almost independent of the numerical aperture of the objective. The shift of the microscope interferogram response due to the phase change on reflection, however, increases with the numerical aperture. Measurements of the interferogram shift are made with a Linnik interference microscope equipped with various numerical-aperture objectives and are reported and compared with theory.

Journal Article↗

PubMed2001

Effect of an electric field on the motility of Entamoeba histolytica examined by multiple-beam interference microscopy.

A recently developed multiple-beam interference microscopic technique has been used to visualize submicroscopic structures of Entamoeba histolytica and their movements in applied external electric fields. The movements were videorecorded and it was found that at low current (120 microA) pseudopods are filled with hyaline ectoplasm. At slightly higher current (about 150 microA), the amoeba stops extending the pseudopods and loosens its attachment to the surface. At higher currents (200 microA), it forms a cyst and remains immobile for a time. Before this stage is reached a narrow ring is formed around the nucleus due to alterations in the proteins to protect it.

Animals↗

PubMed1978

The surface structure of ageing human articular cartilage: a study by reflected light interference microscopy (RLIM).

The surface contours of the weight-bearing area of the left lateral femoral condyle have been examined in vitro in 26 cadavers by reflected light interference microscopy over the age range 0--47 years. The presence of 4 orders of surface irregularity was confirmed. In addition, measurement of the depth, diameter and frequency of the tertiary order of surface hollows showed that while none of these variables was sex-associated, age-related changes in the variables were present. Multiple regression analysis showed that these changes could be considered in terms of periods of maturation (0--21 years) and ageing (22--50 years) of the cartilage. Focal changes in the surface were observed with advancing age. These changes appeared to be regressive and possibly represent an early stage in cartilage fibrillation.

Adolescent↗

PubMed1989

Fluid distribution in pork, measured by x-ray diffraction, interference microscopy and centrifugation compared to paleness measured by fiber optics.

Moderately PSE (pale, soft, exudative) and moderately DFD (dark, firm, dry) pork was examined by x-ray diffraction for interfilament separation, by differential interference contrast microscopy for interfiber area, and was centrifuged to measure water holding capacity (WHC). Internal reflectance spectra were measured by fiber optics. For PSE to DFD pork, filament separation ranged from 39 to 48 nm, interfiber area from 42 to 3%, and WHC from 49 to 64%, respectively. The correlation of reflectance with interfilament separation varied considerably with wavelength (reaching r = -.83 at 680 nm, P less than .005). The correlation of reflectance with interfiber area was more uniform across the spectrum (reaching r = .90 at 450 nm, P less than .005), as was the correlation of reflectance with WHC (reaching r = -.80 at 400 nm, P less than .005). At 24 h postmortem, fiber-optic spectrophotometry may be used as a rapid, nondestructive method to predict WHC and potential fluid losses from commercial pork with a moderate range from PSE to DFD. Interfiber area was correlated negatively with filament lattice area and WHC, but no significant correlation was found between filament lattice area and WHC. Filament separation was decreased only slightly by centrifugation. These results indicate that at 24 h postmortem the extra fluid released from PSE pork already has been lost from the myofilament lattice and is awaiting release from compartments downstream such as interfiber and interfascicular spaces.

Animals↗

PubMed1985

Morphology of Giardia agilis: observation by scanning electron microscopy and interference reflexion microscopy.

The flagellated protozoan, Giardia agilis, was isolated from tadpole small intestine and examined by scanning electron microscopy and interference reflexion microscopy. The general morphology of the G. agilis trophozoite is similar to G. muris and G. duodenalis, but with modifications that reflect its elongated form. Interference reflexion microscopic analysis of attachment of G. agilis reveals a pattern of focal contacts by the lateral crest of the ventral disc, the ventrolateral flange, the lateral shield, and by numerous microvillus-like appendages found along the lateral border of the trophozoite. The pattern of focal contacts was observed to be dynamic; trophozoites were observed to make and break the focal contacts in a relatively short time and to glide along the surface of the substratum without breaking focal contacts.

Animals↗

PubMed2004

[New method for quantification of cell swelling using infrared differential interference microscopy].

We have developed a novel device for the quantification of swelling of cells in acute brain slices. We can also carry out detailed real-time monitoring of hippocampal cells. The device we developed is based on an infrared differential interference contrast microscopy (IR-DIC) and a custom-made real-time computerized image analysis system for the quantification of the morphological dynamics of cells in slice preparations. We applied the coefficient of variation (CV) of light intensity in IR-DIC images to quantify the change in morphological dynamics. There were notable close relationships among the edema formations, the light transmittance, extent of changes in CV, and features of fEPSP during ischemic insult. We also applied this method for the evaluation of neuroprotective effects of mannitol. The dose-dependent improvement on the deteriorated hippocampal slices could be obtained by administration of mannitol (10, 50, and 100 mM) after 10-min ischemia. The present results indicate that CV is a reliable quantification index for edema formation of brain tissue and confirm that applying CV for the analysis in addition to the light transmittance analysis presents additional important information on brain tissue sweling.

Animals↗

PubMed1999

Axonal neurofilaments are nonessential elements of toxicant-induced reductions in fast axonal transport: video-enhanced differential interference microscopy in peripheral nervous system axons.

Neurofilament modification and accumulation, occurring in toxicant-induced neuropathies, has been proposed to compromise fast axonal transport and contribute to neurological symptoms or pathology. The current study compares the effects of the neurotoxicants acrylamide (ACR) and 2,5-hexanedione (2,5-HD) on the quantity of fast, bidirectional vesicular traffic within isolated mouse sciatic nerve axons from transgenic mice lacking axonal neurofilaments (Eyer and Peterson, Neuron 12, 1-20, 1994) and nontransgenic littermates possessing neurofilaments. Fast anterograde and retrograde membrane bound organelle (MBO) traffic was quantitated within axons, before and after toxicant exposure, using video-enhanced differential interference contrast (AVEC-DIC) microscopy. Addition of 0.7 mM ACR to the buffer bathing the nerve produced a time-dependent reduction in bidirectional transport with a similar time to onset and magnitude in both transgenic and nontransgenic mice. 2,5-HD (4 mM) exposure reduced bidirectional vesicle traffic by a similar amount in both transgenic and nontransgenic animals. The time to onset of the transport reduction was less and the magnitude of the reduction was greater with 2,5-HD compared to ACR. A single 10-min exposure to ACR or 2,5-HD produced a similar reduction in transport to that produced by prolonged (1 h) exposure. Nonneurotoxic propionamide or 3,4-hexanedione (3,4-HD) produced no changes in bidirectional transport in either transgenic or nontransgenic animals. We conclude that ACR or 2,5-HD produces a rapid, saturable, nonreversible, neurotoxicant-specific reduction in fast bidirectional transport within isolated peripheral nerve axons. These actions are mediated through direct modification of axonal component(s), which are independent of toxicant-induced modifications of, or accumulations of, neurofilaments.

Acrylamide↗

PubMed2006

Stroboscopic white-light interference microscopy.

The principle of stroboscopic motion freezing of oscillating objects extends directly to interference microscopes that use coherence as part of the measurement principle. Analysis shows, however, that the fringe contrast loss for out-of-plane motion in stroboscopic interferometry is a wavelength-dependent phenomenon, which can alter the apparent nominal center wavelength of the white-light source. As in monochromatic systems, the key adjustable parameter is the duty cycle, equal to the product of the vibrational frequency and the pulse width. This theoretical study provides detailed graphs of expected errors as a function of the duty cycle, including fringe contrast loss, apparent wavelength shift, and measurement error.

Algorithms↗

PubMed1988

[Interference microscopy of human dental enamel at various age periods].

Investigation of the human tooth enamel by means of interference contrast makes it possible to reveal certain peculiarities of the prismatic structure in various age groups. For example, in children the enamel has specific porosity of the superficial layers, that is peculiar for teeth with a delayed eruption. With age the enamel becomes more "homogeneous", amount and size of the pores decrease, aprismatic areas in the superficial layer are found more often. Erased teeth are characterized with formation of microdefects, destruction of prisms on the enamel surface and at the same time with increased mineralization of the subsuperficial layer. Thus, age changes of the enamel are of adaptive character--consolidation of structural elements promotes increasing resistivity of the teeth to influence of pathological factors.

Adolescent↗

PubMed1998

Image Formation in Femtosecond Confocal Interference Microscopy

: Three-dimensional image formation in an interference confocal scanning microscope under ultrashort pulsed beam illumination is investigated in this study. The novelty of this new image system is that it keeps advantages in femtosecond interferometry but also provides a femtosecond-resolved three-dimensional image without necessarily using an ultrafast detector. For a 5-fs pulsed beam illumination, spatial resolution in the axial and transverse directions in this system is improved by approximately 45% and 15%, respectively, compared with that in the case of continuous wave illumination. However, strong chromatic aberration caused by an ultrashort pulsed beam can result in a degradation of spatial and temporal resolution, whereas weak chromatic aberration may lead to an improvement in transverse resolution.

Journal Article↗

PubMed2004

Signal modeling for low-coherence height-scanning interference microscopy.

We propose a computationally efficient theoretical model for low-coherence interferometric profilers that measure surface heights by scanning the optical path difference of the interferometer. The model incorporates both geometric and spectral effects by means of an incoherent superposition of ray bundles through the interferometer spanning a range of wavelengths, incident angles, and pupil plane coordinates. This superposition sum is efficiently performed in the frequency domain, followed by a Fourier transform to generate the desired simulated interference signal. Example applications include white-light interferometry, high-numerical-aperture microscopy with a near-monochromatic light source, and interference microscopy for thickness and topography analysis of thin-film structures and other complex surface features.

Journal Article↗

PubMed1959

Protein concentration in normal mouse lymphocytes, thymocytes, and mouse and human leukemic cells as measured by interference microscopy.

The apparent protein content of a single spherical lymphocyte or a similar cell, as well as its diameter, can be measured by a modified and stabilized AO-Baker interference microscope which is either fitted with an AO half-shade eyepiece or connected to a photomultiplier. This paper gives results for the apparent protein content and the diameter of normal mouse lymphocytes and thymocytes; these do not differ significantly from each other. It also gives values for the apparent protein concentration and the diameter of the lymphocytes of mice of the same strain which have developed leukemia or lymphoma; these values are significantly different from those of normal mice. Related data are given for the apparent protein content and for the diameter of normal human lymphocytes and the white cells found in myeloblastic leukemia and in stem cell leukemia in man; here the values differ significantly from those of the normal human lymphocyte. The rule seems to be that the abnormal white cells become more watery and larger as the disease advances.

Animals↗