Wavelength selection in measuring red blood cell aggregation based on light transmittance

The reversible aggregation of red blood cells (RBC) is of current basic science and clinical interest. Using a flow channel and light transmittance (LT) through RBC suspensions, we have examined the effects of wavelength (500 to 900 nm) on the static and dynamic aspects of RBC aggregation for normal blood and suspensions with reduced or enhanced aggregation; the effects of oxygenation were also explored. Salient observations include: 1. significant effects of wavelength on aggregation parameters reflecting the extent of aggregation (i.e., number of RBC per aggregate); 2. no significant effects of wavelength on parameters reflecting the time course of RBC aggregation; 3. a prominent influence of hemoglobin oxygen saturation on both extent and time-course related aggregation parameters measured at wavelengths less than 700 nm, but only on the time-course at 800 nm; and 4. the power of parameters in detecting a given alteration of RBC aggregation is affected by wavelength, in general being greater at higher wavelengths. It is recommended that light sources with wavelengths around 800 nm be used in instruments for measuring RBC aggregation via LT.     

Optical attenuation characteristics of breast tissues at visible and near-infrared wavelengths.

Optical experiments are described for measuring the attenuation characteristics of breast tissues at visible and near-infrared wavelengths. Total attenuation coefficients post mortem were measured directly in thin tissue sections. They are usually within the range from 10 to 30 mm-1, are rather higher in fat than in fibroglandular specimens and decrease with increasing wavelength. The scattering phase function is strongly forward-peaked with the mean cosine of scattering in the range from 0.85 to 0.97 and appearing more forward-peaked in fat than in fibroglandular tissue. The reduced scattering coefficient is of the order of 1 mm-1 in all tissues. Absorption coefficients were measured indirectly in optically thick sections. They are typically between 0.1 and 0.5 mm-1 at wavelengths around 580 nm and an order of magnitude lower at 850 nm. At 580 nm and shorter wavelengths the absorption in carcinoma is significantly higher than in adjacent uninvolved tissue. Significant differences were observed in the first-order derivatives of the transmission spectra of carcinoma and surrounding tissues at certain infrared wavelengths. Transmission spectra measured in vivo across the wavelength range from 500 to 860 nm have a similar form to the spectra of excised samples. Linear absorption coefficients are generally of the same order of magnitude as those found in vitro although they are lower at green wavelengths.     

Expression of alpha-smooth muscle actin as special and morphometric assessment in the small intestine during the postnatal development in hamster

Immunoreactivity of alpha-smooth muscle actin (ASMA), along, with morphometric values in the hamster small intestine, was examined during postnatal development. Twelve healthy hamsters were divided into three age groups of 2·5 months (group 1), 3 months (group 2), and 4 months (group 3). For morphometrical study, routine tissue processing was carried out by autotechnicon tissue processor, the processed tissues were embedded and sectioned serially into 5-μm thick sections. The sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed by primary antibodies against alpha-smooth muscle actin (ASMA). All samples showed ASMA expression in the muscularis mucosa and circular and longitudinal layers of muscularis propria. The small intestine samples in group 1 showed minimal ASMA expression in total muscular layers. Alpha-smooth muscle actin labeling was increased in groups 2 and 3 in the muscularis mucosa and both layers of inner and outer circular muscle as compared to group 1. A more intense ASMA expression was observed in the longitudinal layers in both groups 2 and 3. The morphometrical results showed that there was a smaller increase in thickness of the mucosa, submucosa, and muscular layers from group 1 to group 2 that was followed by a larger increase between groups 2 and 3. There were significant increases in thickness of all morphometrical parameters (mucosa, submucosa, and muscularis) in groups 2 and 3 in comparison with group 1, and also group 3 compared with group 2. In groups 2 and 3, thickness of the submucosa and muscular layers were significantly higher in the jejunum compared with the ileum.     

Analysis of tissue optical coefficients using an approximate equation valid for comparable absorption and scattering.

New photosensitizers activated by longer wavelengths than 630 nm light used with Photofrin II are under evaluation by various groups for the treatment of malignancies. Any increase in tumour volume destroyed by these agents as compared to Photofrin II will be partly determined by tissue penetrance at the longer wavelengths. Attenuation coefficients were measured for various tissues at 630 nm and the more penetrative near infrared wavelength of 789 nm. A new model of light propagation in tissue is shown to be accurate for arbitrary ratios of absorption and scattering, by comparison with a rigorous solution to the transport equation. Absorption and transport scattering coefficients of tissues at 630 and 789 nm were obtained by fitting this model to optical attenuation measurements. In vitro tissues included bovine heart, kidney and tongue, pig liver and fat, and chicken muscle; in vivo tissues included Dunning R3327-AT and R3327-H tumours. The penetration depth was found to be 1.35-2.25 times greater at 789 than 630 nm, depending on tissue type. The greatest differences in penetration between the two wavelengths were in the highly pigmented tissues. These substantial increases in penetration in the infrared may be important in future applications of photodynamic therapy.     

Approximation of Mie scattering parameters in near-infrared tomography of normal breast tissue in vivo

A method for estimating Mie theory scattering parameters from diffuse light tomography measurements in breast tissue is discussed. The approach provides an estimate of the mean particle size and number density given assumptions about the index of refraction change expected in lipid-membrane-bound scatterers. When using a sparse number of wavelengths in the reduced scattering spectra, the parameter extraction technique is limited to representing a continuous distribution of scatterer sizes that appears to be dominated by an exponential decrease with increasing particle size. The fitting method is tested on simulated data and then on Intralipid-based tissue-phantom data, giving a mean particle size of 93+/-17 nm, which is in excellent agreement with expectations. The approach is also applied retrospectively to breast tissue spectra acquired from normal healthy volunteers, where the average particle size and number density were found to be in the range of 20 to 1400 nm. Grouping of the data based on radiographic breast density, as a surrogate measure of tissue composition yielded values of 20 to 65, 25 to 200, 140 to 1200, and 150 to 1400 nm, respectively, for the four BI-RADS (American College of Radiology Breast Imaging Reporting and Data System) density classifications of extremely dense, heterogeneously dense, scattered, and fatty. These results are consistent with the microscopic characteristics of each breast type given the expected progression from predominantly collagenous connective tissue (extremely dense category) to increasing proportions of glandular epithelium and fat (intermediate density categories) to predominantly fat (fatty category).     

Spectral sensitivity of wild-type and mutant Drosophila melanogaster larvae

Wild-type (Canton-S) Drosophila melanogaster larvae are generally repelled by white light. Mutant larval photokinesis A (lphA) larvae are less strongly repelled than controls. Mutant Larval photokinesis B (LphB) larvae are unresponsive to light, as are larvae from LI2, an isofemale line whose progenitors were recently derived from a natural population. To characterize the behavior of larvae from the mutant stocks and the isofemale line more precisely, we determined the range of wavelengths that repel wild-type (Canton-S) D. melanogaster larvae and identified wavelengths to which larvae are most sensitive. In comparison to adult flies, Canton-S larvae are much less sensitive to white light and respond to a narrower range of wavelengths. The wavelengths to which Canton-S larvae are maximally sensitive are 500 nm (green), 420 nm (indigo), and 380 nm (ultraviolet). Mutant lphA larvae respond abnormally to green and indigo light but are as strongly repelled by ultraviolet light as controls. In contrast, mutant LphB larvae and larvae from the LI2 isofemale line are unresponsive to green, indigo, or ultraviolet light. Thus, lphA larvae have a wavelength-specific defect, while LphB and LI2 larvae are generally unresponsive to wavelengths that repel wild-type larvae.     

Poly(cyclotriphosphazene‐co‐tris(4‐hydroxyphenyl)ethane) Microspheres with Intrinsic Excitation Wavelength Tunable Multicolor Photoluminescence

Functional photoluminescent materials with a tunable multicolor emission without incorporation of metals is the current focus on luminescent materials due to their multifaceted applications. Herein, novel poly(cyclotriphosphazene‐co‐tris(4‐hydroxyphenyl)ethane) microspheres are synthesized with size distribution of ≈0.8–1.6 µm by reacting hexachlorocyclotriphosphazene and tris(4‐hydroxyphenyl)ethane at different molar ratios under facile conditions. The thermogravimetric analysis shows that the microspheres are highly thermally stable with 10% weight loss at around 500 °C. Fluorescence microscopy results show that the microspheres emit three blue, green, and red colors with an emission maximum at 487, 530, and 623 nm under different excitation wavelengths of 365, 420, and 546 nm, respectively. Furthermore, under UV and visible (365 and 420 nm) excitations, microspheres demonstrate extraordinary photobleaching stability and highly intense luminescence for expending time and repeating excitations. The microspheres also demonstrate semiconductor‐like optical absorption at 235–285 nm with a tail extended to 700 nm. This exploration provides an insight for the preparation of intrinsic superior wavelength tunable multicolor emission materials from phosphazene. These microspheres are potential candidates for anti‐counterfeits, solar cells, full spectrum fluorescence emitters, organic light emitting diodes, biological, catalysis applications and unlock new areas of research on multicolor photoluminescence.     

Near-infrared autofluorescence imaging for detection of cancer

Near-infrared autofluorescence imaging of tissues under long-wavelength laser excitation in the green and red spectral region complemented by cross-polarized elastic light scattering was explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation. A set of images for each tissue sample was recorded that consisted of two autofluorescence images obtained under 532- and 632.8-nm excitation and light-scattering images obtained under linearly polarized illumination at 700, 850, and 1000 nm. These images were compared with the histopathology of the tissue sample. The experimental results indicated that for various tissue types, the intensity of the autofluorescence integrated over the 700 to 1000-nm spectral region was considerably different in cancer tissues than in that of the contiguous non-neoplastic tissues. This difference provided the basis for the detection of cancer and delineation of the tumor margins. Variations on the relative intensity were observed among different tissue types and excitation wavelengths.     

Optical properties of normal and diseased human breast tissues in the visible and near infrared

The optical absorption and scattering coefficients have been determined for specimens of normal and diseased human breast tissues over the range of wavelengths from 500 to 1100 nm. Total attenuation coefficients were measured for thin slices of tissue cut on a microtome. The diffuse reflectance and transmittance were measured for 1.0 mm thick samples of these tissues, using standard integrating sphere techniques. Monte Carlo simulations were performed to derive the scattering and absorption coefficients, as well as the mean cosine of the scattering angle. The results indicate that scatter exceeds absorption by at least two orders of magnitude. Absorption is most significant at wavelengths below 600 nm. The scattering coefficients lie in the range 30-90 mm-1 at 500 nm, and fall smoothly with increasing wavelength to between 10 and 50 mm-1 at 1100 nm. The scattering coefficient for adipose tissue differs, in that it is invariant with wavelength over this spectral range. For all tissues examined, the scattered light is highly forward peaked, with the mean cosine of the scattering angle in the range 0.945-0.985. Systematic differences between the optical properties of some tissue types are demonstrated.     

Visualization of human heart conduction system by means of fluorescence spectroscopy

The conduction system of the heart is a specific muscular tissue, where a heartbeat signal originates and initiates the depolarization of the ventricles. The muscular origin makes it complicated to distinguish the conduction system from the surrounding tissues. A surgical intervention can lead to the accidental harm of the conduction system, which may eventually result in a dangerous obstruction of the heart functionality. Therefore, there is an immense necessity for developing a helpful method to visualize the conduction system during the operation time. The specimens for the spectroscopic studies were taken from nine diverse human hearts. The localization of distinct types of the tissue was preliminary marked by the pathologist and approved histologically after the spectral measurements. Variations in intensity, as well as in shape, were detected in autofluorescence spectra of different heart tissues. The most distinct differences were observed between the heart conduction system and the surrounding tissues under 330 and 380 nm excitation. The spectral region around 460 nm appeared to be the most suitable for an unambiguous differentiation of the human conduction system avoiding the absorption peak of blood. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, was also demonstrated.     

Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography

The feasibility of ultrahigh resolution optical coherence tomography (UHR OCT) to image ex vivo and in vitro brain tissue morphology on a scale from single neuron cells to a whole animal brain was investigated using a number of animal models. Sub-2-microm axial resolution OCT in biological tissue was achieved at different central wavelengths by separately interfacing two state-of-the-art broad bandwidth light sources (titanium:sapphire, Ti:Al2O3 laser, lambdac=800 nm, Deltalambda=260 nm, Pout=50 mW and a fiber laser light source, lambdac=1350 nm, Deltalambda=470 nm, Pout=4 mW) to free-space or fiber-based OCT systems, designed for optimal performance in the appropriate wavelength regions. The ability of sub-2-microm axial resolution OCT to visualize intracellular morphology was demonstrated by imaging living ganglion cells in cultures. The feasibility of UHR OCT to image the globular structure of an entire animal brain as well as to resolve fine morphological features at various depths in it was tested by imaging a fixed honeybee brain. Possible degradation of OCT axial resolution with depth in optically dense brain tissue was examined by depositing microspheres through the blood stream to various depths in the brain of a living rabbit. It was determined that in the 1100 to 1600-nm wavelength range, OCT axial resolution was well preserved, even at depths greater than 500 microm, and permitted distinct visualization of microspheres 15 microm in diameter. In addition, the OCT image penetration depth and the scattering properties of gray and white brain matter were evaluated in tissue samples from the visual cortex of a fixed monkey brain.     

Histologic and anatomic correlation of the infraorbital region

The infra-orbital area has not been well studied although it represents an interesting area in plastic surgery because of its implication in malarplasty reduction or transplantation for facial tissue repair. This work comprises an anatomic dissection layer by layer and a histologic study describing the various structures of this area. It was performed on 10 heads taken from cadavers (mean age 70 years old). The anatomic and histologic study found four layers: a subcutaneous fat layer, a muscular layer, a deep fat layer and a deep muscular layer. The superficial fat layer constitutes the convexity of the cheek and can be removed in plastic surgery reduction or reconstituted after atrophy following nasolabial musculo cutaneous flaps.     

Horizontal segregation of color information in the middle layers of foveal striate cortex

The present study examines the chromatic organization of foveal striate cortex in the awake monkey by means of a series of microelectrode penetrations made as perpendicular as possible to the layers. The study includes 79 penetrations and 261 cells, of which 218 were tested systematically for color selectivity. Detailed analyses are conducted on a subset of 41 penetrations, which included 164 color-tested cells, an average of four cells per penetration. The penetrations were divided into two major categories on the basis of orientation selectivity testing. One group of penetrations contained at least one nonoriented cell in the first 600 microns of microelectrode trajectory (upper layers), whereas the other group of penetrations contained only oriented cells in the first 600 microns of microelectrode trajectory. The two groups were called N (nonoriented) and O (oriented), respectively. Analysis of the color properties of cells in N and O penetrations revealed that middle layer cells in N penetrations showed poor responses to white light, and color preferences for endspectral wavelengths, i.e., red or blue. Middle layer cells in O penetrations, by contrast, responded well to white light and to midspectral wavelengths. There were two subgroups of N penetrations, characterized by predominantly red (NR) or blue (NB) sensitivity in the middle layers. O penetrations could likewise be divided up into three subgroups (OG, OY, OW), characterized, respectively, by predominant sensitivity to greenish wavelengths (490-540 nm), yellowish wavelengths (550-600 nm), or white (i.e., all colors). Despite the identification of five subgroups, similarities between NR and OY, between NB and OG, and between OY and OW subgroups might be consistent with a continuum. The middle layers of N penetrations contained a unique class of cells with excitatory responses restricted to the two spectral ends, endspectral double-peak cells. A model is proposed for the color organization of layer 4 in foveal striate cortex, with red and blue zones in register with alternate cytochrome oxidase "blobs" of layers 2 and 3, white zones in register with interblob centers, and yellow and green zones in between.     

Multispectral imaging of tissue absorption and scattering using spatial frequency domain imaging and a computed-tomography imaging spectrometer

We present an approach for rapidly and quantitatively mapping tissue absorption and scattering spectra in a wide-field, noncontact imaging geometry by combining multifrequency spatial frequency domain imaging (SFDI) with a computed-tomography imaging spectrometer (CTIS). SFDI overcomes the need to spatially scan a source, and is based on the projection and analysis of periodic structured illumination patterns. CTIS provides a throughput advantage by simultaneously diffracting multiple spectral images onto a single CCD chip to gather spectra at every pixel of the image, thus providing spatial and spectral information in a single snapshot. The spatial-spectral data set was acquired 30 times faster than with our wavelength-scanning liquid crystal tunable filter camera, even though it is not yet optimized for speed. Here we demonstrate that the combined SFDI-CTIS is capable of rapid, multispectral imaging of tissue absorption and scattering in a noncontact, nonscanning platform. The combined system was validated for 36 wavelengths between 650-1000 nm in tissue simulating phantoms over a range of tissue-like absorption and scattering properties. The average percent error for the range of absorption coefficients (μa) was less than 10% from 650-800 nm, and less than 20% from 800-1000 nm. The average percent error in reduced scattering coefficients (μs') was less than 5% from 650-700 nm and less than 3% from 700-1000 nm. The SFDI-CTIS platform was applied to a mouse model of brain injury in order to demonstrate the utility of this approach in characterizing spatially and spectrally varying tissue optical properties.     

Measurement of oxygen saturation in venous blood by dynamic near infrared spectroscopy

A method for the measurement of oxygen saturation in the venous blood, SvO2, based on optical measurements of light absorption in the infrared region is presented. The method consists of applying relatively low external pressure of 25 mm Hg on the forearm, thereby increasing the venous blood volume in the tissue, and comparing the light absorption before and after the external pressure application. SvO2 has been determined from light absorption measurements in two wavelengths, before and after the pressure application, using a formula derived for two adjacent wavelengths. The method has been applied to the hands and fingers of 17 healthy male subjects, using wavelengths of 767 and 811 nm. SaO2, the oxygen saturation for arterial blood, was also obtained from photoplethysmographic measurements in these two wavelengths (pulse oximetry) using the same formula. The mean (+/- SD) value of SaO2 was 94.5% (+/- 3.0). The mean value of SvO2 was 86.2% (+/- 4.1) for the finger and 80.0% (+/- 8.2) for the hand. These SvO2 values are reasonable for the finger and the hand where arterio-venous anastomoses exist. The method enables the measurement of SvO2 in the limbs, a parameter which is related to tissue blood flow and oxygen consumption.     

Histomorphometric comparison of the human,swine, and ovine collecting systems

The ovine kidney has been recently determined to be a better model than the swine kidney for the study of collecting system healing after partial nephrectomy. However, there is no histological study comparing the collecting systems of these species. To compare human, swine, and ovine collecting systems using histomorphometry. The collecting systems of 10 kidneys from each species (human, swine, and ovine) were processed for histomorphometry. The thickness of the three layers (mucosal connective tissue, submucosal muscular, and adventitial connective tissue) were measured. The densities of smooth muscle fibers, elastic system fibers, and cells were also measured. Additionally, blood vessel density in the adventitial connective tissue was measured. Analysis of the collecting systems from the three species presented several differences. The adventitial connective tissue from the swine samples was thicker, with more blood vessels and smooth muscle fibers, compared with that from the human and ovine samples. Swine also had higher density of elastic fibers on the submucosal muscular layer. Ovine and human collecting systems shared several similar features, such as blood vessel and elastic fiber density in all layers and the density of cellular and muscular fibers in the submucosal muscular and adventitial connective tissue layers. The collecting system of the ovine kidney is more similar to that of the human kidney compared with that of the swine kidney. This may explain the differences between the healing mechanisms in swine and those in humans and sheep after partial nephrectomy. Anat Rec, 299:967–972, 2016. © 2016 Wiley Periodicals, Inc.     

Mucosal nerves and smooth muscle relationships with gastric glands of the opossum: An ultrastructural and three-dimensional reconstruction study

The precise anatomical relation by which autonomic nerve endings contact gastric epithelial cells to enhance the rate of gastric secretions is not fully understood. The aim of the present study was to clarify this issue by using the technique of serial section reconstruction of areas of the gastric mucosa. The work also explored the possibility of a functional role for a system of smooth muscle strands in the gastric mucosa that emanate from the muscularis mucosa, run in the lamina propria, and are associated in a unique manner with the gastric glands. Electron microscopic serial sections of the gastric mucosa were performed to visualize the entire limiting membrane of gastric epithelial cells to determine any nerve associations (especially varicose endings) with these cells. Evaluation of serial sections of five separate parietal cells showed that their basal membrane did not come in close contact (nearest distance 500 nm) with any nerve axon or varicosity. Moreover, the axons passing in the area of these cells ultimately showed varicose endings associated with smooth muscle cells in the adjacent connective tissue (often separated by only 20 nm), with mast cells or with vascular elements. Additionally, the lateral membrane of these five parietal cells did not contact any endocrine cell in the epithelium, although other parietal cells in the area were adjacent to endocrine cells. Chief cells in the immediate area also did not form any close associations with nerve varicosities. Random analysis of 5,000 additional epithelial cells in these sections showed no close associations to nerve elements with significant accumulations of neurosecretory vesicles (varicosities). Because of the observed existence of innervation to the smooth muscle strands in the area of the gastric glands, serial 1-μm epoxy sections of the gastric mucosa were prepared, and profiles of smooth muscle and gastric glands were entered into a computer-assisted reconstruction system. Three-dimensional reconstruction techniques were employed to reveal the existence of a unique association between the mucosal smooth muscle strands and the gastric glands. The muscle strands arose from the muscularis mucosa at regular intervals and became branched to form an intricate wrap around a series of gastric glands that empty into one gastric pit. Branching of the muscle strands initially occurred at the point where they approached the base of the glands and then emanated into the connective tissue around the glands in a crossing pattern, ending at the base of the gastric pit. Although muscarinic agents have been shown to directly stimulate parietal cells to secrete acid, these findings have led us to postulate that autonomic nerve stimulation may also aid gastric secretion both by stimulation of mast cells and by glandular excretion mediated via mucosal muscular contractions.     

Histopathological changes in the skin and subcutaneous tissues of mouse legs after treatment with hyperthermia

The right hind legs of mice wee heated in a waterbath at 44 degrees C. The animals were killed at various time intervals after exposure. Tissue damage was studied histologically. After 15 min exposure light and reversible changes were seen including oedema and some neutrophilic inflammatory infiltration immediately after treatment. After 30 min exposure an extensive inflammatory infiltrate and strong oedema were seen during the first days after treatment. Adjacent to areas in the skin with strong oedema extensive muscular necrosis was observed. The muscular tissue regenerated almost completely in three weeks. After 60 min heating the histological picture was dominated by massive necrosis of muscle, subcutaneous fat tissue and skin during the first week after treatment followed by local ulceration. From about the 7th day after treatment regeneration of the epithelium started and granulation tissue could be observed in the margin of the ulceration. Healing of the skin was completed at about day 21 after treatment. Our results indicate that heat induced tissue damage in some tissues is due to a direct effect on the cells composing the tissue (e.g., fat cells in subcutaneous fat) but that, in most other tissues (e.g., muscle and skin) it is a consequence of damage to the vasculature.     

Photoplethysmography

When the microvascular blood perfusion in human skin is measured by photoplethysmography (PPG), infra-red light (800-960 nm) is normally used as the light source. The PPG signal, which consists of a pulsatile (AC) and a slowly fluctuating (DC) component, was studied at different optical wavelengths utilising optical fibres for guiding the light to and from the skin surface. Finger and forearm skin was examined and high and low skin blood perfusion was brought about by local water-induced temperature provocation. The analysis of the measurement results provided evidence that the use of shorter wavelengths in PPG (AC) for monitoring skin perfusion changes could be applicable. The use of different optical wavelengths also raises the possibility of recording perfusing changes at different depths in the superficial tissue. The sweat water content in stratum corneum of human skin will probably determine the total amount of reflected and backscattered radiation reaching the photodetector. This is important when the skin perfusion is changed by alterations in the environmental temperature conditions activating the sweat glands in tissue. Temperature-dependent optical characteristics of blood-free skin tissue may explain the limited ability of the DC component of PPG to monitor skin perfusion changes.     

Histological analysis of esophageal muscular layers from 27 autopsy cases with mixed connective tissue disease (MCTD)

Esophageal symptoms in mixed connective tissue disease (MCTD) have been investigated radiologically. We investigated the esophageal lesions in MCTD histopathologically, and analyzed relationships between these lesions and autoantibodies extracted from the serum of MCTD patients. Esophageal tissues from 27 MCTD patients submitted to autopsy were examined. We compared histopathological features of the esophagus in different wall layers from the mucosa, submucosa, and muscular layer to the adventitia, and in the upper, middle, and lower portions of esophagus. The most striking change observed was severe atrophy and occasional loss of smooth muscle cells in the muscular layer, followed by fibrosis. These muscular changes were particularly prominent in the inner layer of the lower esophagus. Immunohistochemically, degenerated muscular tissues of the esophagus were positive for anti-IgG and anti-C3 antibodies, but not for anti-IgM antibodies. IgG fractions extracted from three MCTD patients were immunohistochemically used to examine whether some antibodies in MCTD patients showed reactivity for esophageal components. The IgG fractions isolated from MCTD patients reacted with smooth muscle from non-connective tissue disease cases, suggesting that some serum antibodies may trigger esophageal changes. These findings suggest that esophageal lesions associated with clinical dysphagia in MCTD may be related to autoantibodies.