Design and use of substrata to measure traction forces exerted by cultured cells.
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Semen from 200 Holstein bulls in an artificial insemination center was examined for the frequency of craters on the surface of sperm heads, as visualized with the aid of differential interference contrast microscopy. Semen from 100 of these bulls was examined in more detail in 2 experiments by staining with eosin-aniline blue to determine the relationship of unstained spermatozoa, and spermatozoa with normal acrosomes with apical ridges to the incidence of craters and fertility. Only 3 of 100 bulls had a substantial incidence of craters (15 to 23%), whereas the average of the other 97 bulls in 2 experiments was 1 to 3%. The percentage of sperm cells with craters was correlated (P < 0.05) with the percentage of unstained spermatozoa (r = -0.29 and sperm cells with normal acrosomes (r = -0.52) but was not significantly correlated (r = -0.24) with the nonreturn rate. One bull with many sperm cells with craters was slaughtered, and the epididymal spermatozoa were examined. The high incidence of sperm cells with craters was limited to one side, with the testis on that side having 2 Sertoli cell tumors. The remaining 2 bulls as well as one other that produced 16% of sperm cells with craters did so only temporarily. Within a few months crater sperm production had decreased and semen quality increased. The condition usually appears to be transitory, presumably due to temporary stress.
A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.
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To assess the value of microscopic analysis of urinary erythrocyte morphology as the initial step in the investigation of patients with isolated symptomless microhaematuria, 316 consecutive patients were grouped according to whether they excreted eumorphic or mixed forms of erythrocytes or only dysmorphic forms. The former group was investigated fully, and urological disease was found in 85% of 123 patients. The 192 patients with exclusively dysmorphic erythrocytes in their urine and normal renal function (benign renal microhaematuria) were assigned to annual follow-up examinations of urinary red cell morphology and renal function, and subjected to invasive diagnostic procedures when a change was noted. In only 2 of the 132 patients followed up for at least 2 years did a new disease develop; this was easily identified at one of the annual examinations. Microscopic analysis of urinary erythrocyte morphology is therefore an effective method for identifying patients with symptomless microhaematuria needing specific diagnostic investigation.
We have used fibrinogen gold conjugates and fluorescein-labelled fibrinogen to examine the presence of fibrinogen receptors on the ventral membrane of spreading platelets and to visualize the redistribution of the receptors during platelet spreading. The movement of fibrinogen gold conjugates was observed in real time using video-enhanced interference reflection microscopy (VEIRM) and the redistribution of fluorescein-labelled fibrinogen was examined using video-intensified epifluorescence microscopy (VIFM). Fibrinogen was picked up by spreading platelets and moved towards the centre of the platelets. The velocity of the movement of fibrinogen-gold conjugates under the ventral membrane ranged from less than 0.1 microm min(-1) to more than 10 microm min(-1). The average velocity in the peripheral web region was more than 6 microm min(-1), and was reduced to around 1 microm min(-1) in the outer and inner filamentous zones. The centralized fibrinogen molecules were internalized and eventually removed from the platelets. Video microscopy in conjunction with colloidal gold particles provides a simple method for real-time observation of the movement of fibrinogen receptors on the ventral membrane of spreading platelets.
OBJECTIVE: To study the corneal microstructure by optical coherence tomography (OCT) after laser in situ keratomileusis (LASIK) for high myopia with and without astigmatism. DESIGN: Nonrandomized self-controlled comparative trial. PARTICIPANTS: Sixty-three consecutive LASIK eyes with spherical equivalent refraction between -6.0 and -17.0 diopters (D) and astigmatism between 0.0 and -5.0 D were prospectively recruited for examination. INTERVENTION: LASIK was performed with the Chiron Hansatome microkeratome (160-microm fixed plate) and Summit Apex Plus excimer laser using a 5.5/6.0/6.5-mm multizone pattern. Proper preoperative calculations were performed to ensure stromal beds thicker than 250 microm. MAIN OUTCOME MEASURES: OCT imaging and measurement of corneal thickness was performed preoperatively. In addition, corneal cap and stromal bed thickness measurements were performed 1 day, 1 month, and 3 months postoperatively. RESULTS: The average central corneal pachymetry was 538.9 +/- 26.2 microm preoperatively. Mean corneal cap thickness measured 124.8 +/- 18.5 microm 1-day postoperatively. Mean stromal bed thickness was 295.2 +/- 37.1 microm on the first postoperative day. Compared with the 1-day postoperative examination, the average stromal bed thickness increased significantly by 5.9 microm (P = 0.001) and 7.2 microm (P = 0.001) at the 1-month and 3-month postoperative examinations, respectively. Mean difference between actual (118.7 +/- 27.8 microm) and predicted (104.1 +/- 20.8 microm) central ablation depths was 14.6 +/- 16.7 microm (P = 0.0001). A weak but statistically significant positive association was found between preoperative refraction and the difference between expected and real ablation depth values (R = 0.26; P = 0.042). Posterior stromal beds were more than 250-microm thick in 58 eyes (89.9%) 1 day postoperatively. This safety requirement improved at the 1-month postoperative examination, when the partial regression accounted for slightly thicker stromal beds and only two cases (3.2%) exhibited posterior stromal tissue thinner than 250 microm. These two cases were seen only for corrections exceeding 12 D (P = 0.04). CONCLUSIONS: OCT appears to be a useful tool for the evaluation of both the qualitative and quantitative anatomic outcome of LASIK. Corrections of higher degrees of ametropia run a higher risk of producing a thinner than expected central cornea. Particularly, corrections greater than 12 D may lead eventually to stromal beds thinner than 250 microm, despite proper preoperative calculations. Because corneal flaps are usually thinner than expected with the microkeratome used herein, adequate posterior corneal stroma is preserved in most instances.
Mapping of the activity of brain by optical intrinsic signal imaging (OISI) provides a two-dimensional activation pattern of visual cortical areas at a resolution of a few hundred microns. However, integration of the intrinsic signal over depth results in loss of finer information about functional organization across the depth. Here, we report the first successful implementation of optical coherence tomography (OCT) at around 30 microm depth resolution to investigate cortical functions of a cat brain in vivo. This technique, named functional OCT (fOCT) provided visually evoked changes in the OCT signal. The fOCT signal shows stimulus specificity that correlates well with that of the intrinsic signals and provides depth resolved layer specific functional information.
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OBJECTIVE: Tympanic membrane perforations may cause hearing impairment and otorhea. It is a common indication for ear surgery. The aim of the study was to test whether stem cells may enhance the healing of fresh tympanic membrane perforations. METHODS: In a first assay, the status of the tympanic membrane at 5 days after myringotomy was tested in five Mongolian gerbils that were treated on one side with embryonic stem cells and on the other side with control substance. In a second assay, nine gerbils were treated in the same way, except that fluorescent-labeled embryonic stem cells were used. The integration of the stem cells into the surface layer of the healing tympanic membrane was assessed with fluorescence microscopy, as well as the differentiation of these cells. RESULTS: In the first assay, all perforations in the treated ears were closed, whereas only two of the untreated ears were closed. The strength of the healed perforation was greater in the stem cell treated tympanic membranes (mean rupture pressure 120 daPa in three treated ears compared to 60 daPa in the one control ear). Two stem cell-treated tympanic membranes remained intact throughout the whole sequence of pressures, whereas only one control tympanic membrane remained intact. In three tympanic membranes in the second assay, a group of fluorescence-doped cells was detected in the region of the perforation. CONCLUSION: These findings indicate that stem cells enhance the healing of tympanic membrane perforations, possibly by differentiation and integration into the tympanic membrane tissue.
Splenic hypofunction has been described in a number of diseases in which autoimmune mechanisms are believed to play a part. It has been reported in association with chronic active hepatitis although this relationship has not been systematically investigated. Twenty patients with chronic active hepatitis and 22 patients with primary biliary cirrhosis, together with an equal number of age-matched controls for each group, were studied. Splenic function was assessed by counting pits on erythrocytes viewed under differential interference contrast microscopy. There was no difference between splenic function in either chronic active hepatitis, or primary biliary cirrhosis, and normal controls. In both groups of patients splenic hypofunction was significantly related to age.
Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.
The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.
Specimens of Virginia opossums (Didelphis virginiana) in Michigan were examined over 1 year to document the presence of Besnoitia darlingi cysts. Cyst morphology, prevalence, seasonal variation, and tissue sites of isolation were studied. Histology and ultrastructural features of the detected cysts and bradyzoites were consistent with B. darlingi. In the opossums, B. darlingi had intracellular tissue cysts. Tissue cysts had a mean diameter of 560 microm and were separated from the host tissue by a thick (5-20 microm) cyst wall. Overall prevalence of B. darlingi cysts in opossums was 10.9% (15/137). Variations in the prevalence were detected during spring (3/17; 17.6%), summer (10/34; 29.4%), and fall (2/60; 3.3%). No cysts were detected in the specimens examined during winter (0/26; 0%). Numerous B. darlingi cysts were detected in ears, conjunctiva, tongue, abdominal muscles, diaphragm, stomach, heart, liver, kidney, lung, and spleen. Cysts were detected mainly in adult female opossums that were debilitated. Ear was the most frequent organ from which the cysts were reported (10/15; 66.7%) when compared individually with other body tissues (P<0.05).