Measurement of wheat germ agglutinin binding with a fluorescence microscope.

Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield-corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA-Alexa 488 and WGA-TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell-bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible.

Hoof accelerations and ground reaction forces of Thoroughbred racehorses measured on dirt, synthetic, and turf track surfaces.

OBJECTIVE: To compare hoof acceleration and ground reaction force (GRF) data among dirt, synthetic, and turf surfaces in Thoroughbred racehorses. ANIMALS: 3 healthy Thoroughbred racehorses. PROCEDURES: Forelimb hoof accelerations and GRFs were measured with an accelerometer and a dynamometric horseshoe during trot and canter on dirt, synthetic, and turf track surfaces at a racecourse. Maxima, minima, temporal components, and a measure of vibration were extracted from the data. Acceleration and GRF variables were compared statistically among surfaces. RESULTS: The synthetic surface often had the lowest peak accelerations, mean vibration, and peak GRFs. Peak acceleration during hoof landing was significantly smaller for the synthetic surface (mean + or - SE, 28.5g + or - 2.9g) than for the turf surface (42.9g + or - 3.8g). Hoof vibrations during hoof landing for the synthetic surface were < 70% of those for the dirt and turf surfaces. Peak GRF for the synthetic surface (11.5 + or - 0.4 N/kg) was 83% and 71% of those for the dirt (13.8 + or - 0.3 N/kg) and turf surfaces (16.1 + or - 0.7 N/kg), respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The relatively low hoof accelerations, vibrations, and peak GRFs associated with the synthetic surface evaluated in the present study indicated that synthetic surfaces have potential for injury reduction in Thoroughbred racehorses. However, because of the unique material properties and different nature of individual dirt, synthetic, and turf racetrack surfaces, extending the results of this study to encompass all track surfaces should be done with caution.