An in-situ fluorescence-based optical extensometry system for imaging mechanically loaded bone

Abstract

The application and quantification of well-controlled tissue strains is required for investigations into mechanisms of tissue adaptation within the musculoskeletal system. Although many commercial and custom extensometry systems exist for large biological samples, integrated loading/strain measurement for small samples is not as readily available. Advanced imaging modules such as laser scanning microscopy provide in situ, minimally invasive tools to probe cellular and molecular processes with high spatiotemporal resolution. Currently, a need exists to devise loading/strain measurement systems that can be integrated with such advanced imaging modules. We describe the development and validation of a fluorescence-based, optical extensometry system directly integrated within a confocal microscopy platform. This system allows in situ measurement of surface strain and is compatible with the direct imaging of cellular processes within small bone samples. This optical extensometry system can accurately and reproducibly measure physiologically relevant surface strains (200 to 3000 microstrain) in beams machined from various well-characterized materials, including bovine femoral cortex, and in intact murine tibia. This simple system provides a powerful tool to further our investigation of the relationships between mechanical loading, fluid and solute transport, and mechanosensation within the musculoskeletal system.

Figure 1

A) Schematic of the optical extensometry system, consisting of an electromagnetic loading system (Electroforce LM1 TestBench) and a confocal microscope (Zeiss LSM510) with standard epifluorescent imaging capabilities. B) Schematic of pattern tracking and strain determination. Fluorescent micro-spheres were used as markers to track strain on the surface of specimens under UV illumination. Unique micro-sphere patterns (shown in grey boxes) were manually selected at the right and left margins of an image and an automated algorithm tracked inter-pattern distance (L_o), displacement (L_i-L_o), and strain (ε) for both baseline and loaded conditions. Displacement and strain are exaggerated for the purpose of illustration.

Figure 2

Four calibration tests to confirm system accuracy. A) Apparent strain between fixed micro-sphere patterns due to in-plane edge distortion within the field of view (FOV). B) Apparent strain due to out-of-plane focus shifting. C) Comparison of the optical and the actuator’s positional sensor measures for large (20µm) incremental translations of labeled beams toward each other. D) Comparison of the two methods for small (2-µm) incremental translations. An initial gauge length of 3.0-mm was used in C & D. All regressions reported in mm units.

Figure 3

Schematics of strain measurements location in A) machined beams and B) murine tibiae under uniaxial compression.

Figure 4

Minimal errors were found for the optically measured displacement and strain. A) In-plane edge distortion; B) Out-of-plane focus shifting; C) Large displacement tests showed the mean and maximal errors are −0.02 and 0.61-µm, respectively; D) Small displacement tests showed the mean and maximal errors are −0.03 and 0.38-µm, respectively. The initial gauge separation was 3.0-mm for panels C & D. LVDT value of zero corresponds to the center of the camera FOV. Linear regression and 95% confidence intervals are shown.

Figure 5

Stress vs. strain curves for beams machined from UHMWPE, cast acrylic, and bovine femoral cortex (n = 8–10 per material) under uniaxial compression.

Figure 4

Strain vs. load curve for adult male B6 mouse tibia under uniaxial compression.