Our lab is located in Biomedical Science Tower #3.
3501 Fifth Avenue, Pittsburgh PA 15213:
Micromechanical Test Devices
We use this device to measure the mechanical properties of isolated ‘bricks’ of embryonic tissue. The nNFMD uses uniaxial unconfined compression to measure the viscoelastic properties of frog embryonic tissues. Briefly, a regularly shaped block of embryonic tissue, prepared by microsurgery, is held under a cantilever and compressed. The cantilever acts as a force transducer, reporting force by amount of deflection of the tip from its base. The tissue resists compression with a force that opposed the one produced by the cantilever. That resistance force, the degree of compression or strain of the tissue, and the cross-sectional area of the tissue along the axis of compression are used to calculate the time-varying viscoelastic properties of the embryonic tissue. The contribution of molecular composition of the tissue can be tested by knock-down or inhibitor studies. The contribution of specific mechanical structures can be tested by removing, scrambling, or reconstructing the structures, or alternatively using molecular genetic approaches to disrupt the formation or sub-elements within the structure. The limitations of this approach is that it requires microsurgical removal and shaping of tissues into regularly shaped blocks. The smallest structure tested by the nNFMD is about 200um x 300um x 600 um and contains approximately 5000 embryonic frog cells and their extracellular matrix.
We use this device to measure the viscoelastic properties of intact embryos and spherical aggregates. This device uses pressure to micro-aspirate a small patch of tissue on the surface of the embryo or aggregate into a wide cylindrical channel (~120 um in diameter) to measure the tissue’s time-varying viscoelastic properties. Any patch of tissue can be micro-aspirated but informative results are generally only found when the tissue is thicker than the diameter of the channel. Very small pressure differences across the face of the channel are generated by lowering the water level in the adjoining reservoir by a small amount (~0.5 to 1 mm). As pressure is lowered a small patch of the embryo is pulled into the cylindrical channel. The amount of tissue that moves into the channel and the pressure difference are used to calculate the viscoelastic properties of the embryonic tissue. The advantages of this approach is that embryos do not need to be ‘taken-apart’ to measure mechanical properties. This non-destructive approach allows the tested embryo to continue developing and allows further testing or assessment. The smallest structure tested by our device is about 120 um diameter channel x 120 um deep and contains between 5 and 40 embryonic frog cells and associated extracellular matrix.
A new device for micrometer scale indentation of moderately stiff biomaterials (range 10 to 100 KPa). Samples are mounted on a moveable XZ-stage and brought into contact with a cantilever force sensor. Position of the sample, the cantilever tip, and deflection allow estimation of the shear modulus of the sample. Two cameras simultaneously record deflection of the cantilever and site of indentation for approximate ‘mapping’ of the tissue surfaces. This device is analogous to an atomic force microscope but can be used to gather modulus data from 10 to 100 µm depths greatly exceeding the shallow 200 nm to 1 um depths interrogated by AFM.
Our most important imaging tools. These stations enable microsurgical manipulations where we can move and isolate cells and tissues from one embryo to another or from an embryo into a novel microenvironment. These dissection stereomicroscopes are used for microsurgery, microinjection, or for constructing small devices. Each of these stations is mounted on a temperature controlled “cold-plate” that can be used to slow development, thus enabling complex microsurgical manipulations.
Laser Scanning Confocal (Leica SP5)
One of our two principle imaging microscopes is used for both live and prepared samples. The motorized stage has been modified to support stable imaging of F-actin dynamics and high speed XZ time-lapse sequences. Supports light-weight microaspiration w/ electrical stimulation. The manual stage can support larger or heavier devices from custom-fabricated tissue stretchers to multi-well Flexcell chambers.
Spinning Disk Confocal Microscope (Yokagawa CSU-X1)
This high speed, wide field tiling confocal imaging microscope is used primarily with live samples. Equipped with an environmental chamber, photoconversion, photoablation, and motorized stage this rig can be used to support long term imaging of whole tissues and embryos, high resolution imaging of cytoskeletal or cell polarity factors, and optogenetics.
Multiposition Time-lapse Stereoscope Rig
Many questions concerning morphogenesis, especially concerning the variability and rates of processes can be answered with lower magnification imaging approaches that do not require fluorescence imaging. We have numerous single stereoscopes equipped with cameras but when large sample sizes are required we use computer controlled XY-stage at the focus of a stereoscope mounted with a CMOS camera. The stage and camera are controlled by NIH ImageJ and the microManager plugin. This rig allows simultaneous acquisition of multiple time-lapse sequences.
Epifluorescence Stereoscope Rig (far left)
A fluorescence light source and filter blocks allow imaging of fluorescently labeled samples or screening of frog embryos expressing a fluorescent protein. A color camera allows recording images from these samples.
Time-lapse Stereoscope Rig (right)
A dissection sterescope with a grey-scale CMOS camera mounted on the video-port allows time-lapse collection of living tissues or documentation of tissues and embryos or demonstration of microsurgical manipulations. In this photo the CMOS has been replaced by a pro-sumer Canon T3i camera to record demonstrations of microsurgical maneuvers.