Substrates For Electron Microscopy Research Articles

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell–cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

Linear immunofluorescence staining of the cutaneous basement membrane zone produced by pemphigoid antibodies: The result of hemidesmosome staining

Bullous pemphigoid autoantibodies bind the basement membrane zone of stratified squamous epithelium in a linear pattern, as shown by indirect immunofluorescence; however, other patterns of staining, such as tubular (with convolutions), cytoplasmic, and membranous, have been noted. Recently, by using indirect immunoelectron microscopy, we have shown that bullous pemphigoid antibodies bind specifically to hemidesmosomes. The purpose of this investigation was to correlate the various patterns of basement membrane zone staining by bullous pemphigoid antibodies by immunofluorescence and to correlate these patterns with the ultrastructural findings. We employed adult human skin, neonatal human foreskin, neonatal mouse skin, and rabbit cornea as substrates for electron microscopy and indirect immunofluorescence using bullous pemphigoid serum. For indirect immunofluorescence, cryosections were obtained at vertical, oblique, and horizontal planes with respect to the basement membrane zone. We show that the immunofluorescence band of basement membrane zone staining results from the coalescence of fluorescence from individual hemidesmosomes. We also show that the pattern of basement membrane zone staining depends on the ultrastructural morphology of the basement membrane zone in each tissue and on the angle of sectioning.