A blind spot in confocal reflection microscopy: the dependence of fiber brightness on fiber orientation in imaging biopolymer networks.
We investigate the dependence of fiber brightness on three-dimensional fiber orientation when imaging biopolymer networks with confocal reflection microscopy (CRM) and confocal fluorescence microscopy (CFM). We compare image data of fluorescently labeled type I collagen networks concurrently acquired using each imaging modality. For CRM, fiber brightness decreases for more vertically oriented fibers, leaving fibers above approximately 50 degrees from the imaging plane entirely undetected. As a result, the three-dimensional network structure appears aligned with the imaging plane. In contrast, CFM data exhibit little variation of fiber brightness with fiber angle, thus revealing an isotropic collagen network. Consequently, we find that CFM detects almost twice as many fibers as are visible with CRM, thereby yielding more complete structural information for three-dimensional fiber networks. We offer a simple explanation that predicts the detected fiber brightness as a function of fiber orientation in CRM.
Jawerth
Louise M
LM
Department of Physics, Harvard University, Cambridge, Massachusetts, USA. ljawerth@fas.harvard.edu
Münster
Stefan
S
Vader
David A
DA
Fabry
Ben
B
Weitz
David A
DA
D016880:Anisotropy; D001704:Biopolymers / Q000737:chemistry; D024042:Collagen Type I / Q000737:chemistry; D005453:Fluorescence; D005782:Gels; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D012399:Rotation; D014741:Video Recording
D001704:Biopolymers; D024042:Collagen Type I; D005782:Gels