Background: Primary cilia length is an important measure of cell and tissue function. While accurate length measurements can be calculated from cells in 2D culture, measurements in tissue or 3D culture are inherently difficult due to optical distortions. This study uses a novel combination of image processing techniques to rectify optical distortions and accurately measure cilia length from 3D images.
Methods: Point spread functions and experimental resolutions were calculated from subresolution microspheres embedded in 3D agarose gels for both wide-field fluorescence and confocal laser scanning microscopes. The degree of axial smearing and spherical aberration was calculated from xy:xz diameter ratios of 3D image data sets of 4 μm microspheres that had undergone deconvolution and/or Gaussian blurring. Custom-made 18 and 50 μm fluorescent microfibers were also used as calibration objects to test the suitability of processed image sets for 3D skeletonization. Microfiber length in 2D was first measured to establish an original population mean. Fibers were then embedded in 3D agarose gels to act as ciliary models. 3D image sets of microfibers underwent deconvolution and Gaussian blurring. Length measurements within 1 standard deviation of the original 2D population mean were deemed accurate. Finally, the combined method of deconvolution, Gaussian blurring and skeletonization was compared to previously published methods using images of immunofluorescently labeled renal and chondrocyte primary cilia.
Results: Deconvolution significantly improved contrast and resolution but did not restore the xy:xz diameter ratio (0.80). Only the additional step of Gaussian blurring equalized xy and xz resolutions and yielded a diameter ratio of 1.02. Following image processing, skeletonization successfully estimated microfiber boundaries and allowed reliable and repeatable measurement of fiber lengths in 3D. We also found that the previously published method of calculating length from 2D maximum projection images significantly underestimated ciliary length.
Conclusions: This study used commercial and public domain image processing software to rectify a long-standing problem of 3D microscopy. We have shown that a combination of deconvolution and Gaussian blurring rectifies optical distortions inherent in 3D images and allows accurate skeletonization and length measurement of microfibers and primary cilia that are bent or curved in 3D space.