Image Restoration for Confocal Microscopy: Improving the Limits of Deconvolution, with Application to the Visualization of the Mammalian Hearing Organ

• Published in: Biophysical journal Vol. 80; no. 5; pp. 2455 - 2470
• Main Authors: Boutet de Monvel, Jacques, Le Calvez, Sophie, Ulfendahl, Mats
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2001; Biophysical Society
• Subjects: Algorithms; Biology; Biophysical Phenomena; Biophysics; Computer Simulation; Ear - physiology; Ears & hearing; Entropy; Fluorescence; Humans; Image Processing, Computer-Assisted; Medicin och hälsovetenskap; Microscopes; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; Models, Statistical; Temperature
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(01)76214-5

Deconvolution algorithms have proven very effective in conventional (wide-field) fluorescence microscopy. Their application to confocal microscopy is hampered, in biological experiments, by the presence of important levels of noise in the images and by the lack of a precise knowledge of the point spread function (PSF) of the system. We investigate the application of wavelet-based processing tools to deal with these problems, in particular wavelet denoising methods, which turn out to be very effective in application to three-dimensional confocal images. When used in combination with more classical deconvolution algorithms, these methods provide a robust and efficient restoration scheme allowing one to deal with difficult imaging conditions. To make our approach applicable in practical situations, we measured the PSF of a Biorad-MRC1024 confocal microscope under a large set of imaging conditions, including in situ acquisitions. As a specific biological application, we present several examples of restorations of three-dimensional confocal images acquired inside an intact preparation of the hearing organ. We also provide a quantitative assessment of the gain in quality achieved by wavelet-aided restorations over classical deconvolution schemes, based on a set of numerical experiments that we performed with test images.