Optical microscopy allows us to study living fluorescent biological samples. Optical sectioning is a technique to obtain three-dimensional (3D) information about the observed object by acquiring a stack of two-dimensional (2D) images at different depths through the sample. However, the specific shape of the 3D optical transfer function of the optical microscope leads to images presenting defects, such as, for example, an apparent elongation along the vertical axis. It is therefore necessary to preprocess the images before any quantitative measurement is performed. This image restoration can be obtained by deconvolution of the acquired 3D image. We have tested several deconvolution algorithms on synthetic images, obtained by convolution of a solid sphere with a measured point spread function. We have compared the restored image with the original one (shape and volume). The linear least-squares method is fast, but artefacts are still present in the restored images. The Carrington method is well adapted to thin objects. The maximum likelihood-expectation maximization method leads to a good reconstruction of the object, but is very time consuming.Résumé. La microscopie optique permet l'étude de spécimens biologiques vivants et fluorescents. La technique par coupes sériées donne des informations tridimensionnelles (3D) sur l'objetétudié par l'acquisition d'une pile d'images bidimensionnellesà différentes profondeurs de focalisationà travers l'échantillon. Les spécificités de la fonction de transfert optique 3D du microscope conduisentà des images présentant des défauts, comme par exemple uneélongation apparente selon l'axe vertical. Il est donc nécessaire de traiter les images avant toute mesure quantitative. On procèdeà une déconvolution de l'image 3D obtenue. Nous avons testé différents algorithmes de déconvolution sur des images de synthèse obtenues par convolution d'une bille pleine avec une fonction de transfert optique mesurée. Nous avons comparé, en forme et en volume, les images restaurées avec l'image d'origine. La méthode 'linear least square' est rapide, mais l'image restaurée présente des artefacts. La méthode de Carrington est bien adaptéeà la restauration d'objets fins. La méthode 'maximum likelihood-expectation maximization' permet une bonne reconstruction des images, mais demande de grands temps de calcul.
The resolution of microscopes is limited by the sizes of their point-spread functions. The invention of confocal, theta, and 4Pi microscopes has permitted the classic Abbe limit to be exceeded. We propose the use of a combination of 4Pi and theta microscopy to decrease resolution by using four illumination objectives and two detection objectives. Using middle numerical aperture, long-working-distance objectives yielded a resolution near 100 nm in the three dimensions, which opens the possibility of exploring large volumes with a high resolution.
International audienceExcept for blind methods, deconvolution of 3-D data sets acquired from a fluorescence microscope requires the knowledge of the point spread (PSF) of the instrument. Unsing the XCOSM package, we show first with simulations and then with recorded data that it is possible to recover from an experimental PSF some parameters, which are very difficult or impossible to measure during the acquisition, like the specimen depth or the immersion medium refractive index. Doing so, we can precise the acquisition protocol, which helps to use the instrument under optimal conditions. Furthermore, the knowledge of the actual acquisition condtions permits to use fo the deconvolution process a computed PSF, which is noiseless and as close as possible to the actual PSF. This helps to reduce errors in quantitative measurements after deconvolution, as shown with computations
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.