Publications about point spread function

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• 1 PhD Thesis and Habilitation
• 2 Conference articles
• 2 Technical and Research Reports

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PhD Thesis and Habilitation

1 - Blind Deconvolution for Confocal Laser Scanning Microscopy. P. Pankajakshan. PhD Thesis, Universite de Nice Sophia Antipolis, December 2009. Keywords : Confocal Laser Scanning Microscopy, Blind Deconvolution, point spread function, Maximum likelihood estimation , total variation regularization. @PHDTHESIS{PankajakshanThesis09, author = {Pankajakshan, P.}, title = {Blind Deconvolution for Confocal Laser Scanning Microscopy}, year = {2009}, month = {December}, school = {Universite de Nice Sophia Antipolis}, url = {http://tel.archives-ouvertes.fr/tel-00474264/fr/}, keyword = {Confocal Laser Scanning Microscopy, Blind Deconvolution, point spread function, Maximum likelihood estimation , total variation regularization} } Résumé : La microscopie confocale à balayage laser, est une technique puissante pour étudier les spécimens biologiques en trois dimensions (3D) par sectionnement optique. Elle permet d’avoir des images de spécimen vivants à une résolution de l’ordre de quelques centaines de nanomètres. Bien que très utilisée, il persiste des incertitudes dans le procédé d’observation. Comme la réponse du système à une impulsion, ou fonction de flou (PSF), est dépendante à la fois du spécimen et des conditions d’acquisition, elle devrait être estimée à partir des images observées du spécimen. Ce problème est mal posé et sous déterminé. Pour obtenir une solution, il faut injecter des connaisances, c’est à dire, a priori dans le problème. Pour cela, nous adoptons une approche bayésienne. L’état de l’art des algorithmes concernant la déconvolution et la déconvolution aveugle est exposé dans le cadre d’un travail bayésien. Dans la première partie, nous constatons que la diffraction due à l’objectif et au bruit intrinsèque à l’acquisition, sont les distorsions principales qui affectent les images d’un spécimen. Une approche de minimisation alternée (AM), restaure les fréquences manquantes au-delà de la limite de diffraction, en utilisant une régularisation par la variation totale sur l’objet, et une contrainte de forme sur la PSF. En outre, des méthodes sont proposées pour assurer la positivité des intensités estimées, conserver le flux de l’objet, et bien estimer le paramètre de la régularisation. Quand il s’agit d’imager des spécimens épais, la phase de la fonction pupille, due aux aberrations sphériques (SA) ne peut être ignorée. Dans la seconde partie, il est montré qu’elle dépend de la difference à l’index de réfraction entre l’objet et le milieu d’immersion de l’objectif, et de la profondeur sous la lamelle. Les paramètres d’imagerie et la distribution de l’intensité originelle de l’objet sont calculés en modifiant l’algorithme AM. Due à la nature de la lumière incohérente en microscopie à fluorescence, il est possible d’estimer la phase à partir des intensités observées en utilisant un modèle d’optique géométrique. Ceci a été mis en évidence sur des données simulées. Cette méthode pourrait être étendue pour restituer des spécimens affectés par les aberrations sphériques. Comme la PSF varie dans l’espace, un modèle de convolution par morceau est proposé, et la PSF est approchée. Ainsi, en plus de l’objet, il suffit d’estimer un seul paramétre libre. Abstract : Confocal laser scanning microscopy is a powerful technique for studying biological specimens in three dimensions (3D) by optical sectioning. It permits to visualize images of live specimens non-invasively with a resolution of few hundred nanometers. Although ubiquitous, there are uncertainties in the observation process. As the system’s impulse response, or point-spread function (PSF), is dependent on both the specimen and imaging conditions, it should be estimated from the observed images in addition to the specimen. This problem is ill-posed, under-determined. To obtain a solution, it is necessary to insert some knowledge in the form of a priori and adopt a Bayesian approach. The state of the art deconvolution and blind deconvolution algorithms are reviewed within a Bayesian framework. In the first part, we recognize that the diffraction-limited nature of the objective lens and the intrinsic noise are the primary distortions that affect specimen images. An alternative minimization (AM) approach restores the lost frequencies beyond the diffraction limit by using total variation regularization on the object, and a spatial constraint on the PSF. Additionally, some methods are proposed to ensure positivity of estimated intensities, to conserve the object’s flux, and to well handle the regularization parameter. When imaging thick specimens, the phase of the pupil function due to spherical aberration (SA) cannot be ignored. It is shown to be dependent on the refractive index mismatch between the object and the objective immersion medium, and the depth under the cover slip. The imaging parameters and the object’s original intensity distribution are recovered by modifying the AM algorithm. Due to the incoherent nature of the light in fluorescence microscopy, it is possible to retrieve the phase from the observed intensities by using a model derived from geometrical optics. This was verified on the simulated data. This method could also be extended to restore specimens affected by SA. As the PSF is space varying, a piecewise convolution model is proposed, and the PSF approximated so that, apart from the specimen, it is sufficient to estimated only one free parameter.

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2 Conference articles

1 - Point-spread function retrieval for fluorescence microscopy. P. Pankajakshan and L. Blanc-Féraud and Z. Kam and J. Zerubia. In Proc. IEEE International Symposium on Biomedical Imaging (ISBI), Publ. IEEE, Org. IEEE, Boston, USA, June 2009. Keywords : fluorescence microscopy, point spread function, EM algorithm, Deconvolution. Copyright : Copyright 2009 IEEE. Published in the 2009 International Symposium on Biomedical Imaging: From Nano to Macro (ISBI 2009), scheduled for June 28 - July 1, 2009 in Boston, Massachusetts, U.S.A. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works, must be obtained from the IEEE. Contact: Manager, Copyrights and Permissions / IEEE Service Center / 445 Hoes Lane / P.O. Box 1331 / Piscataway, NJ 08855-1331, USA. Telephone: + Intl. 908-562-3966. @INPROCEEDINGS{ppankajakshan09a, author = {Pankajakshan, P. and Blanc-Féraud, L. and Kam, Z. and Zerubia, J.}, title = {Point-spread function retrieval for fluorescence microscopy}, year = {2009}, month = {June}, booktitle = {Proc. IEEE International Symposium on Biomedical Imaging (ISBI)}, publisher = {IEEE}, organization = {IEEE}, address = {Boston, USA}, pdf = {http://hal.inria.fr/docs/00/39/55/34/PDF/pankajakshan.pdf}, keyword = {fluorescence microscopy, point spread function, EM algorithm, Deconvolution} } Abstract : In this paper we propose a method for retrieving the Point-Spread Function (PSF) of an imaging system given the observed images of fluorescent microspheres. Theoretically calculated PSFs often lack the experimental or microscope specific signatures while empirically obtained data are either over sized or (and) too noisy. The effect of noise and the influence of the microsphere size can be mitigated from the experimental data by using a Maximum Likelihood Expectation Maximization (MLEM) algorithm. The true experimental parameters can then be estimated by fitting the result to a model based on the scalar diffraction theory. The algorithm was tested on some simulated data and the results obtained validate the usefulness of the approach for retrieving the PSF from measured data.

2 - Blind deconvolution for diffraction-limited fluorescence microscopy. P. Pankajakshan and B. Zhang and L. Blanc-Féraud and Z. Kam and J.C. Olivo-Marin and J. Zerubia. In Proc. IEEE International Symposium on Biomedical Imaging (ISBI), pages 740-743, Paris, France, May 2008. Keywords : Confocal microscopy, Blind Deconvolution, point spread function, Richardson-Lucy algorithm, total variation regularization. Copyright : This work has been submitted to the IEEE for possible publication. Copyright may be transferred without notice, after which this version may no longer be accessible. @INPROCEEDINGS{ppankajakshan08a, author = {Pankajakshan, P. and Zhang, B. and Blanc-Féraud, L. and Kam, Z. and Olivo-Marin, J.C. and Zerubia, J.}, title = {Blind deconvolution for diffraction-limited fluorescence microscopy}, year = {2008}, month = {May}, booktitle = {Proc. IEEE International Symposium on Biomedical Imaging (ISBI)}, pages = {740-743}, address = {Paris, France}, pdf = {ftp://ftp-sop.inria.fr/ariana/Articles/2008_ppankajakshan08a.pdf}, keyword = {Confocal microscopy, Blind Deconvolution, point spread function, Richardson-Lucy algorithm, total variation regularization} } Abstract : Optical Sections of biological samples obtained from a fluorescence Confocal Laser Scanning Microscopes (CLSM) are often degraded by out-of-focus blur and photon counting noise. Such physical constraints on the observation are a result of the diffraction-limited nature of the optical system, and the reduced amount of light detected by the photomultiplier respectively. Hence, the image stacks can benefit from postprocessing restoration methods based on deconvolution. The parameters of the acquisition system’s Point Spread Function (PSF) may vary during the course of experimentation, and so they have to be estimated directly from the observation data. We describe here an alternate minimization algorithm for the simultaneous blind estimation of the specimen 3D distribution of fluorescent sources and the PSF. Experimental results on real data show that the algorithm provides very good deconvolution results in comparison to theoretical microscope PSF models.

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2 Technical and Research Reports

1 - Space non-invariant point-spread function and its estimation in fluorescence microscopy. P. Pankajakshan and L. Blanc-Féraud and Z. Kam and J. Zerubia. Research Report 7157, INRIA, December 2009. Keywords : Confocal Laser Scanning Microscopy, point spread function, Bayesian estimation, MAP estimation, Deconvolution, fluorescence microscopy. @TECHREPORT{ppankajakshan09c, author = {Pankajakshan, P. and Blanc-Féraud, L. and Kam, Z. and Zerubia, J.}, title = {Space non-invariant point-spread function and its estimation in fluorescence microscopy}, year = {2009}, month = {December}, institution = {INRIA}, type = {Research Report}, number = {7157}, url = {http://hal.archives-ouvertes.fr/inria-00438719/en/}, keyword = {Confocal Laser Scanning Microscopy, point spread function, Bayesian estimation, MAP estimation, Deconvolution, fluorescence microscopy} } Résumé : Dans ce rapport de recherche, nous rappelons brièvement comment la nature limitée de diffraction de l'objectif d'un microscope optique, et le bruit intrinsèque peuvent affecter la résolution d'une image observée. Un algorithme de déconvolution aveugle a été proposé en vue de restaurer les fréquences manquants au delà de la limite de diffraction. Cependant, sous d'autres conditions, l'approximation du systéme imageur l'imagerie sans aberration n'est plus valide et donc les aberrations de la phase du front d'onde émergeant d'un médium ne sont plus ignorées. Dans la deuxième partie de ce rapport de recherche, nous montrons que la distribution d'intensité originelle et la localisation d'un objet peuvent être retrouvées uniquement en obtenant de la phase du front d'onde réfracté, à partir d'images d'intensité observées. Nous démontrons cela par obtention de la fonction de ou a partir d'une microsphère imagée. Le bruit et l'influence de la taille de la microsphère peuvent être diminués et parfois complètement supprimes des images observées en utilisant un estimateur maximum a posteriori. Néanmoins, a cause de l'incohérence du système d'acquisition, une récupération de phase a partir d'intensités observées n'est possible que si la restauration de la phase est contrainte. Nous avons utilisé l'optique géométrique pour modéliser la phase du front d'onde réfracté, et nous avons teste l'algorithme sur des images simulées. Abstract : In this research report, we recall briefly how the diffraction-limited nature of an optical microscope's objective, and the intrinsic noise can affect the observed images' resolution. A blind deconvolution algorithm can restore the lost frequencies beyond the diffraction limit. However, under other imaging conditions, the approximation of aberration-free imaging, is not applicable, and the phase aberrations of the emerging wavefront from a specimen immersion medium cannot be ignored any more. We show that an object's location and its original intensity distribution can be recovered by retrieving the refracted wavefront's phase from the observed intensity images. We demonstrate this by retrieving the point-spread function from an imaged microsphere. The noise and the influence of the microsphere size can be mitigated and sometimes completely removed from the observed images by using a maximum a posteriori estimate. However, due to the incoherent nature of the acquisition system, phase retrieval from the observed intensities will be possible only if the phase is constrained. We have used geometrical optics to model the phase of the refracted wavefront, and tested the algorithm on some simulated images.

2 - Parametric blind deconvolution for confocal laser scanning microscopy-proof of concept. P. Pankajakshan and L. Blanc-Féraud and B. Zhang and Z. Kam and J.C. Olivo-Marin and J. Zerubia. Research Report 6493, INRIA, April 2008. Keywords : Confocal Laser Scanning Microscopy, Bayesian restoration, Blind Deconvolution, point spread function, Richardson-Lucy algorithm, Total variation. Copyright : ARIANA/INRIA @TECHREPORT{ppankajakshan08b, author = {Pankajakshan, P. and Blanc-Féraud, L. and Zhang, B. and Kam, Z. and Olivo-Marin, J.C. and Zerubia, J.}, title = {Parametric blind deconvolution for confocal laser scanning microscopy-proof of concept}, year = {2008}, month = {April}, institution = {INRIA}, type = {Research Report}, number = {6493}, url = {https://hal.inria.fr/inria-00269265}, pdf = {http://hal.inria.fr/docs/00/27/02/92/PDF/report.pdf}, keyword = {Confocal Laser Scanning Microscopy, Bayesian restoration, Blind Deconvolution, point spread function, Richardson-Lucy algorithm, Total variation} } Résumé : Nous proposons une méthode de restauration itérative d’images de fluorescence CLSM et d’estimation paramétrique de la fonction de flou (PSF) du système d’acquisition. Le CLSM est un microscope qui balaye un échantillon en 3D et utilise une sténopée pour rejeter la lumière en dehors du point de focalisation. Néanmoins, la qualité des images souffre de deux limitations physiques. La première est due à la diffraction due au système optique et la seconde est due à la quantité réduite de lumière détectée par le tube photo-multiplicateur (PMT). Ces limitations induisent respectivement un flou et du bruit de comptage de photons. Les images peuvent alors bénéficier d’un post-traitement de restauration fondé sur la déconvolution. Le problème à traiter est l’estimation simultanée de la distribution 3D de l’échantillon des sources fluorescentes et de la PSF du microscope (i.e. de déconvolution aveugle). En utilisant un modèle de processus physique d’acquisition d’images microscopiques (CLSM), on réduit le nombre de paramètres libres décrivant la PSF et on introduit des contraintes. On introduit aussi des connaissances a priori sur l’échantillon ce qui permet de stabiliser le processus d’estimation et de favoriser la convergence. Des expériences sur des données synthétiques montrent que la PSF peut être estimée avec précision. Des expériences sur des données réelles montrent de bons resultats de déconvolution en comparaison avec le modèle théorique de la PSF du microscope. Abstract : We propose a method for the iterative restoration of fluorescence Confocal Laser Scanning Microscope (CLSM) images with parametric estimation of the acquisition system’s Point Spread Function (PSF). The CLSM is an optical fluorescence microscope that scans a specimen in 3D and uses a pinhole to reject most of the out-of-focus light. However, the quality of the image suffers from two primary physical limitations. The first is due to the diffraction-limited nature of the optical system and the second is due to the reduced amount of light detected by the photomultiplier tube (PMT). These limitations cause blur and photon counting noise respectively. The images can hence benefit from post-processing restoration methods based on deconvolution. An efficient method for parametric blind image deconvolution involves the simultaneous estimation of the specimen 3D distribution of fluorescent sources and the microscope PSF. By using a model for the microscope image acquisition physical process, we reduce the number of free parameters describing the PSF and introduce constraints. The parameters of the PSF may vary during the course of experimentation, and so they have to be estimated directly from the observation data. We also introduce a priori knowledge of the specimen that permits stabilization of the estimation process and favorizes the convergence. Experiments on simulated data show that the PSF could be estimatedwith a higher degree of accuracy and those done on real data show very good deconvolution results in comparison to the theoretical microscope PSF model.

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