III WVC 2007 - Iris.sel.eesc.sc.usp.br - USP

WVC'2007 - III Workshop de Visão Computacional, 22 a 24 de Outubro de 2007, São José do Rio Preto, SP.Improving the Richardson-Lucy Algorithm in Deconvolution MicroscopyThrough Poisson Noise ReductionHomem, M. R. P.; Zorzan, M. R.; Mascarenhas, N. D. A.Universidade Federal de São Carlos, Departamento de ComputaçãoVia Washington Luís, Km 235, CP 676, CEP: 13.565-905, São Carlos, SP, Brazilmurillo rodrigo@dc.ufscar.br; marcelozorzan@yahoo.com.br; nelson@dc.ufscar.brAbstractComputational optical sectioning microscopy is a techniqueto obtain three-dimensional images of microscopic biologicalspecimens. It consists in obtaining a set of twodimensionaloptical sections of an object where each sectionis acquired by means of a light microscope using fluorescencetechniques. However, due to limiting factors in theimaging systems, microscopic images are always degradedby the microscope optics and also by the detection process.Each observed section is a blurred version of the actual imageand it also has contributions of light from other outof-focusplanes. In this sense, it is important to search forcomputational algorithms that are able to improve the qualityof the three-dimensional observations. The Richardson-Lucy algorithm is one of the most important methods forimage deconvolution in optical sectioning microscopy andit is often regarded as the algorithm that is able to producethe best results in that application. In this work, we demonstratethat the restored images can be further improved byfirst removing the Poisson noise in the images before applyingthe iterative Richardson-Lucy algorithm. We show thatbetter results are achieved in a similar number of iteractionsand they also have a better quality following the improvementin signal to noise ratio criteria.1. IntroductionThe proper three-dimensional (3D) visualization of cellulararchitectures in biological applications has been consideredin the last twenty years [1]. It is substantially important,because the cell structure and its function are known tobe strongly correlated.Computational optical-sectioning microscopy (COSM)is recognized as an important tool to reconstruct 3D imagesfrom optical two-dimensional (2D) sections of a fluorescentlystained biological specimen [22]. Considering thatthe specimen is translucid, this technique consists in movingthe focal plane of the microscope while a set of 2D imagesare acquired and recorded. In this sense, stacking theset of 2D images forms a 3D image.Commonly, confocal and conventional light microscopesare used in COSM [25]. The former modality is able to producehigh-quality images because only the light from the regionnear the in-focus plane is detected. However, the costof a confocal equipment is substantially high. In addition,in this modality, the light efficiency and also the sensitivityare less than in a conventional microscope, which canbe a problem in experiments where the light efficiency is animportant concern. On the other hand, a conventional microscope(also known as widefield microscope) is cheaperthan a confocal one and it is particularly valuable for workwith living cells, because it avoids specimen damage due tothe laser light used in confocal microscopy.However, in both modalities, the quality of the recordeddata is limited by the optical system. This procedure hasthe disadvantage that each optical slice (or the 2D image) isblurred by out-of-focus information. Indeed, each slice hascontributions of light from other out-of-focus planes. Theblurring effects come from light diffraction due to the finiteaperture of the microscope lens [10]. Particularly, it is importantto note that in conventional microscopy the blurringeffects are higher than in confocal microscopy [18].Futhermore, it can be shown that the optical transferfunction of a fluorescence microscope is zero valued formost of the frequencies in the Fourier domain [22]. Then,it removes the image content in the regions where it haszero values. Also, in the region where it has non-zero valuesit works as a low-pass filter and smooth the image.Besides the blurring effects, there are several sourcesof noise that decrease the quality of the images in COSM[18, 24]. It can be shown that the predominant one influorescence microscopy is due to the low level of photonscount. Indeed, the exposure time in fluorescence microscopyneeds to be frequently short. It implies that eachimage is acquired under low level of photons count and then133