In this work, for the first time, the quality of restoration in wide-field microscopy images after deconvolution was analyzed as a function of different Point Spread Functions using one deconvolution method, on a specimen of known size and on a biological specimen. The empirical Point Spread Function determination can significantly depend on the numerical aperture, refractive index of the embedding medium, refractive index of the immersion oil and cover slip thickness. The influence of all of these factors is shown in the same article and using the same microscope. We have found that the best deconvolution results are obtained when the empirical PSF utilized is obtained under the same conditions as the specimen. We also demonstrated that it is very important to quantitatively check the process' outcome using several quality indicators: Full-Width at Half-Maximum, Contrast-to-Noise Ratio, Signal-to-Noise Ratio and a Tenengrad-based function. We detected a significant improvement when using an indicator to measure the focus of the whole stack. Therefore, to qualitatively determinate the best deconvolved image between different conditions, one approach that we are pursuing is to use Tenengrad-based function indicators in images obtained using a wide-field microscope.

© 2011 Optical Society of Korea

1. D. Agard, "Optical sectioning microscopy: cellular architecture in three dimensions," Annu. Rev. Biophys. Bioeng. 13, 191-219 (1984). [CrossRef]
2. J. C. McNally, T. Karpova, J. Cooper, and J. A. Conchello, "Three-dimensional imaging by deconvolution microscopy," Methods 19, 373-385 (1999). [CrossRef]
3. P. Sarder and A. Nehorai, "Deconvolution methods for 3D fluorescence microscopy images," IEEE Sig. Proc. Mag. 23, 32-45 (2006). [CrossRef]
4. Y. Hiraoka, J. W. Sedat, and D. A. Agard, "Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy," Biophys. J. 57, 325-333 (1990). [CrossRef]
5. S. F. Gibson and F. Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 1, 154-166 (1992).
6. M. Abramowitz, K. R. Spring, H. E. Keller, and M. W. Davidson, "Basic principles of microscope objectives," Biotechniques 33, 772-781 (2002).
7. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer Press, New York, USA, 1995), Chapter 7.
8. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer Press, New York, USA, 1995), Chapter 20.
9. J. B. Sibarita, "Deconvolution microscopy," Adv. Biochem. Eng. Biotechnol. 95, 201-243 (2005).
10. J. G. McNally, C. Preza, J. A. Conchello, and L. J. Jr. Thomas, "Artifacts in computational optical-sectioning microscopy," J. Opt. Soc. Am. A 11, 1056-1067 (1994). [CrossRef]
11. B. A. Scalettar, J. R. Swedlow, J. W. Sedat, and D. A. Agard, "Dispersion, aberration and deconvolution in multi-wavelength fluorescence images," J. Microsc. 1, 50-60 (1996).
12. M. Kozubek, "Theoretical versus experimental resolution in optical microscopy," Microsc. Res. Tech. 2, 157-166 (2001).
13. J. Adur and J. Schlegel, "Design, development and construction of an advance micrometric system for microscopes," Bioengineering Degree Thesis, Faculty of Engineering- Bioengineering, Entre Ríos National University, Argentina (1997).
14. J. E. Diaz-Zamboni, "Software to users of desconvolution digital microscopy," Bioengineering Degree Thesis, Faculty of Engineering-Bioengineering, Entre Ríos National University, Argentina (2004).
15. W. A. Carrington, K. E. Fogarty, and F. S. Fay, "3D fluorescence imaging of single cells using image restoration," in Noninvasive Techniques in Cell Biology, K. Foskett and S. Grinstein, eds. (Wiley-Liss, New York, USA, 1990).
16. K. L. Gosner, "A simplified table for stanging anuran embryos and larval with noter on identification," Herpetologica 16, 183-190 (1960).
17. M. F. Izaguirre, D. Larrea, J. F. Adur, J. E. Diaz-Zamboni, N. B. Vicente, C. D. Galetto, and V. H. Casco, "E-cadherin role in epithelial architecture maintenance," Cell. Commun. Adhes. 2, 1-12 (2010).
18. S. Grgic, M. Grgic, and M. Mrak, "Reliability of objective picture quality measures," J. Elect. Engineering 55, 3-10 (2004).
19. Y. Yao, B. R. Abidi, and M. A. Abidi, "Extreme zoom surveillance: system design and image restoration," J. Multimedia 2, 20-31 (2007).
20. K. R. Castleman, Digital Image Processing (Prentice Hall, New Jersey, USA, 1996), Chapter 9.
21. C. Preza and J. Conchello, "Image estimation accounting for point-spread function depth variation in threedimensional fluorescence microscopy," Proc. SPIE 4964, 135-142 (2003). [CrossRef]
22. J. A. Conchello and J. W. Lichtman, "Optical sectioning microscopy," Nat. Methods 12, 920-931 (2005).
23. J. F. Adur, "Determinación de las propiedades ópticas de un sistema de epifluorescencia y su utilización en estudios de microscopía cuantitativa 3D," Biomedical Engineer Magister Thesis, Faculty of Engineering-Bioengineering, Entre Ríos National University, Argentina (2006).
24. S. Shin and Y. Yu, "Three-dimensional information and refractive index measurement using a dual-wavelength digital holographic microscope," J. Opt. Soc. Korea 13, 173-177 (2009). [CrossRef]
25. B. Lee, N. Shin, K. Jeong, M. J. Park, B. G. Kim, J. H. Yoo, D. G. Kim, K. H. Yun, K. Lee, K. H. Kim, D. K. Kim, and S. H. Park, "Nondestructive optical measurement of refractive-index profile of graded-index lenses," J. Opt. Soc. Korea 13, 468-471 (2009). [CrossRef]
26. Y. Urata, S. J. Parmelee, D. A. Agard, and J. W. Sedat, "A three-dimensional structural dissection of Drosophila polytene chromosomes," J. Cell. Biol. 2, 279-295 (1995).
27. T. C. Wei, D. H. Shin, and B. G. Lee, "Resolution-enhanced reconstruction of 3D object using depth-reversed elemental images for partially occluded object recognition," J. Opt. Soc. Korea 13, 139-145 (2009). [CrossRef]
28. A. Diaspro, F. Federici, and M. Robello, "Influence of refractive-index mismatch in high-resolution threedimensional confocal microscopy," Appl. Opt. 41, 685-690 (2002). [CrossRef]
29. J. W. Shaevitz and D. A. Fletcher, "Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function," J. Opt. Soc. Am. A 24, 2622-2627 (2007). [CrossRef]
30. H. Wei and J. Zhongliang, "Evaluation of focus measures in multi-focus image fusion," Pattern Recog. Lett. 28, 493-500 (2007). [CrossRef]
31. N. B. Vicente, J. E. Diaz-Zamboni, J. F. Adur, M. F. Izaguirre, C. D. Galetto, and V. H. Casco, "Development of a semiautomatic algorithm for deconvolution and quantification of three-dimensional microscopy images," A. Microscópia 19, 328-336 (2010).
32. X. Lai, Z. Lin, E. S. Ward, and R. J. Ober, "Noise suppression of point spread functions and its influence on deconvolution of three-dimensional fluorescence microscopy image sets," J. Microsc. 217, 93-108 (2005). [CrossRef]
33. A. Marian, F. Charriere, T. Colomb, F. Montfort, J. Kuhn, P. Marquet, and C. Depeursinge, "On the complex threedimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography," J. Microsc. 225, 156-169 (2007). [CrossRef]

A. Microscópia

• N. B. Vicente, J. E. Diaz-Zamboni, J. F. Adur, M. F. Izaguirre, C. D. Galetto, and V. H. Casco, "Development of a semiautomatic algorithm for deconvolution and quantification of three-dimensional microscopy images," A. Microscópia 19, 328-336 (2010).

Adv. Biochem. Eng. Biotechnol.

• J. B. Sibarita, "Deconvolution microscopy," Adv. Biochem. Eng. Biotechnol. 95, 201-243 (2005).

Annu. Rev. Biophys. Bioeng.

• D. Agard, "Optical sectioning microscopy: cellular architecture in three dimensions," Annu. Rev. Biophys. Bioeng. 13, 191-219 (1984). [CrossRef]

Appl. Opt.

• A. Diaspro, F. Federici, and M. Robello, "Influence of refractive-index mismatch in high-resolution threedimensional confocal microscopy," Appl. Opt. 41, 685-690 (2002). [CrossRef]

Biophys. J.

• Y. Hiraoka, J. W. Sedat, and D. A. Agard, "Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy," Biophys. J. 57, 325-333 (1990). [CrossRef]

Biotechniques

• M. Abramowitz, K. R. Spring, H. E. Keller, and M. W. Davidson, "Basic principles of microscope objectives," Biotechniques 33, 772-781 (2002).

Cell. Commun. Adhes.

• M. F. Izaguirre, D. Larrea, J. F. Adur, J. E. Diaz-Zamboni, N. B. Vicente, C. D. Galetto, and V. H. Casco, "E-cadherin role in epithelial architecture maintenance," Cell. Commun. Adhes. 2, 1-12 (2010).

Herpetologica

• K. L. Gosner, "A simplified table for stanging anuran embryos and larval with noter on identification," Herpetologica 16, 183-190 (1960).

IEEE Sig. Proc. Mag.

• P. Sarder and A. Nehorai, "Deconvolution methods for 3D fluorescence microscopy images," IEEE Sig. Proc. Mag. 23, 32-45 (2006). [CrossRef]

J. Cell. Biol.

• Y. Urata, S. J. Parmelee, D. A. Agard, and J. W. Sedat, "A three-dimensional structural dissection of Drosophila polytene chromosomes," J. Cell. Biol. 2, 279-295 (1995).

J. Elect. Engineering

• S. Grgic, M. Grgic, and M. Mrak, "Reliability of objective picture quality measures," J. Elect. Engineering 55, 3-10 (2004).

J. Microsc.

• B. A. Scalettar, J. R. Swedlow, J. W. Sedat, and D. A. Agard, "Dispersion, aberration and deconvolution in multi-wavelength fluorescence images," J. Microsc. 1, 50-60 (1996).
• X. Lai, Z. Lin, E. S. Ward, and R. J. Ober, "Noise suppression of point spread functions and its influence on deconvolution of three-dimensional fluorescence microscopy image sets," J. Microsc. 217, 93-108 (2005). [CrossRef]
• A. Marian, F. Charriere, T. Colomb, F. Montfort, J. Kuhn, P. Marquet, and C. Depeursinge, "On the complex threedimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography," J. Microsc. 225, 156-169 (2007). [CrossRef]

J. Multimedia

• Y. Yao, B. R. Abidi, and M. A. Abidi, "Extreme zoom surveillance: system design and image restoration," J. Multimedia 2, 20-31 (2007).

J. Opt. Soc. Am. A

• J. G. McNally, C. Preza, J. A. Conchello, and L. J. Jr. Thomas, "Artifacts in computational optical-sectioning microscopy," J. Opt. Soc. Am. A 11, 1056-1067 (1994). [CrossRef]
• S. F. Gibson and F. Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 1, 154-166 (1992).
• J. W. Shaevitz and D. A. Fletcher, "Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function," J. Opt. Soc. Am. A 24, 2622-2627 (2007). [CrossRef]

J. Opt. Soc. Korea

• T. C. Wei, D. H. Shin, and B. G. Lee, "Resolution-enhanced reconstruction of 3D object using depth-reversed elemental images for partially occluded object recognition," J. Opt. Soc. Korea 13, 139-145 (2009). [CrossRef]
• S. Shin and Y. Yu, "Three-dimensional information and refractive index measurement using a dual-wavelength digital holographic microscope," J. Opt. Soc. Korea 13, 173-177 (2009). [CrossRef]

Journal of the Optical Society of Korea

• B. Lee, N. Shin, K. Jeong, M. J. Park, B. G. Kim, J. H. Yoo, D. G. Kim, K. H. Yun, K. Lee, K. H. Kim, D. K. Kim, and S. H. Park, "Nondestructive optical measurement of refractive-index profile of graded-index lenses," J. Opt. Soc. Korea 13, 468-471 (2009). [CrossRef]

Methods

• J. C. McNally, T. Karpova, J. Cooper, and J. A. Conchello, "Three-dimensional imaging by deconvolution microscopy," Methods 19, 373-385 (1999). [CrossRef]

Microsc. Res. Tech.

• M. Kozubek, "Theoretical versus experimental resolution in optical microscopy," Microsc. Res. Tech. 2, 157-166 (2001).

Nat. Methods

• J. A. Conchello and J. W. Lichtman, "Optical sectioning microscopy," Nat. Methods 12, 920-931 (2005).

Pattern Recog. Lett.

• H. Wei and J. Zhongliang, "Evaluation of focus measures in multi-focus image fusion," Pattern Recog. Lett. 28, 493-500 (2007). [CrossRef]

Proc. SPIE

• C. Preza and J. Conchello, "Image estimation accounting for point-spread function depth variation in threedimensional fluorescence microscopy," Proc. SPIE 4964, 135-142 (2003). [CrossRef]

Other

• J. F. Adur, "Determinación de las propiedades ópticas de un sistema de epifluorescencia y su utilización en estudios de microscopía cuantitativa 3D," Biomedical Engineer Magister Thesis, Faculty of Engineering-Bioengineering, Entre Ríos National University, Argentina (2006).
• J. Adur and J. Schlegel, "Design, development and construction of an advance micrometric system for microscopes," Bioengineering Degree Thesis, Faculty of Engineering- Bioengineering, Entre Ríos National University, Argentina (1997).
• J. E. Diaz-Zamboni, "Software to users of desconvolution digital microscopy," Bioengineering Degree Thesis, Faculty of Engineering-Bioengineering, Entre Ríos National University, Argentina (2004).
• W. A. Carrington, K. E. Fogarty, and F. S. Fay, "3D fluorescence imaging of single cells using image restoration," in Noninvasive Techniques in Cell Biology, K. Foskett and S. Grinstein, eds. (Wiley-Liss, New York, USA, 1990).
• K. R. Castleman, Digital Image Processing (Prentice Hall, New Jersey, USA, 1996), Chapter 9.
• J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer Press, New York, USA, 1995), Chapter 7.
• J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer Press, New York, USA, 1995), Chapter 20.

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