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    Digital Holography Micros

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    Rimple Poonia Dabas
    Digital holographic microscopy provides a non-invasive technique for quantitative visualization of living cells with sub-wavelength accuracy. It involves recording the interference pattern of light scattered from the sample with a reference beam using a CCD, and numerically reconstructing the hologram using methods like Fresnel or convolution approaches. Issues like zero order removal, twin image elimination, and autofocusing need to be addressed for clear reconstructions. Digital holographic microscopy allows for quantitative phase-contrast imaging without optical aberrations.

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    Digital Holography Micros

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    Rimple Poonia Dabas
    122 views27 pages
    Digital holographic microscopy provides a non-invasive technique for quantitative visualization of living cells with sub-wavelength accuracy. It involves recording the interference pattern of light scattered from the sample with a reference beam using a CCD, and numerically reconstructing the hologram using methods like Fresnel or convolution approaches. Issues like zero order removal, twin image elimination, and autofocusing need to be addressed for clear reconstructions. Digital holographic microscopy allows for quantitative phase-contrast imaging without optical aberrations.

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    Digital Holography Microscopy

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    Digital holographic microscopy provides a non-invasive technique for quantitative visualization of living cells with sub-wavelength accuracy. It involves recording the interference pattern of light scattered from the sample with a reference beam using a CCD, and numerically reconstructing the hologram using methods like Fresnel or convolution approaches. Issues like zero order removal, twin image elimination, and autofocusing need to be addressed for clear reconstructions. Digital holographic microscopy allows for quantitative phase-contrast imaging without optical aberrations.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    122 views27 pages

    Digital Holography Micros

    Uploaded by

    Rimple Poonia Dabas
    Digital holographic microscopy provides a non-invasive technique for quantitative visualization of living cells with sub-wavelength accuracy. It involves recording the interference pattern of light scattered from the sample with a reference beam using a CCD, and numerically reconstructing the hologram using methods like Fresnel or convolution approaches. Issues like zero order removal, twin image elimination, and autofocusing need to be addressed for clear reconstructions. Digital holographic microscopy allows for quantitative phase-contrast imaging without optical aberrations.

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    of 27

    DIGITAL HOLOGRAPHIC MICROSCOPY

    PRESENTED BY : RIMPLE POONIA 2010PHA2765

    UNDER THE GUIDANCE OF: PROF. JOBY JOSEPH

    Holography:
    Digital holography: recording by Charge Coupled

    Devices(CCDs). Reconstruction by numerical methods

    Recording and reconstruction

    Ulf Schnars,and Werner P O Juptner Ulf Schnars,and Werner P O Juptner : Digital : Digital recording and numerical reconstruction of hologram recording and numerical reconstruction of hologram

    Reconstruction methods:
    Fresnel approach: diffraction at any

    aperture given by Fresnel Kirchoff integral

    , (, )

    exp

    ( )2 +(

    )2 + 2

    Using approximation : and values, and values small compared to d we have for real image
    = + 2
    2

    + 2

    1 )2 + 8 3

    +. .

    For Fourth term

    1 8

    2 )2 + 2

    2 2 = + + 2 2 Using in above equation 2 2 , = exp i exp + 2 2 + 2 2 + x, y h x, y exp i exp

    One fourier transform involved


    Minimum distance requirement

    Pixel distance in reconstructed images varies with the

    reconstruction distance as =

    Convolution approach:
    Numerical processing of Fresnel Kirchhoff integral is time

    consuming Reconstruction formula with superposition integral , = , , (, , , ) With impulse response


    , , , =

    2 2 2 2 exp + + 2 + 2 + 2

    System characterised by , , , = ,

    Convolution theorem is applied Three Fourier transforms involved:


    , = 1 . .

    Digital holographic microscopy


    Lateral resolution of holographic reconstruction is given

    1 1 = [1 + ] 2 where =distance between source point and hologram plane in recording process =distance between source point and hologram plane in reconstruction process 1 and 2 wavelenghts during recording and reconstruction For desired magnification reconstruction distance = . and 1 = 2

    by

    Source point needs to be placed at 1 1 1 1 = [ + ]

    Reference wave needs to be placed at


    , 2 = exp( 2 +

    Reconstructions

    Fresnel approach

    d=37cm Convolution approach

    Fresnel approach

    d=37cm Convolution approach

    Interesting Property

    Zero order removal

    Intensity at the hologram plane

    Reconstructed wavefront at hologram plane

    Average intensity subtraction

    Average intensity of all pixels in hologram

    High pass filtering-steps involved


    Read the hologram

    Convert from spatial domain to frequency domain with FFT2 and shift the zero components with FFTSHIFT

    Define a circular mask and position it at the center of the Fourier transformed image

    Return to spatial domain with IFFT2 and IFFTSHIFT2

    Save as tiff image

    High pass filtering

    Average subtraction

    Spatial filtering-steps involved


    Read the hologram

    Convert from spatial domain to frequency domain with FFT2 and shift the zero components with FFTSHIFT

    Define masks numerically according to the spectrum of the hologram and multiply with the Fourier transform obtained above

    Return to spatial domain with IFFT2 and IFFTSHIFT2

    Filtered hologram is obtained which is numerically reconstructed

    Spatial filtering

    Spectrum of the hologram

    Twin image elimination

    FIR METHOD

    Autofocusing evaluation functions


    In autofocusing determination and maximization of image

    contrast needed For amplitude objects-focused image at maximal focus value For phase objects-minimum absorption-focused image at minimal focus value Evaluation functions: Variance of the grey function based on the statistical analysis of the gray value distribution Sharp structures in a focused image result in a higher contrast than in a smooth defocused image
    1 =

    ,
    =1 =1

    Weighted Fourier spectral analysis quantifies the sharpness of the edges as a focused image

    contains more fine details than defocused image


    =
    1 =1 =1 log[1 +

    (, )]

    Standard deviation correlation function

    The reconstructed image field is given by


    , , = And intensity

    2 + 2 (, , 0)

    2 + 2

    I , , , z = ( , , , z)

    Pin hole

    Beam splitter1

    NF mirror

    laser

    lens object

    MO

    Beam splitter2 mirror

    CCD

    Result

    At d=30 cm

    At d=45 cm

    At d=60cm

    REFERENCES
    B. Kemper, and G. Bally, "Digital holographic microscopy for live cell applications and

    technical inspection," Appl.Opt. 47(2), A52-A61 (2008). Collier R. J., Burckhardt C. B., and Lin L.H., Optical Holography, (Academic Press, New York, 1971). Christopher J Mann, L Yu and K Kim* Movies of cellular and sub-cellular motion by digital holographic microscopy, (BioMedical Engineering OnLine2006, 5:21) Cuche E., Marquet P., and Depeursinge C., Spatial filtering for zero-order and twin image elimination in digital off axis holography, Appl.Opt.39 (2000) 4070-4075. Cuche E, Marquet P, and Depeursinge C,Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms,( Appl. Opt , Vol. 38, 1999) Dubois F, Schockaert C,Callens N and Yourassowsky C, Focus plane detection criteria in digital holography microscopy by amplitude analysis (Optical Society of America, 2006) Gabor D, A new microscope principle, Nature, 161 (1948) 777-778. Goodman J. W, Introduction to Fourier Optics, (McGraw-Hill, New York, 1996). Hariharan P., Optical Holography,:Principle, techniques, and applications, (Cambridge University Press, Cambridge ,1983).

    Sucerquia J, Xu W, Stephan K. Jericho,Klages P,Manfred H. Jericho, and H. Jrgen Kreuzer, Digital in-line holographic microscopy (Appl. Opt Vol. 45, 2006) Kreis Th, Adams M., and Jptner W., Methods of digital holography: a comparison, Proc. SPIE 3098 (1997) 224 233. Kreis T.M., and Juptner W., Suppression of dc term in digital holography, Opt.Engn. 36 (1997) 2367-2370. Langehanenberg P, G von Bally and B. Kemper, Autofocusing in Digital Holographic Microscopy. 3D Research (3DR Review), Vol.2, No. 1 (2011) Lihong Ma, Hui Wang, Yong Li & Haijun Zhang):Elimination of zero-order diffraction and conjugate image in off-axis digital holography, Journal of Modern Optics, 56:21, 2377-2383,(2009) Lee W. H., Computer Generated Holograms, Techniques and Applications, Progress in Optics, 16 (1978) 120-232. Liu W,Kang X , Xiaoyuan He . Filter method used to eliminate the zero-order image,( SPIE Vol. 7375, 2009) Lohmann A. W., Mendlovic David, and Shabtay Gal, Significance of phase and amplitude in the Fourier domain , J. Opt. Soc. Am. A, 14 (1997) 2901-2904. M. Liebling, M. Unser, Autofocus for digital Fresnel holograms by use of a Fresnelet-sparsity criterion,( J. Opt.Soc. Am. A 21: 24242430,2009) M. K. Kim, Principles and techniques of digital holographic microscopy (SPIE Rev. 1 2010. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, C. Depeursinge,Digital holographic microscopy: a non invasive contrast imaging technique allowing quantitative visualization of living cells with sub wavelength accuracy, (Opt. Lett. 30, 468 -470,2005) Schnars U. and Juptner W. Direct recording of holograms by a CCD target and numerical reconstruction, Appl. Opt. 33 (1994) 79181. Schnars U. and Juptner W., Digital Holography, (Springer Verlag, New York, 2005) Wang H, Aili Qin, Min Huang,Autofocus method for digital holographic reconstruction of microscopic object,. ( IEEE,2009) Wang H, Aili Qin, Min et al,Autofocus method for amplitude and pure phase objects reconstruction in digital holography.( Proc. of SPIE Vol. 7513,2009) Wang H , Lihong M Yong Li,Haijun Zhang, Elimination of zero-order diffraction and conjugate image in off-axis digital holography (Journal of Modern Optics, 56:21, 2377-2383, 2009)

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