Digital Holography Microscopy

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DIGITAL HOLOGRAPHICMICROSCOPY

PRESENTED BY : UNDER THE

RIMPLE POONIA GUIDANCE OF:

2010PHA2765 PROF. JOBY JOSEPH


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Reconstruction methods:

Fresnel approach: diffraction at anyaperture given by Fresnel Kirchoff integral

,

, (,)

( )( )Using approximation : and values, and values small compared to d we have for real

image

2

2

18

) 3 . .


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For Fourth term

)+

2

2

Using in above equation

, exp i2

exp

x,y h x,y exp i exp 2


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One fourier transform involved

Minimum distance requirement Pixel distance in reconstructed images varies with the

reconstruction distance as


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Convolution approach:

Numerical processing of Fresnel Kirchhoff integral is timeconsuming

Reconstruction formula with superposition integral

, , , (, , , ) With impulse response

, , , + + + +

System characterised by , , , , Convolution theorem is applied

Three Fourier transforms involved:

, . .


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Digital holographic microscopy

Lateral resolution of holographic reconstruction is given

by

[1

]

where =distance between source point and hologramplane in recording process

=distance between source point and hologram plane inreconstruction process

and wavelenghts during recording and reconstructionFor desired magnification reconstruction distance

. and


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Source point needs to be

placed at

[ 1 1

1]

Reference wave needs to be

placed at

, exp( 2


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Fresnel

approach

Convolution

approach

d=37cm

Reconstructions


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Fresnel

approach

Convolution

approach

d=37cm


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Interesting Property


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Intensity at the hologram plane

Reconstructed wavefront at hologram plane

Zero order removal


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Average intensity subtraction

Average intensity of all pixels in hologram


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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


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High pass filtering

Average subtraction


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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


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Spatial filtering

Spectrum of the hologram


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Twin image elimination


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FIR METHOD


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Autofocusing evaluation functions

In autofocusing determination and maximization of image

contrast needed

For amplitude objects-focused image at maximal focusvalue

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 ,

=

=


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Weighted Fourier spectral analysis

quantifies the sharpness of the edges as a focused image

contains more fine details than defocused image

log[1(,)]== Standard deviation correlation functionThereconstructed image field is given by

, , (,,0)

And intensity

I , , , z ( , , , z)


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laser

Pin

hole

lens

Beam splitter1NF

mirror

mirror

object

MO

Beam

splitter2

CCD


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Result


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At d=30 cm

At d=45 cm At d=60cm


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

technical inspection," Appl.Opt. 47(2), A52-A61 (2008).

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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).


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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 JptnerW., Methods of digital holography: a comparison, Proc. SPIE 3098 (1997) 224

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Kreis T.M., and JuptnerW., Suppression of dc term in digital holography, Opt.Engn. 36 (1997) 2367-2370.

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(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)

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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.

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Schnars U. and JuptnerW. Direct recording of holograms by a CCD target and numerical reconstruction, Appl. Opt.33 (1994) 79181.

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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)

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holography (Journal of Modern Optics, 56:21, 2377-2383, 2009)

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Digital Holography Microscopy