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)