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Extended focused image in microscopy by digital holography

Pietro Ferraro, Simonetta Grilli, Domenico Alfieri, Sergio De Nicola, Andrea Finizio, Giovanni Pierattini, Bahram Javidi, Giuseppe Coppola, and Valerio Striano

Open Access Open Access

Abstract

In microscopy, high magnifications are achievable for investigating micro-objects but the paradigm is that higher is the required magnification, lower is the depth of focus. For an object having a three-dimensional (3D) complex shape only a portion of it appears in good focus to the observer who is essentially looking at a single image plane. Actually, two approaches exist to obtain an extended focused image, both having severe limitations since the first requires mechanical scanning while the other one requires specially designed optics. We demonstrate that an extended focused image of an object can be obtained through digital holography without any mechanical scanning or special optical components. The conceptual novelty of the proposed approach lies in the fact that it is possible to completely exploit the unique feature of DH in extracting all the information content stored in hologram, amplitude and phase, to extend the depth of focus.

©2005 Optical Society of America

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Figures (7)
Fig. 1.
Fig. 1. Qualitative drawing of the working principle of the EFI method. Stack of in-focus images (sequentially numbered) corresponding to different portions of the imaged object are stuck together to get an overall in-focus image (on the right).

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Fig. 2.
Fig. 2. Optical set-up of the digital holographic microscope.

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Fig. 3.
Fig. 3. Numerical reconstruction of the hologram of the cantilever beam (a) SEM image (b) hologram (c) wrapped phase map (d) 3D profile of the cantilever.

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Fig. 4.
Fig. 4. Conceptual flow chart describing how the EFI image is obtained by a Digital Holography approach.

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Fig. 5.
Fig. 5. Comparison of the microscopy and DH reconstruction method (a) in focus image of the base of the cantilever obtained by the microscope (b) Amplitude reconstruction of the base of the cantilever by DH method (c) In–focus image of the tip of the cantilever obtained by the microscope (d) Amplitude reconstruction of the tip of the cantilever by DH method (e) EFI image of the cantilever (f) reconstructed amplitude image of the cantilever by DH.

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Fig. 6.
Fig. 6. Movie (5.5 MB) of a sequence of images obtained by means of the optical microscope by changing the distance of microscope objective-MEMS and the reconstructed amplitude images of the same MEMS obtained by a single digital hologram but reconstructed numerically at different distances.

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Fig. 7.
Fig. 7. Comparison between conventional EFI technique and holographic EFI method (a) Conventional EFI image after stacking together in-focus images by microscope of portions of the cantilever beam (b) Holographic EFI of the cantilever by DH method as obtained by stacking of 50 reconstructed amplitude images from the 3D amplitude volume (c) combination of 3D plot of phase map and holographic EFI of the cantilever. Holographic EFI is obtained by only one image.

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Equations (3)

Equations on this page are rendered with MathJax. Learn more.

OPD x y = λ 2 π φ x y
Δ q x y = − M 2 Δ p x y
Δ q x y = − M 2 Δ φ x y 4 π λ
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