High-resolution quantitative phase-contrast microscopy by digital holography
TL;DR: Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy, and the angular spectrum method of calculating holographic optical field is seen to have significant advantages including tight control of spurious noise components.
Abstract: Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating holographic optical field is seen to have significant advantages including tight control of spurious noise components. Holographic phase images are obtained with 0.5 μm diffraction-limited lateral resolution and largely immune from the coherent noise common in other holographic techniques. The phase profile is accurate to about 30 nm of optical thickness. Images of SKOV-3 ovarian cancer cells display intracellular and intranuclear organelles with clarity and quantitative accuracy.
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TL;DR: Digital holography is an emerging field of new paradigm in general imaging applications as discussed by the authors, and a review of a subset of the research and development activities in digital holographic microscopy techniques and applications is presented.
Abstract: Digital holography is an emerging field of new paradigm in general imaging applications. We present a review of a subset of the research and development activities in digital holography, with emphasis on microscopy techniques and applications. First, the basic results from the general theory of holography, based on the scalar diffraction theory, are summarized, and a general description of the digital holographic microscopy process is given, including quantitative phase microscopy. Several numerical diffraction methods are described and compared, and a number of representative configurations used in digital holography are described, including off-axis Fresnel, Fourier, image plane, in-line, Gabor, and phase-shifting digital holographies. Then we survey numerical techniques that give rise to unique capabilities of digital holography, including suppression of dc and twin image terms, pixel resolution control, optical phase unwrapping, aberration compensation, and others. A survey is also given of representative application areas, including biomedical microscopy, particle field holography, micrometrology, and holographic tomography, as well as some of the special techniques, such as holography of total internal reflection, optical scanning holography, digital interference holography, and heterodyne holography. The review is intended for students and new researchers interested in developing new techniques and exploring new applications of digital holography.
672 citations
Cites background or methods from "High-resolution quantitative phase-..."
...Image plane digital holography has been useful in a number of different application areas, including biological microscopy [47,103], where it is important to be able to monitor the live specimen being imaged....
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...Quantitative phase microscopy by digital holography (DH-QPM) has been applied to image various cell types, including SKOV-3 ovarian cancer cells [47,54], fibroblast cells [19], testate amoeba [180], diatom skeletons [181], and red blood cells [53]....
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TL;DR: Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the marker-free analysis of living cells.
Abstract: Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the marker-free analysis of living cells. The digital holographic feature of (subsequent) numerical focus adjustment makes possible applications for multifocus imaging. An overview of digital holographic microscopy methods is described. Applications of digital holographic microscopy are demonstrated by results obtained from livings cells and engineered surfaces.
668 citations
TL;DR: Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells.
Abstract: Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or “dry mass” of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells.
473 citations
TL;DR: A sub-pixel shifting based super-resolution algorithm is implemented to effectively recover much higher resolution digital holograms of the objects, permitting sub-micron spatial resolution to be achieved across the entire sensor chip active area.
Abstract: We demonstrate lensfree holographic microscopy on a chip to achieve approximately 0.6 microm spatial resolution corresponding to a numerical aperture of approximately 0.5 over a large field-of-view of approximately 24 mm2. By using partially coherent illumination from a large aperture (approximately 50 microm), we acquire lower resolution lensfree in-line holograms of the objects with unit fringe magnification. For each lensfree hologram, the pixel size at the sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, we implement a sub-pixel shifting based super-resolution algorithm to effectively recover much higher resolution digital holograms of the objects, permitting sub-micron spatial resolution to be achieved across the entire sensor chip active area, which is also equivalent to the imaging field-of-view (24 mm2) due to unit magnification. We demonstrate the success of this pixel super-resolution approach by imaging patterned transparent substrates, blood smear samples, as well as Caenoharbditis Elegans.
454 citations
TL;DR: In this article, a review summarizes recent advances in volumetric particle detection and 3D flow velocimetry involving holography and discusses the debilitating depth-of-focus problem, along with methods that have been implemented to circumvent it.
Abstract: The quantification of three-dimensional (3D) flow structures and particle dynamics is crucial for unveiling complex interactions in turbulent flows. This review summarizes recent advances in volumetric particle detection and 3D flow velocimetry involving holography. We introduce the fundamental principle of holography and discuss the debilitating depth-of-focus problem, along with methods that have been implemented to circumvent it. The focus of this review is on recent advances in the development of in-line digital holography in general, and digital holographic microscopy in particular. A mathematical background for the numerical reconstruction of digital holograms is followed by a summary of recently introduced 3D particle tracking and velocity measurement techniques. The review concludes with sample applications, including 3D velocity measurements that fully resolve the flow in the inner part of a turbulent boundary layer, the diffusion of oil droplets in high–Reynolds number turbulence, and predator-p...
400 citations
Cites methods from "High-resolution quantitative phase-..."
...Provided this refractive index is known, this procedure could be used to measure the thickness of a cell (e.g., Mann et al. 2005, Marquet et al. 2005)....
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References
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TL;DR: A new application of digital holography for phase-contrast imaging and optical metrology and an application to surface profilometry shows excellent agreement with contact-stylus probe measurements.
Abstract: We present a new application of digital holography for phase-contrast imaging and optical metrology. This holographic imaging technique uses a CCD camera for recording of a digital Fresnel off-axis hologram and a numerical method for hologram reconstruction. The method simultaneously provides an amplitude-contrast image and a quantitative phase-contrast image. An application to surface profilometry is presented and shows excellent agreement with contact-stylus probe measurements.
1,202 citations
TL;DR: A digital holographic microscope, in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells, is developed, based on an original numerical algorithm presented in detail elsewhere.
Abstract: We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [
Cuche
, Appl. Opt.38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.
1,135 citations
TL;DR: A new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope is presented, able to recover phase even in the presence of amplitude modulation.
Abstract: We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.
545 citations
TL;DR: In this paper, the authors applied phase-shifting digital holography to microscopy by deriving the complex amplitude of light scattered from microscopic 3D objects through a microscope objective by video camera recording, phase shifting analysis, and computer reconstruction.
Abstract: We applied phase-shifting digital holography to microscopy by deriving the complex amplitude of light scattered from microscopic three-dimensional objects through a microscope objective by video camera recording, phase-shifting analysis, and computer reconstruction. This method requires no mechanical movement and provides a flexible display and quantitative evaluation of the reconstructed images. A theory of image formation and experimental verification with specimens are described.
525 citations
TL;DR: By use of the Fourier decomposition of a low-coherence optical image field into two spatial components that can be controllably shifted in phase with respect to each other, a new high-transverse-resolution quantitative-phase microscope has been developed.
Abstract: By use of the Fourier decomposition of a low-coherence optical image field into two spatial components that can be controllably shifted in phase with respect to each other, a new high-transverse-resolution quantitative-phase microscope has been developed. The technique transforms a typical optical microscope into a quantitative-phase microscope, with high accuracy and a path-length sensitivity of lambda/5500, which is stable over several hours. The results obtained on epithelial and red blood cells demonstrate the potential of this instrument for quantitative investigation of the structure and dynamics associated with biological systems without sample preparation.
466 citations