Dual plane in-line digital holographic microscopy

Department of Physics, University of Massachusetts, Boston, Massachusetts 02125, USA.
Optics Letters (Impact Factor: 3.29). 10/2010; 35(20):3426-8. DOI: 10.1364/OL.35.003426
Source: PubMed


We report a dual plane in-line digital holographic microscopy technique that exploits the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. Two interferograms are recorded at different planes to eliminate the conjugate image. The experimental results demonstrate successful reconstruction of phase objects as well as of amplitude objects. The two interferograms can be recorded simultaneously, using two CCD or CMOS sensors, in order to increase the acquisition rate. This enhanced acquisition rate, together with the improved reconstruction capability of the proposed method, may find applications in biomedical research for visualization of rapid dynamic processes at the cellular level.

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    • "Hence a single interferogram is suffice to reconstruct the object information, ideal for studying dynamic process such as live cells in growth media. In-line DHM like phase-shifting DHM the angle between the object and the reference beam is set to zero [40] [53] [54] [55] [56] [57] [58] [59]. It is worth noting that while other advanced optical imaging techniques have also demonstrated great utility in similar biological models [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70], this DHM approach requires on relatively low cost and widely available optical components. "
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    ABSTRACT: While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.
    Proceedings of SPIE - The International Society for Optical Engineering 02/2014; 8947. DOI:10.1117/12.2040515 · 0.20 Impact Factor
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    • "Each additional lensfree diffraction measurement captured at a different object height brings a new set of equations to help us solve this underdetermined phase recovery problem without the use of any object-support constraint or spatial masks. In the literature, there are various methods that one can use to retrieve the lost phase information from two or more diffraction intensity measurements, such as transport-of-intensity equation (TIE) based methods [‎40, ‎41, ‎57, ‎58], iterative methods which use these diffraction measurements as successive amplitude constraints [‎36, ‎59] and other non-iterative methods [‎60, ‎61]. Furthermore, multiple defocused images are also utilized in phase diversity methods, where phase aberrations of incoherent imaging systems can be characterized [‎62-‎64]. "
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    ABSTRACT: Lensfree in-line holographic microscopy offers sub-micron resolution over a large field-of-view (e.g., ~24 mm2) with a cost-effective and compact design suitable for field use. However, it is limited to relatively low-density samples. To mitigate this limitation, we demonstrate an on-chip imaging approach based on pixel super-resolution and phase recovery, which iterates among multiple lensfree intensity measurements, each having a slightly different sample-to-sensor distance. By digitally aligning and registering these lensfree intensity measurements, phase and amplitude images of dense and connected specimens can be iteratively reconstructed over a large field-of-view of ~24 mm2 without the use of any spatial masks. We demonstrate the success of this multi-height in-line holographic approach by imaging dense Papanicolaou smears (i.e., Pap smears) and blood samples.
    Optics Express 01/2012; 20(3):3129-43. DOI:10.1364/OE.20.003129 · 3.49 Impact Factor
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    ABSTRACT: Microorganisms, cells and thin tissue sections are transparent and not visible to view in ordinary microscope. Techniques such as phase contrast and Normarski/Differential interference contrast microscopy transform the phase variation information into intensity distribution to reveal the details of internal structures. Similarly fluorescence microscope uses intrinsic or extrinsic chromophores to reveal specific and hidden details. Advances achieved in recent years have greatly improved the versatility of microscopes to obtain more insightful information about different physiological functions that occur at cellular level. Understanding the cell response, involving both structural and functional changes within the cell, dictates ability to image cell structure and function at the same time. We report a novel optical Fourier phase contrast multimodal optical microscopy technique for real time display of phase and fluorescence features of biological specimens at the same time. It combines the principles of (a) Fourier phase contrast microscopy which exploits monochromaticity, intensity and phase coherence of the laser beam via optical Fourier transform and photoinduced birefringence of dye doped liquid crystal for phase contrast imaging, and (b) common-path multimodal optical microscopy for co-registered imaging of phase and fluorescence features of biological specimens in real time using a single optical path, single light source, and single camera with no requirement of image registration. Further the instrument also enables co-registered imaging of fluorescence and spatial filtering facilitating simultaneous display of structural and functional information. This comprehensive microscope has the capability of simultaneously providing both structural and functional information in a streamlined simplified design and may find applications in highthroughput screening and automated microscopy.
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