Quantitative DIC microscopy using an off-axis self-interference approach

G. R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Optics Letters (Impact Factor: 3.29). 07/2010; 35(14):2370-2. DOI: 10.1364/OL.35.002370
Source: PubMed


Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

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    • "This is very well know in the field of classical image processing where appropriate kernels are adopted for performing spatial derivative with these aims. Actually, research in PCI field is basically divided in two branches: (i) direct recovering of the quantitative PCI map by interferometric and/or holographic methods [4–6] or (ii) QPM computation starting from classical DIC experimental analysis [7,8]. Several groups are still working on recovering quantitative information from DIC with the two following motivations. "
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    ABSTRACT: Differential image contrast (DIC), through the numerical managing and manipulation of complex wavefronts obtained by digital holography (DH), is investigated. We name the approach Dynamical Differential Holographic Image Contrast (DDHIC). DDHIC dispenses from special optics and/or complex setup configurations with moveable components, as usually occurs in classical DIC, that is not well-suited for investigating objects experiencing dynamic evolution during the measurement. In fact, the technique presented here, is useful for floating samples since it allows, from a single recording, to set a posteriori the best conditions for DIC imaging in conjunction with the numerical focusing feature of DH. By DDHIC, the movies can be easily built-up to offering dynamic representation of phase-contrast along all directions, thus improving the visualization. Furthermore, the dynamic representation is useful for making the proper choice of other key parameters of DIC such as the amount of shear and the bias, with the aim to optimize the visualized phase-contrast imaging as favorite representation for bio-scientists. Investigation is performed on various biological samples.
    Full-text · Article · Feb 2011 · Biomedical Optics Express
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    • "It is hard to disentangle the contribution of biomolecule spatial distribution and laser speckle noise with the current setup. This problem could potentially be alleviated with phase measurement based on a spatially incoherent source which has much less speckle noise [14]. "
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    ABSTRACT: Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples.
    Full-text · Article · Sep 2010 · Biomedical Optics Express
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    ABSTRACT: Microscopy is one of the main research and application areas of digital holography. Direct access to the phase as well as amplitude profiles makes quantitative phase microscopy by digital holography (DH-QPM) particularly powerful and versatile. A number of techniques of DH are developed especially for microscopy imaging and these are made possible because of the particular imaging characteristics of DH. Digital holographic and interferometric principles are the basis of many other techniques of QPM with novel capabilities. A survey is given of the wide and very active field of research in DHM techniques and applications. We begin with a brief background on optical microscopy.
    No preview · Chapter · Jan 2011
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