Quantitative phase-contrast microscopy

Summary

FORCETOC Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Translucent objects, like a living human cell, absorb and scatter small amounts of light. This makes translucent objects much easier to observe in ordinary light microscopes. Such objects do, however, induce a phase shift that can be observed using a phase contrast microscope. Conventional phase contrast microscopy and related methods, such as differential interference contrast microscopy, visualize phase shifts by transforming phase shift gradients into intensity variations. These intensity variations are mixed with other intensity variations, making it difficult to extract quantitative information. Quantitative phase contrast methods are distinguished from conventional phase contrast methods in that they create a second so-called phase shif

Official source

https://en.wikipedia.org/wiki/Quantitative_phase-contrast_microscopy

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Introduction to the different contrast enhancing methods in optical microscopy. Basic hands-on experience with optical microscopes at EPFL's BioImaging and Optics Facility. How to investigate biological samples? How to obtain high quality images?

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Digital holographic reflectometry

Ahmet Ata Akatay, Tristan Colomb, Christian Depeursinge, Jonas Kühn, Nicolas Pavillon

Digital holographic microscopy (DHM) is an interferometric technique that allows real-time imaging of the entire complex optical wavefront (amplitude and phase) reflected by or transmitted through a sample. To our knowledge, only the quantitative phase is exploited to measure topography, assuming homogeneous material sample and a single reflection on the surface of the sample. In this paper, dual-wavelength DHM measurements are interpreted using a model of reflected wave propagation through a three-interfaces specimen (2 layers deposited on a semi-infinite layer), to measure simultaneously topography, layer thicknesses and refractive indices of micro- structures. We demonstrate this DHM reflectometry technique by comparing DHM and profilometer measurement of home-made SiO2/Si targets and Secondary Ion Mass Spectrometry (SIMS) sputter craters on specimen including different multiple layers.

2010

In the past decade, quantitative phase imaging gave a new dimension to optical microscopy, and the recent extension of digital holography techniques to nonlinear microscopy appears very promising, for the phase of nonlinear signal provides additional information, inaccessible to incoherent imaging schemes. Last year, we have demonstrated how the SHG phase makes possible real-time nanometric 3D-tracking of SHG emitters, such as nanoparticles (Shaffer et al., Opt. Express 18, p.17392-17403, 2010). Here, we investigate the phase of second harmonic generated by a label-free biological specimen—more precisely collagen fibers forming the connective tissue of a mouse dermis—and discuss of its interpretation. Notably, we show how the SHG phase, qualitatively acting as an indicator of phase-matching conditions, tends to indicate that second harmonic generation, in collagen, is dominated by coherent SHG scattering.

2011

Digital holographic microscopy at fundamental and second harmonic wavelengths

Christian Depeursinge, Etienne Shaffer

Optical second harmonic generation, thanks to its coherent nature, is a suitable signal for interferometric measurements, such as digital holography: a well-established imaging technique that allows recovering of the complex diffraction wavefields from which it is possible to extract both amplitude- contrast and quantitative phase images. We believe that application of digital holography to non-linear optical fields might ultimately be the key to phase- related functional imaging. In a first approach, we report here on second harmonic generation digital holographic microscopy, and present its application to (1) discrimination of nanoparticles of different nature - here polystyrene microspheres and barium titanate (BaTiO3) nanoparticles - and to (2) 3D-mapping of a distribution of BaTiO3 nanoparticles.

SPIE2009