LED based large field of view off-axis quantitative phase contrast microscopy by hologram multiplexing.

In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources for large field of view quantitative phase imaging of biological samples. To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple Fresnel Biprisms is used for hologram multiplexing, enhancing the field of view and increasing the signal to noise ratio. Biprisms offers the ease of obtaining interference patterns by automatically matching the path length between the two off-axis beams. The use of low temporally coherent sources reduces the speckle noise and the cost, and the form factor of the setup. The developed technique was implemented using both visible and UV LEDs and tested on polystyrene microspheres and human erythrocytes.

Wavefront division digital holographic microscopy.

Digital holographic microscopy is the state of the art quantitative phase imaging of micro-objects including living cells. It is an ideal tool to image and quantify cell thickness profiles with nanometer thickness resolution. Digital holographic techniques usually are implemented using a two-beam setup that may be bulky and may not be field portable. Self-referencing techniques provide compact geometry but suffer from a reduction of the field of view. Here, we discuss the development of a wavefront division digital holographic microscope providing the full field of view with a compact system. The proposed approach uses a wavefront division module consisting of two lenses. The developed microscope is tested experimentally by measuring the physical and mechanical properties of red blood cells.

Wide field of view common-path lateral-shearing digital holographic interference microscope.

Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images. Self-referencing geometry is the simplest of the common-path techniques, in which a portion of the object beam itself acts as the reference, leading to compact setups using fewer optical elements. However, it has reduced field of view, and the reference may contain object information. Here, we describe the development of a common-path digital holographic microscope, employing a shearing plate and converting one of the beams into a separate reference by employing a pin-hole. The setup is as compact as self-referencing geometry, while providing field of view as wide as that of a two-beam microscope. The microscope is tested by imaging and quantifying the morphology and dynamics of human erythrocytes.

Highly stable digital holographic microscope using Sagnac interferometer.

Interferometric microscopy has grown into a very potent tool for quantitative phase imaging of biological samples. Among the interfermetric methods, microscopy by digital holography is one of the most effective techniques, especially for studying dynamics of cells. Imaging of cell fluctuations requires digital holographic setups with high temporal stability. Common path setups in which the object and the reference beams encounter the same set of optical elements provide better temporal stability compared to two-beam setups. Here, we present a compact, easy-to-implement, common path digital holographic microscope based on Sagnac interferometer geometry. The microscope is implemented using a diode laser module employing a CCD array or a webcam sensor to record holograms. The system was tested for three-dimensional imaging capability, numerical focusing ability, and temporal stability. Sub-nanometer temporal stability without external vibration isolation components was obtained in both cases. The higher temporal stability makes the microscope compatible to image cell fluctuations, which is demonstrated by imaging the oscillation of the cell membrane of human red blood cells.

Quantitative phase-contrast imaging with compact digital holographic microscope employing Lloyd's mirror.

Digital holographic microscopy (DHM) is one of the most effective techniques used for quantitative phase imaging of cells. Here we present a compact, easy to implement, portable, and very stable DHM setup employing a self-referencing Lloyd's mirror configuration. The microscope is constructed using a diode laser source and a CMOS sensor, making it cost effective. The reconstruction of recorded holograms yields the amplitude and phase information of the object. The temporal stability of the presented technique was found to be around 0.9 nm without any vibration compensation, which makes it ideal for studying cell profile changes. This aspect of the technique is demonstrated by studying membrane fluctuations of red blood cells.

High-resolution quantitative phase microscopic imaging in deep UV with phase retrieval.

High-resolution three-dimensional (3D) microscopic imaging requires the use of short wavelengths. Quantitative 3D imaging techniques, such as digital holographic microscopy, require interference between the object beam and a known reference background for the extraction of phase information. At shorter wavelengths, due to short coherence lengths, it may be difficult to implement a two-beam off-axis setup. Thus, a single-beam technique, which provides complete phase information, may be better suited for short wavelengths. This Letter describes the development of a quantitative microscopy technique at 193 nm using multiple intensity samplings and phase retrieval.

Quantitative cell imaging using single beam phase retrieval method.

Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.

Shape and deformation measurements of 3D objects using volume speckle field and phase retrieval.

Shape and deformation measurement of diffusely reflecting 3D objects are very important in many application areas, including quality control, nondestructive testing, and design. When rough objects are exposed to coherent beams, the scattered light produces speckle fields. A method to measure the shape and deformation of 3D objects from the sequential intensity measurements of volume speckle field and phase retrieval based on angular-spectrum propagation technique is described here. The shape of a convex spherical surface was measured directly from the calculated phase map, and micrometer-sized deformation induced on a metal sheet was obtained upon subtraction of the phase, corresponding to unloaded and loaded states. Results from computer simulations confirm the experiments.

Measurement of parameters of simple lenses using digital holographic interferometry and a synthetic reference wave.

The use of digital holographic intrerferometry in the testing of simple thin lenses is explored. Focal length, radius of curvature, and refractive index are the lens parameters that can be determined using this method. The digital holograms using the lens under test are recorded at various positions of the test lens using off-axis geometry. This is combined with a digitally computed plane wavefront to determine the curvature of the light beam emerging from the test lens. Focal length is the position of the test lens where a single fringe results. The radius of curvature of the test lens is also determined similarly using a long focal length lens to concentrate a collimated beam onto the test lens. The nonuniformities on the lens surface could also be found by using this method. The implementation of the method is shown by using computer simulations in the case of biconvex lenses. The method can be utilized to measure the parameters of plano-convex and concave lenses also.

Determination of refractive indices of biconvex lenses by use of a Michelson interferometer.

Measurements of lens parameters such as focal length, radius of curvature, and refractive index are important. We describe a measurement method that utilizes a Michelson interferometer to determine parameters of thin, convex lenses. The real fringe system formed by a Michelson interferometer is used to determine the focal lengths and the radii of curvature of the lenses. The refractive index of the lens material is determined from the thin-lens formula. We were able to determine the refractive indices to an accuracy as great as 99.97%. A detailed theoretical and experimental analysis is given.

Diffusivity studies of transparent liquid solutions by use of digital holographic interferometry.

Real-time digital holography is used to study the diffusion process in transparent liquid solutions. Holograms of an object diffusively reflecting through an experimental cell containing diffusing solutions are recorded at different time instances. The recording medium is a CCD chip. The holographic interference of the object at two time instances is carried out numerically in a PC and is used to determine the diffusion coefficient. Holographic interferometric fringes can be displayed on a PC monitor in near real time. The software developed for this method determines the diffusion coefficients automatically. The calculated diffusion coefficients obtained with this method matched well with literature values.

Collimation testing with optically active materials.

A novel method to test the collimation of laser beams with optically active mediums and a pair of crossed polarizers is presented. Optically active materials rotate the plane of polarization of incident plane-polarized light. A decollimated laser beam passing through such a material will experience a greater effective thickness than a collimated laser beam, resulting in greater outputs. In this method the output intensity variation is related to the amount of decollimation of the incident beam, and the method does not require any referencing or fringe analysis and is easy to implement.