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... Visual identification of malarial red blood cells (RBCs) may be unreliable if there is a lack of sufficiently trained technicians plus poor quality microscopes and reagents used for staining the blood smears. Non-invasive techniques based on interferometry [4]–[16], such as digital holographic microscopy [4]–[11] and optical coherence microscopy [12], [13], provide ...
Malaria is one of the most widespread diseases, particularly in Asia and Africa. Correct diagnosis of malaria is necessary for its proper treatment. A compact automated tool for malaria identification will greatly benefit healthcare professionals in these regions. We propose a method that has the potential to automatically detect malaria-infected red blood cells (RBCs). This method combines the simplicity and robustness of lateral shearing interferometry with the flexibility of statistical methods to achieve the classification of diseased RBCs. Shearing interferograms generated using a glass plate in a common path setup were Fourier analyzed to retrieve the gradient phase and amplitude information of the cell. Then, multiple features based on the complex amplitude information of the cells are measured automatically and used to differentiate healthy and malaria-infected cells. Multivariate statistical inference algorithm of the experimental data shows that there is a difference between the populations of healthy and malaria-infected RBCs by using the measured RBC features.
In the search for higher resolution, modern day imaging systems frequently employ objective lenses with a high numerical aperture. Propagation of light through such lenses introduces a spatial variation in the polarisation across the beam profile, whilst the inherently large propagation angles also necessitates inclusion of additional transverse and axial electric field components in modelling. A full treatment of polarisation effects including such considerations has implications at all stages in the image formation process, namely; illumination, scattering from the sample, imaging and detection. This tutorial review considers each stage in turn and details the theories required for rigorous modelling and analysis. In particular a generalisation of the well known Jones calculus and ray tracing methods are shown to conveniently and accurately allow rigorous studies of high numerical aperture confocal and conventional polarised light microscopes, imaging samples of arbitrary complexity. Generalisation of the illumination to partially coherent, partially polarised systems is also briefly given. Whilst rigorous modelling techniques can prove time consuming a number of simplifications and approximations can be adopted, allowing computational gains to be achieved. Discussion in this vein is hence also presented.
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.
In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc.
We study the effect of primary aberrations on the 3-D polarization of the electric field in a focused lowest order radially polarized beam. A full vector diffraction treatment of the focused beams is used. Attention is given to the effects of primary spherical, astigmatic, and comatic aberrations on the local polarization, Strehl ratio, and aberration induced degradation of the longitudinal field at focus.
Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.
The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.
We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spectrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.