RESUMEN
Many disease states are associated with cellular biomechanical changes as markers. Label-free phase microscopes are used to quantify thermally driven interface fluctuations, which allow the deduction of important cellular rheological properties. Here, the spatio-temporal coherence of light was used to implement a high-speed reflection phase microscope with superior depth selectivity and higher phase sensitivity. Nanometric scale motion of cytoplasmic structures can be visualized with fine details and three-dimensional resolution. Specifically, the spontaneous fluctuation occurring on the nuclear membrane of a living cell was observed at video rate. By converting the reflection phase into displacement, the sensitivity in quantifying nuclear membrane fluctuation was found to be about one nanometer. A reflection phase microscope can potentially elucidate biomechanical mechanisms of pathological and physiological processes.
RESUMEN
Interferometric microscopy (IM) can provide complex field information of the biological samples with high spatial and temporal resolution with virtually no photodamage. Measuring wavelength-dependent information in particular has a wide range of applications from cell and tissue refractometry to the cellular biophysical measurements. IM measurements at multiple wavelengths are typically associated with a loss in temporal resolution, field of view, stability, sensitivity, and may involve using expensive equipment such as tunable filters or spatial light modulators. Here, we present a novel and simple design for an interferometric microscope that provides single-shot off-axis interferometric measurements at two wavelengths by encoding the two spectral images at two orthogonal spatial frequencies that allows clean separation of information in the Fourier space with no resolution loss. We demonstrated accurate simultaneous quantification of polystyrene bead refractive indices at two wavelengths.
Asunto(s)
Aumento de la Imagen/métodos , Microscopía de Interferencia/tendencias , Refractometría/métodos , Diseño de Equipo , Luz , Microscopía de Interferencia/métodosRESUMEN
Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 µm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM. In this paper, we develop such a model based on the diffraction tomography theory and the speckle statistics. Using this model, we calculate the system response function, which is used to further obtain the depth resolution limit in reflection-mode DSIM. Theoretically calculated depth resolution limit is in an excellent agreement with experiment results. We envision that our physical model will not only help in understanding the imaging process in DSIM, but also enable better designing such systems for depth-resolved measurements in biological cells and tissues.
RESUMEN
Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/farmacología , Eritrocitos/efectos de los fármacos , Hidroxiurea/farmacología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/patología , Biomarcadores/sangre , Recuento de Células Sanguíneas , Transfusión Sanguínea , Deformación Eritrocítica , Eritrocitos/metabolismo , Eritrocitos/patología , Hemoglobina Fetal , Humanos , Microscopía de Interferencia , Oxígeno/farmacologíaRESUMEN
Sensitivity of the amplitude and phase measurements in interferometric microscopy is influenced by factors such as instrument design and environmental interferences. Through development of a theoretical framework followed by experimental validation, we show photon shot noise is often the limiting factor in interferometric microscopy measurements. Thereafter, we demonstrate how a state-of-the-art camera with million-level electrons full well capacity can significantly reduce shot noise contribution resulting in a stability of optical path length down to a few picometers even in a near-common-path interferometer.
RESUMEN
We have developed an interferometric optical microscope that provides three-dimensional refractive index map of a specimen by scanning the color of three illumination beams. Our design of the interferometer allows for simultaneous measurement of the scattered fields (both amplitude and phase) of such a complex input beam. By obviating the need for mechanical scanning of the illumination beam or detection objective lens; the proposed method can increase the speed of the optical tomography by orders of magnitude. We demonstrate our method using polystyrene beads of known refractive index value and live cells.
Asunto(s)
Tomografía Óptica/métodos , Animales , Color , Análisis de Fourier , Células Madre Hematopoyéticas/citología , Humanos , Imagenología Tridimensional , Microscopía , RefractometríaRESUMEN
A simple and practical method to measure three-dimensional (3-D) refractive index (RI) distributions of biological cells is presented. A common-path self-reference interferometry consisting of a compact set of polarizers is attached to a conventional inverted microscope equipped with a beam scanning unit, which can precisely measure multiple 2-D holograms of a sample with high phase stability for various illumination angles, from which accurate 3-D optical diffraction tomograms of the sample can be reconstructed. 3-D RI tomograms of nonbiological samples such as polystyrene microspheres, as well as biological samples including human red blood cells and breast cancer cells, are presented.
Asunto(s)
Tomografía Óptica/métodos , Línea Celular Tumoral , Eritrocitos/citología , Humanos , Microscopía , PoliestirenosRESUMEN
We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 µm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells.