Three-dimensional wide-field pump-probe structured illumination microscopy: erratum.

We found errors in Eqs. (5), (6), (7), (8), (23), (25), Figs. 2, 3, 4, 5, 10, and Discussion of our article "Three-dimensional wide-field pump-probe structured illumination microscopy." Here we publish the revised equations, figures, and discussion. In general, the corrections do not affect the essential conclusion and image reconstruction quality improves with these corrections.

Large population cell characterization using quantitative phase cytometer.

A major challenge in cellular analysis is the phenotypic characterization of large cell populations within a short period of time. Among various parameters for cell characterization, the cell dry mass is often used to describe cell size but is difficult to be measured directly with traditional techniques. Here, we propose an interferometric approach based on line-focused beam illumination for high-content precision dry mass measurements of adherent cells in a non-invasive fashion-we call it quantitative phase cytometry (QPC). Besides dry mass, abundant cellular morphological features such as projected area, sphericity, and phase skewness can be readily extracted from the QPC interferometric data. To validate the utility of our technique, we demonstrate characterizing a large population of ∼104 HeLa cells. Our reported QPC system is envisioned as a promising quantitative tool for label-free characterization of a large cell count at single cell resolution. © 2017 International Society for Advancement of Cytometry.

Three-dimensional wide-field pump-probe structured illumination microscopy.

We propose a new structured illumination scheme for achieving depth resolved wide-field pump-probe microscopy with sub-diffraction limit resolution. By acquiring coherent pump-probe images using a set of 3D structured light illumination patterns, a 3D super-resolution pump-probe image can be reconstructed. We derive the theoretical framework to describe the coherent image formation and reconstruction scheme for this structured illumination pump-probe imaging system and carry out numerical simulations to investigate its imaging performance. The results demonstrate a lateral resolution improvement by a factor of three and providing 0.5 µm level axial optical sectioning.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy.

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.

Pushing phase and amplitude sensitivity limits in interferometric microscopy.

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.

Surface-plasmon-coupled emission microscopy with a spiral phase plate.

The surface plasmon-coupled emission microscope provides high sensitivity for surface imaging. However, it suffers from a distorted donut-shape point-spread function (PSF). Here we report an effective yet simple method to correct for the distortion by introducing a spiral phase plate. This modification converts the donut PSF into one that is single lobed, which is preferable for imaging. The optical performance of the system is characterized and compared with previous publications. This technique provides more than twofold lateral resolution enhancement.

Wide-field extended-resolution fluorescence microscopy with standing surface-plasmon-resonance waves.

The resolution of conventional surface-plasmon-resonance (SPR) imaging has been limited by the diffraction nature of light. A wide-field extended-resolution optical imaging technique, standing-wave SPR fluorescence (SW-SPRF) microscopy, has been developed. Based on evanescent SPR standing waves, SW-SPRF provides lateral resolution approaching 100 nm and offers the advantages of significant signal enhancement and background-noise reduction. SW-SPRF has the potential for sensitive biomolecular detection, nanoscale imaging, and lithographic applications.

Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens.

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.

Investigation of the point spread function of surface plasmon-coupled emission microscopy.

Surface plasmon-coupled emission microscopy (SPCEM) was proposed as a high sensitivity technique that makes use of a thin layer of metal deposited on glass slides to efficiently excite fluorophores and to collect the emission light. However, the theoretical aspect of SPCEM imaging has not been well studied. We propose a model for SPCEM and show, through theoretical analysis and empirical results, that the point spread function of SPCEM is irregular and has an annular-like structure, significantly different from the familiar point spread function of the conventional wide-field microscopy. This result is due to the highly polarized and anisotropic emission caused by the metal layer.