Three-dimensional imaging in reflection phase microscopy with minimal axial scanning.

Reflection phase microscopy is a valuable tool for acquiring three-dimensional (3D) images of objects due to its capability of optical sectioning. The conventional method of constructing a 3D map is capturing 2D images at each depth with a mechanical scanning finer than the optical sectioning. This not only compromises sample stability but also slows down the acquisition process, imposing limitations on its practical applications. In this study, we utilized a reflection phase microscope to acquire 2D images at depth locations significantly spaced apart, far beyond the range of optical sectioning. By employing a numerical propagation, we successfully filled the information gap between the acquisition layers, and then constructed complete 3D maps of objects with substantially reduced number of axial scans. Our experimental results also demonstrated the effectiveness of this approach in enhancing imaging speed while maintaining the accuracy of the reconstructed 3D structures. This technique has the potential to improve the applicability of reflection phase microscopy in diverse fields such as bioimaging and material science.

The effect of pupil transmittance on axial resolution of reflection phase microscopy.

A reflection phase microscope (RPM) can be equipped with the capability of depth selection by employing a gating mechanism. However, it is difficult to achieve an axial resolution close to the diffraction limit in real implementation. Here, we systematically investigated the uneven interference contrast produced by pupil transmittance of the objective lens and found that it was the main cause of the practical limit that prevents the axial resolution from reaching its diffraction limit. Then we modulated the power of illumination light to obtain a uniform interference contrast over the entire pupil. Consequently, we could achieve an axial resolution fairly close to the diffraction limit set by the experimental conditions.

Full ocular biometry through dual-depth whole-eye optical coherence tomography.

We propose a new method of determining the optical axis (OA), pupillary axis (PA), and visual axis (VA) of the human eye by using dual-depth whole-eye optical coherence tomography (OCT). These axes, as well as the angles "α" between the OA and VA and "κ" between PA and VA, are important in many ophthalmologic applications, especially in refractive surgery. Whole-eye images are reconstructed based on simultaneously acquired images of the anterior segment and retina. The light from a light source is split into two orthogonal polarization components for imaging the anterior segment and retina, respectively. The OA and PA are identified based on their geometric definitions by using the anterior segment image only, while the VA is detected through accurate correlation between the two images. The feasibility of our approach was tested using a model eye and human subjects.

Reflection phase microscopy using spatio-temporal coherence of light.

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.

High-resolution, dual-depth spectral-domain optical coherence tomography with interlaced detection for whole-eye imaging.

Dual-depth spectral-domain optical coherence tomography (SD-OCT) enables high-resolution in vivo whole-eye imaging. Two orthogonally polarized beams from a source are focused simultaneously on two axial positions of the anterior segment and the retina. For the detector arm, a 1×2 ultrafast optical switch sequentially delivers two spectral interference signals to a single spectrometer, which extends the in-air axial depth range up to 9.44 mm. An off-pivot complex conjugate removal technique doubles the depth range for all anterior segment imaging. The graphics-processing-unit-based parallel signal processing algorithm supports fast two- and three-dimensional image displays. The obtained high-resolution anterior and retinal images are measured biometrically. The dual-depth SD-OCT system has an axial resolution of ∼6.4 µm in air, and the sensitivity is 91.79 dB at 150 µm from the zero-delay line.

Spectral domain optical coherence tomography with balanced detection using single line-scan camera and optical delay line.

We propose a spectral domain optical coherence tomography (SD-OCT) system that uses a single line-scan detection scheme for balanced detection. Two phase-opposed spectra, generated by two optical fiber couplers, were detected by using a spectrometer with fast optical switching. A 2.69 km optical fiber was introduced to provide a proper time delay to prevent phase errors caused by the difference in measurement time between the two opposing spectra and unstable output voltages for controlling the galvano-scanner. Hence, a phase difference of π was obtained between the spectra over the sample depth without a phase error, which improved sensitivity by approximately 6 dB compared to that of conventional SD-OCT. We directly showed and compared the OCT images before and after applying the proposed balanced detection method in a phantom and in vivo sample.