Spatio-temporal optical coherence tomography provides full thickness imaging of the chorioretinal complex.

Despite the rapid development of optical imaging methods, high-resolution in vivo imaging with penetration into deeper tissue layers is still a major challenge. Optical coherence tomography (OCT) has been used successfully for non-invasive human retinal volumetric imaging in vivo, advancing the detection, diagnosis, and monitoring of various retinal diseases. However, there are important limitations of volumetric OCT imaging, especially coherent noise and the limited axial range over which high resolution images can be acquired. The limited range prevents simultaneous measurement of the retina and choroid with adequate lateral resolution. In this article, we address these limitations with a technique that we term spatio-temporal optical coherence tomography (STOC-T), which uses light with controlled spatial and temporal coherence and advanced signal processing methods. STOC-T enabled the acquisition of high-contrast and high-resolution coronal projection images of the retina and choroid at arbitrary depths.

High-Throughput Monitoring of Bacterial Cell Density in Nanoliter Droplets: Label-Free Detection of Unmodified Gram-Positive and Gram-Negative Bacteria.

Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a high-throughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.

In vivo imaging of the human cornea with high-speed and high-resolution Fourier-domain full-field optical coherence tomography.

Corneal evaluation in ophthalmology necessitates cellular-resolution and fast imaging techniques that allow for accurate diagnoses. Currently, the fastest volumetric imaging technique is Fourier-domain full-field optical coherence tomography (FD-FF-OCT), which uses a fast camera and a rapidly tunable laser source. Here, we demonstrate high-resolution, high-speed, non-contact corneal volumetric imaging in vivo with FD-FF-OCT that can acquire a single 3D volume with a voxel rate of 7.8 GHz. The spatial coherence of the laser source was suppressed to prevent it from focusing on a spot on the retina, and therefore, exceeding the maximum permissible exposure (MPE). The inherently volumetric nature of FD-FF-OCT data enabled flattening of curved corneal layers. The acquired FD-FF-OCT images revealed corneal cellular structures, such as epithelium, stroma and endothelium, as well as subbasal and mid-stromal nerves.

Digital holography with multidirectional illumination by LCoS SLM for topography measurement of high gradient reflective microstructures.

In this paper we present a method for topography measurement of high gradient reflective microstructures that overcomes the limited numerical aperture (NA) of a digital holographic (DH) system working in reflection. We consider a case when a DH system is unable to register the light reflected from the full sample area due to insufficient NA. To overcome this problem, we propose digital holography in a microscope configuration with an afocal imaging system and a modified object arm in the measurement setup. The proposed modification includes application of a spatial light modulator (SLM) based on liquid crystal on silicon (LCoS) technology for multidirectional plane wave illumination. The variable off-axis illumination enables characterization of the sample regions that cannot be imaged by the limited NA of a classical DH system utilizing on-axis illumination. In the proposed method, the final object topography is merged from a set of captured object waves corresponding to various illumination directions using a novel automatic algorithm. The proposed technique is experimentally validated by full-field measurement of a silicon mold with a high gradient of shape.

Absolute shape measurement of high NA focusing microobjects in digital holographic microscope with arbitrary spherical wave illumination.

In this paper a new high NA shape measurement technique working with an arbitrary spherical wave illumination is presented. The main contribution of this work are formulas, derived from exact reflection and refraction laws for both the reflection and the transmission configurations, which enable accurate shape calculations in systems with an arbitrary location of the illuminating point source. The proposed algorithms permit measurement of multiple samples of arbitrary shapes using a single hologram. An accuracy of this method is confirmed with numerical simulations, which show superiority of this approach over a standard procedure utilizing paraxial approximation. The method is validated experimentally using a reflective measurement of a microlens topography, whose NA in reflection is 0.7. Furthermore, a new measurement configuration is presented that extends the capabilities of transmission systems for characterization of high gradient shapes.

High-precision topography measurement through accurate in-focus plane detection with hybrid digital holographic microscope and white light interferometer module.

High-precision topography measurement of micro-objects using interferometric and holographic techniques can be realized provided that the in-focus plane of an imaging system is very accurately determined. Therefore, in this paper we propose an accurate technique for in-focus plane determination, which is based on coherent and incoherent light. The proposed method consists of two major steps. First, a calibration of the imaging system with an amplitude object is performed with a common autofocusing method using coherent illumination, which allows for accurate localization of the in-focus plane position. In the second step, the position of the detected in-focus plane with respect to the imaging system is measured with white light interferometry. The obtained distance is used to accurately adjust a sample with the precision required for the measurement. The experimental validation of the proposed method is given for measurement of high-numerical-aperture microlenses with subwavelength accuracy.

Degeneration of Fraunhofer diffraction on bacterial colonies due to their light focusing properties examined in the digital holographic microscope system.

The degeneration of Fraunhofer diffraction conditions in the optical system with converging spherical wave illumination for bacteria species identification based on diffraction patterns is analyzed by digital holographic methods. The obtained results have shown that the colonies of analyzed bacteria species act as biological lenses with the time-dependent light focusing properties, which are characterized and monitored by means of phase retrieval from sequentially captured digital holograms. This significantly affects the location of Fraunhofer patterns observation plane, which is continuously shifted across optical axis in time.

Digital holographic microscope for measurement of high gradient deep topography object based on superresolution concept.

In this Letter, a novel concept based on superresolution technique that enables the measurement of high gradient and deep topography objects using digital holographic (DH) microscopy is introduced. The major problem of DH systems is limited NA that prohibits the metrological characterization of object features of high frequencies. The proposed technique has the ability to extend spatial frequency spectrum of the measured topography by applying multidirectional plane wave illumination, which is experimentally realized with a grating. The technique recovers sample topography from the set of object waves with different object spectra that are converted into a set of topographies by using an algorithm which takes into account refraction. Application of this novel approach is experimentally validated by characterization of high gradient topography objects with maximum angle of tangent 65°.

High-numerical-aperture microlens shape measurement with digital holographic microscopy.

In this Letter, we introduce an algorithm that overcomes limitations in shape measurement by holographic microscopic methods in cases of micro-optical elements with high NA, such as microlenses. The presented algorithm provides a simple method for shape reconstruction from interferometrically measured phase. The algorithm is based on the analysis of local ray transition through the measured object. We develop algorithms for holographic configurations working in transmission and reflection. The accuracy of the developed algorithms is proved by experiments and extensive simulations. We present an experiment in a holographic Mach-Zehnder configuration where we have measured and successfully reconstructed the height distribution of spherical and cylindrical microlenses with NA up to 0.3.