Fast topographic optical imaging using encoded search focal scan.

A central quest in optics is to rapidly extract quantitative information from a sample. Existing topographical imaging tools allow non-contact and three-dimensional measurements at the micro and nanoscales and are essential in applications including precision engineering and optical quality control. However, these techniques involve acquiring a focal stack of images, a time-consuming process that prevents measurement of moving samples. Here, we propose a method for increasing the speed of topographic imaging by orders of magnitude. Our approach involves collecting a reduced set of images, each integrated during the full focal scan, whilst the illumination is synchronously modulated during exposure. By properly designing the modulation sequence for each image, unambiguous reconstruction of the object height map is achieved using far fewer images than conventional methods. We describe the theoretical foundations of our technique, characterise its performance, and demonstrate sub-micrometric topographic imaging over 100 µm range of static and dynamic systems at rates as high as 67 topographies per second, limited by the camera frame rate. The high speed of the technique and its ease of implementation could enable a paradigm shift in optical metrology, allowing the real-time characterisation of large or rapidly moving samples.

Single-shot time-folded fluorescence lifetime imaging.

Fluorescence lifetime imaging is an important tool in bioimaging that allows one to detect subtle changes in cell dynamics and their environment. Most time-domain approaches currently involve scanning a single illumination point across the sample, which can make imaging dynamic scenes challenging, while single-shot "rapid lifetime determination" can suffer from large uncertainties when the lifetime is not appropriately sampled. Here, we propose a time-folded fluorescence lifetime imaging microscopy (TFFLIM) approach, whereby a time-folding cavity provides multiple spatially sheared replicas of the lifetime, each shifted temporally with respect to a fixed time gate. This provides a robust, single-shot FLIM approach that we experimentally validate across a broad lifetime range on fluorescent beads and Convallaria samples.

Single-shot optical surface profiling using extended depth of field 3D microscopy.

The measurement of three-dimensional samples at high speed is essential for many applications, either due to the requirement for measuring samples that change fast over time, or due to the requirement of reducing the scanning time, and therefore inspection cost, in industrial environments. Conventionally, the measurement of surface topographies at high resolution typically requires an axial scanning of the sample. We report the implementation of a technique able to reconstruct surface topographies at high resolution, only from the acquisition of a single camera shot, dropping the need to perform an axial scan. A system prototype is reported and assessed as an ultra-fast optical surface profiler. We propose robust calibration and operation methods and algorithms to reconstruct surface topographies of optically-rough samples, and compare the experimental results with a reference confocal optical profiler.

Multi-aperture imaging for flat cameras.

Imaging with high angular resolution requires large apertures and long focal lengths. This has prevented the integration of telephoto lenses into thin devices such as modern mobile phones. We report a camera module employing multiple rotated rectangular apertures and folding of the optical system into the plane of the camera, enabling an order-of-magnitude reduction in depth compared to traditional telephoto lenses. Multiple images are fused in the frequency domain to yield a single high-resolution image equivalent to that yielded by a single circular aperture. The diameter of this equivalent aperture may be several times wider than the depth of the camera module. We propose two architectures and present illustrative optical designs to demonstrate the concept. Simulations of raytraced image acquisition and computational image reconstruction demonstrate the potential for high-quality, high-resolution imaging from thin, flat lens modules.

Publisher Correction: Holistic Monte-Carlo optical modelling of biological imaging.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Precise 3D particle localization over large axial ranges using secondary astigmatism.

We propose an analytical pupil phase function employing cropped secondary astigmatism for extended-depth nanoscale 3D-localization microscopy. The function provides high localization precision in all three dimensions, which can be maintained over extended axial ranges, customizable up to two orders of magnitude relative to the conventional, diffraction-limited imaging. This enables, for example, capturing nanoscale dynamics within a whole cell. The flexibility and simplicity in the implementation of the proposed phase function make its adoption in localization-based microscopy attractive. We demonstrate and validate its application to real-time imaging of 3D fluid flow over a depth of 40 µm with a numerical aperture of 0.8.

Measurement of absorption in scattering media using objective laser speckle: application to blood oximetry.

Multi-spectral imaging enables non-invasive sensing of chemical concentrations in biological tissue based on measurement of optical absorption, but invariably in the presence of high levels of scatter. Absorption is normally inferred from measurement of contrast of biological features, such as the vasculature, and so accuracy is degraded by the poorly characterized modulation-transfer function of the imaging optics and overlying tissue. We report how experimental characterization of the spectral variation of the tissue point-spread function and associated objective speckle pattern can be used to characterize the absorption spectrum and chromophore concentration, with a particular emphasis on determination of the ratio of oxygenated to deoxygenated hemoglobin within blood. Absorption measurements are determined purely by the geometry of the experiment, without degradation due to optical aberrations and associated light scatter. The technique offers enhanced and low-cost determination of in vitro or in vivo chromophore characterizations, including blood-gas analysis.

Holistic Monte-Carlo optical modelling of biological imaging.

The invention and advancement of biological microscopy depends critically on an ability to accurately simulate imaging of complex biological structures embedded within complex scattering media. Unfortunately no technique exists for rigorous simulation of the complete imaging process, including the source, instrument, sample and detector. Monte-Carlo modelling is the gold standard for the modelling of light propagation in tissue, but is somewhat laborious to implement and does not incorporate the rejection of scattered light by the microscope. On the other hand microscopes may be rigorously and rapidly modelled using commercial ray-tracing software, but excluding the interaction with the biological sample. We report a hybrid Monte-Carlo optical ray-tracing technique for modelling of complete imaging systems of arbitrary complexity. We make the software available to enable user-friendly and rigorous virtual prototyping of biological microscopy of arbitrary complexity involving light scattering, fluorescence, polarised light propagation, diffraction and coherence. Examples are presented for the modelling and optimisation of representative imaging of neural cells using light-sheet and micro-endoscopic fluorescence microscopy and imaging of retinal vasculature using confocal and non-confocal scanning-laser ophthalmoscopes.

Computational localization microscopy with extended axial range.

A new single-aperture 3D particle-localization and tracking technique is presented that demonstrates an increase in depth range by more than an order of magnitude without compromising optical resolution and throughput. We exploit the extended depth range and depth-dependent translation of an Airy-beam PSF for 3D localization over an extended volume in a single snapshot. The technique is applicable to all bright-field and fluorescence modalities for particle localization and tracking, ranging from super-resolution microscopy through to the tracking of fluorescent beads and endogenous particles within cells. We demonstrate and validate its application to real-time 3D velocity imaging of fluid flow in capillaries using fluorescent tracer beads. An axial localization precision of 50 nm was obtained over a depth range of 120µm using a 0.4NA, 20× microscope objective. We believe this to be the highest ratio of axial range-to-precision reported to date.

Combined high contrast and wide field of view in the scanning laser ophthalmoscope through dual detection of light paths.

We demonstrate a multimode detection system in a scanning laser ophthalmoscope (SLO) that enables simultaneous operation in confocal, indirect, and direct modes to permit an agile trade between image contrast and optical sensitivity across the retinal field of view to optimize the overall imaging performance, enabling increased contrast in very wide-field operation. We demonstrate the method on a wide-field SLO employing a hybrid pinhole at its image plane, to yield a twofold increase in vasculature contrast in the central retina compared to its conventional direct mode while retaining high-quality imaging across a wide field of the retina, of up to 200 deg and 20 µm on-axis resolution.

Multi-aperture foveated imaging.

Foveated imaging, such as that evolved by biological systems to provide high angular resolution with a reduced space-bandwidth product, also offers advantages for man-made task-specific imaging. Foveated imaging systems using exclusively optical distortion are complex, bulky, and high cost, however. We demonstrate foveated imaging using a planar array of identical cameras combined with a prism array and superresolution reconstruction of a mosaicked image with a foveal variation in angular resolution of 5.9:1 and a quadrupling of the field of view. The combination of low-cost, mass-produced cameras and optics with computational image recovery offers enhanced capability of achieving large foveal ratios from compact, low-cost imaging systems.

Compact multi-aperture imaging with high angular resolution.

Previous reports have demonstrated that it is possible to emulate the imaging function of a single conventional lens with an N×N array of identical lenslets to provide an N-fold reduction in imaging-system track length. This approach limits the application to low-resolution imaging. We highlight how using an array of dissimilar lenslets, with an array width that can be much wider than the detector array, high-resolution super-resolved imaging is possible. We illustrate this approach with a ray-traced design and optimization of a long-wave infrared system employing a 3×3 array of freeform lenslets to provide a fourfold reduction in track length compared to a baseline system. Simulations of image recovery show that recovered image quality is comparable to that of the baseline system.

Super-resolution imaging using a camera array.

The angular resolution of many commercial imaging systems is limited, not by diffraction or optical aberrations, but by pixilation effects. Multiaperture imaging has previously demonstrated the potential for super-resolution (SR) imaging using a lenslet array and single detector array. We describe the practical demonstration of SR imaging using an array of 25 independent commercial-off-the-shelf cameras. This technique demonstrates the potential for increasing the angular resolution toward the diffraction limit, but without the limit on angular resolution imposed by the use of a single detector array.