Most reported photoacoustic ocular imaging work to date uses small animals, such as mice and rats, the eyeball sizes of which are less than one-third of those of humans, posing challenges for clinical translation. Here we developed a novel integrated photoacoustic microscopy (PAM) and optical coherence tomography (OCT) system for dual-modality chorioretinal imaging of larger animals, such as rabbits. The system has quantified lateral resolutions of 4.1 µm (PAM) and 3.8 µm (OCT), and axial resolutions of 37.0 µm (PAM) and 4.0 µm (OCT) at the focal plane of the objective. Experimental results in living rabbits demonstrate that the PAM can noninvasively visualize individual depth-resolved retinal and choroidal vessels using a laser exposure dose of ~80 nJ below the American National Standards Institute (ANSI) safety limit 160 nJ at 570 nm; and the OCT can finely distinguish different retinal layers, the choroid, and the sclera. This reported work may be a major step forward in clinical translation of the technology.

Single molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive-index-mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here, we show a method to obtain accurate, depth dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane.

Abstrcat:We report an automated optically sectioning fluorescence lifetime imaging (FLIM) multiwell plate reader for high content analysis (HCA) in drug discovery and accelerated research in cell biology. The system utilizes a Nipkow disc confocal microscope and performs unsupervised FLIM with autofocus, automatic setting of acquisition parameters and automated localisation of cells in the field of view. We demonstrate its applications to test dye solutions, fixed and live cells and FLIM-FRET.Fluorescence lifetime imaging (FLIM) is a powerful and robust tool, able to contrast different chemical species and sense variations in fluorophore micro-environments [1]. In general, it is a robust quantitative imaging modality that is insensitive to artefacts arising from, e.g. fluorophore concentration, scattering, inner filter effects, etc, that can compromise intensity measurements. FLIM is widely applied to cell biology, particularly to study protein-protein interactions using Forster Resonance Energy Transfer (FRET) [2], as well as to read-out fluorescence-based sensors e.g. di-4-ANEPPDHQ to probe lipid order in the cellular membrane [3], and to provide labelfree contrast in biological tissue, e.g. [4]. These capabilities have many potential applications for high content analysis (HCA) but, until recently, a major obstacle to the application of FLIM in this context has been the speed of data acquisition. For cell biology experiments, the most commonly used commercial FLIM systems are based on time correlated single photon counting (TCSPC) implemented in a laser scanning confocal or multiphoton microscope. While this approach has been combined with a multiwell plate reader [5], this instrument did not acquire images but delivered a single lifetime measurement per well. The imaging speed of TCSPC FLIM is limited by the constraints of single photon counting detection and by the nonlinear photobleaching and photodamage that ensues as the power of the scanning laser beam is increased. Wide-field FLIM achieves much faster imaging rates with lower photobleaching due to the parallel pixel interrogation. An automated instrument for unsupervised FLIM was recently reported exploiting frequency domain fluorescence lifetime measurements, which provided an elegant demonstration of the application to FRET and the new opportunities afforded by statistical analysis of such FLIM array data [6]. This system did not, however, provide optical sectioning, which is important for quantitative imaging and is inherent in laser scanning confocal/multiphoton

Single-walled carbon nanotubes (SWCNTs) illuminated by white light should appear colored due to resonance Rayleigh scattering. However, true-color imaging of SWCNTs on substrates has not been reported, because of the extremely low scattering intensity of SWCNTs and the strong substrate scattering. Here we show that Rayleigh scattering can be greatly enhanced by the interface dipole enhancement effect. Consequently colorful SWCNTs on substrates can be directly imaged under an optical microscope by wide field supercontinuum laser illumination, which facilitates high throughput chirality assignment of individual SWCNTs. This approach, termed "Rayleigh imaging microscopy", is not restricted to SWCNTs, but widely applicable to a variety of nanomaterials, which enables the colorful nanoworld to be explored under optical microscopes.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.