Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror.

This paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a high numerical aperture (NA) objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40×/0.80 NA water-immersion objective through a defocus range of -50-50 µm at 26.3 sweeps s-1. We achieved imaging with a mean Strehl metric of >0.6 over a field of view in the sample of 200 × 200 µm2 over a defocus range of 77 µm. We describe an optimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associated with the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used in light-sheet fluorescence microscopes to achieve video-rate volumetric imaging.

Multivariate analysis of Brillouin imaging data by supervised and unsupervised learning.

Brillouin imaging relies on the reliable extraction of subtle spectral information from hyperspectral datasets. To date, the mainstream practice has been to use line fitting of spectral features to retrieve the average peak shift and linewidth parameters. Good results, however, depend heavily on sufficient signal-to-noise ratio and may not be applicable in complex samples that consist of spectral mixtures. In this work, we thus propose the use of various multivariate algorithms that can be used to perform supervised or unsupervised analysis of the hyperspectral data, with which we explore advanced image analysis applications, namely unmixing, classification and segmentation in a phantom and live cells. The resulting images are shown to provide more contrast and detail, and obtained on a timescale ∼102 faster than fitting. The estimated spectral parameters are consistent with those calculated from pure fitting.

Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure.

We report a flexible light-sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination-detection modes: the first uses angle-dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video-rate volumetric imaging; the second combines digitally-scanned light-sheet illumination with an axially-swept light-sheet waist and stage-scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination-detection mode achieves dual spectral-channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time-lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination-detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t-tubule network in a fixed cardiomyocyte cell.

Detection of proteoglycan loss from articular cartilage using Brillouin microscopy, with applications to osteoarthritis.

The degeneration of articular cartilage (AC) occurs in osteoarthritis (OA), which is a leading cause of pain and disability in middle-aged and older people. The early disease-related changes in cartilage extra-cellular matrix (ECM) start with depletion of proteoglycan (PG), leading to an increase in tissue hydration and permeability. These early compositional changes are small (<10%) and hence difficult to register with conventional non-invasive imaging technologies (magnetic resonance and ultrasound imaging). Here we apply Brillouin microscopy for detecting changes in the mechanical properties and composition of porcine AC. OA-like degradation is mimicked by enzymatic tissue digestion, and we compare Brillouin microscopy measurements against histological staining of PG depletion over varying digestion times and enzyme concentrations. The non-destructive nature of Brillouin imaging technology opens new avenues for creating minimally invasive arthroscopic devices for OA diagnostics and therapeutic monitoring.

Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation.

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue's mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.

Semi-random multicore fibre design for adaptive multiphoton endoscopy.

This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image - measured as the half-maximum area of the two-photon point spread function divided by the area imaged - to be ~3200.

In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation.

This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to approximately 65 µm s-1 . Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.

Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection.

This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.

Assessing corneal biomechanics with Brillouin spectro-microscopy.

A new Brillouin spectro-microscope was designed and built to investigate the mechanical properties of bovine and human corneas. This instrument integrates a single-stage virtually imaged phased array spectrometer with a novel adaptive-optics interferometric filter to achieve unprecedented rejection of the elastic background signal. As a result, highly-resolved, reproducible data from both thin and thick collagen-based materials were obtained. In particular, this technique is capable of rigorously measuring the relative stiffness of different areas of human corneas, thus providing a true non-contact method to characterise the fundamental mechanical features of both live and fixed biological tissue samples.

Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre.

Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.3 dB m(-1) over the range 600-850 nm and the GVD was -180 ± 70 fs(2)  m(-1) at 800 nm. Using an average fibre output power of ∼20 mW and pulse repetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmitted through a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersion compensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within 10% of low power values up to the maximum fibre output power achievable with the laser system used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupled to a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengths from 740 nm to 860 nm without any need for adjustments to the set-up.

Quantification of plaque stiffness by Brillouin microscopy in experimental thin cap fibroatheroma.

Plaques vulnerable to rupture are characterized by a thin and stiff fibrous cap overlaying a soft lipid-rich necrotic core. The ability to measure local plaque stiffness directly to quantify plaque stress and predict rupture potential would be very attractive, but no current technology does so. This study seeks to validate the use of Brillouin microscopy to measure the Brillouin frequency shift, which is related to stiffness, within vulnerable plaques. The left carotid artery of an ApoE(-/-)mouse was instrumented with a cuff that induced vulnerable plaque development in nine weeks. Adjacent histological sections from the instrumented and control arteries were stained for either lipids or collagen content, or imaged with confocal Brillouin microscopy. Mean Brillouin frequency shift was 15.79 ± 0.09 GHz in the plaque compared with 16.24 ± 0.15 (p < 0.002) and 17.16 ± 0.56 GHz (p < 0.002) in the media of the diseased and control vessel sections, respectively. In addition, frequency shift exhibited a strong inverse correlation with lipid area of -0.67 ± 0.06 (p < 0.01) and strong direct correlation with collagen area of 0.71 ± 0.15 (p < 0.05). This is the first study, to the best of our knowledge, to apply Brillouin spectroscopy to quantify atherosclerotic plaque stiffness, which motivates combining this technology with intravascular imaging to improve detection of vulnerable plaques in patients.

Fibre-coupled multiphoton microscope with adaptive motion compensation.

To address the challenge of sample motion during in vivo imaging, we present a fibre-coupled multiphoton microscope with active axial motion compensation. The position of the sample surface is measured using optical coherence tomography and fed back to a piezo actuator that adjusts the axial location of the objective to compensate for sample motion. We characterise the system's performance and demonstrate that it can compensate for axial sample velocities up to 700 µm/s. Finally we illustrate the impact of motion compensation when imaging multiphoton excited autofluorescence in ex vivo mouse skin.

Investigation of the confocal wavefront sensor and its application to biological microscopy.

Wavefront sensing in the presence of background light sources is complicated by the need to restrict the effective depth of field of the wavefront sensor. This problem is particularly significant in direct wavefront sensing adaptive optic (AO) schemes for correcting imaging aberrations in biological microscopy. In this paper we investigate how a confocal pinhole can be used to reject out of focus light whilst still allowing effective wavefront sensing. Using a scaled set of phase screens with statistical properties derived from measurements of wavefront aberrations induced by C. elegans specimens, we investigate and quantify how the size of the pinhole and the aberration amplitude affect the transmitted wavefront. We suggest a lower bound for the pinhole size for a given aberration strength and quantify the optical sectioning provided by the system. For our measured aberration data we find that a pinhole of size approximately 3 Airy units represents a good compromise, allowing effective transmission of the wavefront and thin optical sections. Finally, we discuss some of the practical implications of confocal wavefront sensing for AO systems in microscopy.

Adaptive phase compensation for ultracompact laser scanning endomicroscopy.

We present an approach to laser scanning endomicroscopy that requires no moving parts and can be implemented with no distal scanners or optics, permitting extremely compact endoscopic probes to be developed. Our approach utilizes a spatial light modulator to correct for phase variations across a fiber imaging bundle and to encode for arbitrary wavefronts at the distal end of the fiber bundle. Thus, it is possible to realize both focusing and beam scanning at the output of the fiber bundle with no distal components. We present proof of principle results to illustrate three-dimensional scanning of the focal spot and exemplar images of a United States Air Force resolution test chart.

Characterization of deformable mirrors for spherical aberration correction in optical sectioning microscopy.

In this paper we describe the wavefront aberrations that arise when imaging biological specimens using an optical sectioning microscope and generate simulated wavefronts for a planar refractive index mismatch. We then investigate the capability of two deformable mirrors for correcting spherical aberration at different focusing depths for three different microscope objective lenses. Along with measurement and analysis of the mirror influence functions we determine the optimum mirror pupil size and number of spatial modes included in the wavefront expansion and we present measurements of actuator linearity and hysteresis. We find that both mirrors are capable of correcting the wavefront aberration to improve imaging and greatly extend the depth at which diffraction limited imaging is possible.

Stokes polarimeter optimization in the presence of shot and Gaussian noise.

An error minimization method is presented for Stokes polarimeters applicable when the detected signals are affected by a combination of shot and Gaussian noise. The expectation of the Stokes vector variance is used as a performance measure. This measure is compared with the condition number of a polarization state analyzer matrix that is commonly used as a figure of merit. We show that a polarimeter with the minimum condition number is not necessarily optimal. The approach is used to optimize existing prism based polarimeters giving improvements in the performance when shot-noise cannot be neglected.

Measuring retinal vessel tortuosity in 10-year-old children: validation of the Computer-Assisted Image Analysis of the Retina (CAIAR) program.

PURPOSE: To examine the agreement of a novel computer program measuring retinal vessel tortuosity with subjective assessment of tortuosity in school-aged children. METHODS: Cross-sectional study of 387 retinal vessels (193 arterioles, 194 veins) from 28 eyes of 14 children (aged 10 years). Retinal digital images were analyzed using the Computer Assisted Image Analysis of the Retina (CAIAR) program, including 14 measures of tortuosity. Vessels were graded (from 0 = none; to 5 = tortuous) independently by two observers. Interobserver agreement was assessed by using kappa statistics. Agreement with all 14 objective measures was assessed with correlation/regression analyses. Intersession repeatability (comparing morning and afternoon sessions) of tortuosity indices was calculated. RESULTS: Interobserver agreement of vessel tortuosity within one grade was high (kappa = 0.97), with total agreement in 56% of grades and 42% differing by +/-1 grade. Tortuosity indices based on subdivided chord length methods showed strong log-linear associations with agreed subjective grades (typically r > 0.6; P < 0.001). An approach that averages the distance from the vessel to chord length along the length of the vessel showed best agreement (r = 0.8; P < 0.0001). Tortuosity measures based on curvature performed less well. Intersession repeatability of the vessel to chord technique was good, differing by values equivalent to <1 in subjective grade. CONCLUSIONS: Tortuosity indices based on changes in subdivided chord lengths showed optimal agreement with subjective assessment. The relation of these indices to ethnicity and cardiovascular risk factors in childhood should be examined further, as these indices may be a useful indicator of early vascular function.

Computerized analysis of retinal vessel width and tortuosity in premature infants.

PURPOSE: To determine, with novel software, the feasibility of measuring the tortuosity and width of retinal veins and arteries from digital retinal images of infants at risk of retinopathy of prematurity (ROP). METHODS: The Computer-Aided Image Analysis of the Retina (CAIAR) program was developed to enable semiautomatic detection of retinal vasculature and measurement of vessel tortuosity and width from digital images. CAIAR was tested for accuracy and reproducibility of tortuosity and width measurements by using computer-generated vessel-like lines of known frequency, amplitude, and width. CAIAR was then tested by using clinical digital retinal images for correlation of vessel tortuosity and width readings compared with expert ophthalmologist grading. RESULTS: When applied to 16 computer-generated sinusoidal vessels, the tortuosity measured by CAIAR correlated very well with the known values. Width measures also increased as expected. When the CAIAR readings were compared with five expert ophthalmologists' grading of 75 vessels on 10 retinal images, moderate correlation was found in 10 of the 14 tortuosity output calculations (Spearman rho = 0.618-0.673). Width was less well correlated (rho = 0.415). CONCLUSIONS: The measures of tortuosity and width in CAIAR were validated using sequential model vessel analysis. On comparison of CAIAR output with assessments made by expert ophthalmologists, CAIAR correlates moderately with tortuosity grades, but less well with width grades. CAIAR offers the opportunity to develop an automated image analysis system for detecting the vascular changes at the posterior pole, which are becoming increasingly important in diagnosing treatable ROP.