Martin Villiger

Extended focus depth for Fourier domain optical coherence microscopy

We report on a new detection scheme for Fourier domain optical coherence microscopy that exhibits high transverse resolution along an axially extended focal range. Nearly constant transverse resolution of approximately 1.5 microm along a focal range of 200 microm is experimentally verified with a maximum sensitivity of 105 dB. A broad-bandwidth Ti:sapphire laser allowed for an axial resolution of 3 microm in air.

A bio-inspired swellable microneedle adhesive for mechanical interlocking with tissue

Achieving significant adhesion to soft tissues while minimizing tissue damage poses a considerable clinical challenge. Chemical-based adhesives require tissue-specific reactive chemistry, typically inducing a significant inflammatory response. Staples are fraught with limitations including high-localized tissue stress and increased risk of infection, and nerve and blood vessel damage. Here, inspired by the endoparasite Pomphorhynchus laevis which swells its proboscis to attach to its host’s intestinal wall, we have developed a biphasic microneedle array that mechanically interlocks with tissue through swellable microneedle tips, achieving ~ 3.5 fold increase in adhesion strength compared to staples in skin graft fixation, and removal force of ~ 4.5 N/cm2 from intestinal mucosal tissue. Comprising a poly(styrene)-block-poly(acrylic acid) swellable tip and non-swellable polystyrene core, conical microneedles penetrate tissue with minimal insertion force and depth, yet high adhesion strength in their swollen state. Uniquely, this design provides universal soft tissue adhesion with minimal damage, less traumatic removal, reduced risk of infection and delivery of bioactive therapeutics.

Resonant Doppler flow imaging and optical vivisection of retinal blood vessels

For Fourier domain optical coherence tomography any sample movement during camera integration causes blurring of interference fringes and as such reduction of sensitivity for flow detection. The proposed method overcomes this problem by phase-matching a reference signal to the sample motion. The interference fringes corresponding to flow signal will appear frozen across the detector whereas those of static sample structures will be blurred resulting in enhanced contrast for blood vessels. An electro-optic phase modulator in the reference arm, driven with specific phase cycles locked to the detection frequency, allows not only for qualitative but also for quantitative flow detection already from the relative signal intensities. First applications to extract in-vivo retinal flow and to visualize 3D vascularization, i.e. optical vivisection, are presented.

Vectorial reconstruction of retinal blood flow in three dimensions measured with high resolution resonant Doppler Fourier domain optical coherence tomography

Resonant Doppler Fourier domain optical coherence tomography (FDOCT) is a functional imaging tool for extracting tissue flow. The method is based on the effect of interference fringe blurring in spectrometer-based FDOCT, where the path difference between structure and reference changes during camera integration. If the reference path length is changed in resonance with the Doppler frequency of the sample flow, the signals of resting structures will be suppressed, whereas the signals of blood flow are enhanced. This allows for an easy extraction of vascularization structure. Conventional flow velocity analysis extracts only the axial flow component, which strongly depends on the orientation of the vessel with respect to the incident light. We introduce an algorithm to extract the vessel geometry within the 3-D data volume. The algorithm calculates the angular correction according to the local gradients of the vessel orientations. We apply the algorithm on a measured 3-D resonant Doppler dataset. For validation of the reproducibility, we compare two independently obtained 3-D flow maps of the same volunteer and region.

Fast three-dimensional imaging of gold nanoparticles in living cells with photothermal optical lock-in Optical Coherence Microscopy

We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 μm lateral and 2 μm axial resolution over a 50 μm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.

Spectral binning for mitigation of polarization mode dispersion artifacts in catheter-based optical frequency domain imaging

Polarization mode dispersion (PMD) has been recognized as a significant barrier to sensitive and reproducible birefringence measurements with fiber-based, polarization-sensitive optical coherence tomography systems. Here, we present a signal processing strategy that reconstructs the local retardation robustly in the presence of system PMD. The algorithm uses a spectral binning approach to limit the detrimental impact of system PMD and benefits from the final averaging of the PMD-corrected retardation vectors of the spectral bins. The algorithm was validated with numerical simulations and experimental measurements of a rubber phantom. When applied to the imaging of human cadaveric coronary arteries, the algorithm was found to yield a substantial improvement in the reconstructed birefringence maps.

Label-free imaging of cerebral β-amyloidosis with extended-focus optical coherence microscopy

We demonstrate label-free imaging of cerebral β-amyloidosis ex vivo and in a living mouse model of Alzheimers disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual β-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the Alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of β-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.

Dark-field optical coherence microscopy

Many solutions have been proposed to produce phase quantitative images of biological cell samples. Among these, Spectral Domain Phase Microscopy combines the fast imaging speed and high sensitivity of Optical Coherence Microscopy (OCM) in the Fourier domain with the high phase stability of common-path interferometry. We report on a new illumination scheme for OCM that enhances the sensitivity for backscattered light and detects the weak sample signal, otherwise buried by the signal from specular reflection. With the use of a Bessel-like beam, a dark-field configuration was realized. Sensitivity measurements for three different illumination configurations were performed to compare our method to standard OCM and extended focus OCM. Using a well-defined scattering and reflecting object, we demonstrated an attenuation of -40 dB of the DC-component and a relative gain of 30 dB for scattered light, compared to standard OCM. In a second step, we applied this technique, referred to as dark-field Optical Coherence Microscopy (dfOCM), to living cells. Chinese hamster ovarian cells were applied in a drop of medium on a coverslide. The cells of ~15 μm in diameter and even internal cell structures were visualized in the acquired tomograms.

Deep tissue volume imaging of birefringence through fibre-optic needle probes for the delineation of breast tumour

Identifying tumour margins during breast-conserving surgeries is a persistent challenge. We have previously developed miniature needle probes that could enable intraoperative volume imaging with optical coherence tomography. In many situations, however, scattering contrast alone is insufficient to clearly identify and delineate malignant regions. Additional polarization-sensitive measurements provide the means to assess birefringence, which is elevated in oriented collagen fibres and may offer an intrinsic biomarker to differentiate tumour from benign tissue. Here, we performed polarization-sensitive optical coherence tomography through miniature imaging needles and developed an algorithm to efficiently reconstruct images of the depth-resolved tissue birefringence free of artefacts. First ex vivo imaging of breast tumour samples revealed excellent contrast between lowly birefringent malignant regions, and stromal tissue, which is rich in oriented collagen and exhibits higher birefringence, as confirmed with co-located histology. The ability to clearly differentiate between tumour and uninvolved stroma based on intrinsic contrast could prove decisive for the intraoperative assessment of tumour margins.

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

Aims/hypothesisStructural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition.MethodsEx vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo.ResultsxfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction.Conclusions/interpretationOur results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.