Andreas Stingl

Ultrahigh resolution Fourier domain optical coherence tomography.

We present, for the first time, in vivo ultrahigh resolution (~2.5 microm in tissue), high speed (10000 A-scans/second equivalent acquisition rate sustained over 160 A-scans) retinal imaging obtained with Fourier domain (FD) OCT employing a commercially available, compact (500x260mm), broad bandwidth (120 nm at full-width-at-half-maximum centered at 800 nm) Titanium:sapphire laser (Femtosource Integral OCT, Femtolasers Produktions GmbH). Resolution and sampling requirements, dispersion compensation as well as dynamic range for ultrahigh resolution FD OCT are carefully analyzed. In vivo OCT sensitivity performance achieved by ultrahigh resolution FD OCT was similar to that of ultrahigh resolution time domain OCT, although employing only 2-3 times less optical power (~300 microW). Visualization of intra-retinal layers, especially the inner and outer segment of the photoreceptor layer, obtained by FDOCT was comparable to that, accomplished by ultrahigh resolution time domain OCT, despite an at least 40 times higher data acquisition speed of FD OCT.

Advances in broad bandwidth light sources for ultrahigh resolution optical coherence tomography

Novel ultra-broad bandwidth light sources enabling unprecedented sub-2 microm axial resolution over the 400 nm-1700 nm wavelength range have been developed and evaluated with respect to their feasibility for clinical ultrahigh resolution optical coherence tomography (UHR OCT) applications. The state-of-the-art light sources described here include a compact Kerr lens mode locked Ti:sapphire laser (lambdaC = 785 nm, delta lambda = 260 nm, P(out) = 50 mW) and different nonlinear fibre-based light sources with spectral bandwidths (at full width at half maximum) up to 350 nm at lambdaC = 1130 nm and 470 nm at lambdaC = 1375 nm. In vitro UHR OCT imaging is demonstrated at multiple wavelengths in human cancer cells, animal ganglion cells as well as in neuropathologic and ophthalmic biopsies in order to compare and optimize UHR OCT image contrast, resolution and penetration depth.

Imaging ex vivo healthy and pathological human brain tissue with ultra-high-resolution optical coherence tomography

The ability of ultra-high-resolution optical coherence tomography (UHR OCT) to discriminate between healthy and pathological human brain tissue is examined by imaging ex vivo tissue morphology of various brain biopsies. Micrometer-scale OCT resolution (0.9x2 microm, axialxlateral) is achieved in biological tissue by interfacing a state-of-the-art Ti:Al2O3 laser (lambda(c)=800 nm, delta lambda=260 nm, and P(out)=120 mW exfiber) to a free-space OCT system utilizing dynamic focusing. UHR OCT images are acquired from both healthy brain tissue and various types of brain tumors including fibrous, athypical, and transitional meningioma and ganglioglioma. A comparison of the tomograms with standard hematoxylin and eosin (H&E) stained histological sections of the imaged biopsies demonstrates the ability of UHR OCT to visualize and identify morphological features such as microcalcifications (>20 microm), enlarged nuclei of tumor cells (approximately 8 to 15 microm), small cysts, and blood vessels, which are characteristic of neuropathologies and normally absent in healthy brain tissue.

Precise thickness measurements of Bowman's layer, epithelium, and tear film.

Purpose. To visualize corneal microstructure such as tear film, epithelium, and Bowmans layer in three dimensions with spectral domain optical coherence tomography (SDOCT) exhibiting 1.3 &mgr;m axial resolution at 100,000 A-scans/s. This enables measurement of epithelial and Bowman layer thickness across an area of 8.4 mm × 8.4 mm and measuring the tear film thickness at the central cornea. Methods. We designed a high-performance SDOCT system, which uses a broad bandwidth TiSapph Laser and a high-speed complementary metal-oxide-semiconductor detector technology, providing a resolution in tissue of 1.3 &mgr;m and an acquisition speed of 100,000 A-scans/s. Such speed and resolution is a prerequisite if precise anatomy is to be determined. The high resolution gives access to corneal microstructure such as the epithelium layer as well as the boundaries of Bowmans layer and stroma. Even more interestingly, the tear film can be distinguished on the surface of the cornea. The Bowmans layer and epithelial thickness for both eyes of nine subjects have been measured out of which two subjects underwent photorefractive keratectomy treatment. Results. Three-dimensional volumes of the human cornea have been recorded in vivo at an A-scan rate of 100,000 scans/s. Epithelial thickness was measured to be 55.8 ± 3.3 &mgr;m and Bowmans layer thickness 18.7 ± 2.5 &mgr;m in normal eyes. Epithelial thickness in the eyes after refractive surgery was measured to be 68.2 ± 5.0 &mgr;m. The Bowman layer was degenerated in these eyes. The average tear film thickness of four eyes was 5.1 ± 0.5 &mgr;m. Conclusions. Using a high-performance SDOCT system with high-imaging speed and ultrahigh resolution, we produced precise thickness maps of the epithelium and for the first time of the Bowmans layer. Such a system will give insight into high-fidelity three-dimensional corneal microstructure helping to precisely plan refractive surgery. It may furthermore yield new perspectives on studying and understanding tear film dynamics.

Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography

The feasibility of ultrahigh resolution optical coherence tomography (UHR OCT) to image ex vivo and in vitro brain tissue morphology on a scale from single neuron cells to a whole animal brain was investigated using a number of animal models. Sub-2-microm axial resolution OCT in biological tissue was achieved at different central wavelengths by separately interfacing two state-of-the-art broad bandwidth light sources (titanium:sapphire, Ti:Al2O3 laser, lambdac=800 nm, Deltalambda=260 nm, Pout=50 mW and a fiber laser light source, lambdac=1350 nm, Deltalambda=470 nm, Pout=4 mW) to free-space or fiber-based OCT systems, designed for optimal performance in the appropriate wavelength regions. The ability of sub-2-microm axial resolution OCT to visualize intracellular morphology was demonstrated by imaging living ganglion cells in cultures. The feasibility of UHR OCT to image the globular structure of an entire animal brain as well as to resolve fine morphological features at various depths in it was tested by imaging a fixed honeybee brain. Possible degradation of OCT axial resolution with depth in optically dense brain tissue was examined by depositing microspheres through the blood stream to various depths in the brain of a living rabbit. It was determined that in the 1100 to 1600-nm wavelength range, OCT axial resolution was well preserved, even at depths greater than 500 microm, and permitted distinct visualization of microspheres 15 microm in diameter. In addition, the OCT image penetration depth and the scattering properties of gray and white brain matter were evaluated in tissue samples from the visual cortex of a fixed monkey brain.

Integrated single- and two-photon light sheet microscopy using accelerating beams

We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode, an increase in the field of view over the use of a standard Gaussian beam by a factor of six is demonstrated. This implementation for light sheet microscopy opens up new possibilities across a wide range of biomedical applications, especially for the study of neuronal processes.

Frequency-doubled diode laser for direct pumping of Ti:sapphire lasers

A single-pass frequency doubled high-power tapered diode laser emitting nearly 1.3 W of green light suitable for direct pumping of Ti:sapphire lasers generating ultrashort pulses is demonstrated. The pump efficiencies reached 75 % of the values achieved with a commercial solid-state pump laser. However, the superior electro-optical efficiency of the diode laser improves the overall efficiency of the Ti:sapphire laser by a factor > 2. The optical spectrum emitted by the Ti:sapphire laser shows a spectral width of 112 nm (FWHM). Based on autocorrelation measurements, pulse widths of less than 20 fs are measured. These results open the opportunity of establishing diode laser pumped Ti:sapphire lasers for e.g. biophotonic applications like retinal optical coherence tomography or pumping of photonic crystal fibers for CARS microscopy.

Colloidal quantum dots suitability for long term cell imaging

Fluorescent semiconductor nanoparticles in tree-dimensional quantum confinement, quantum dots (QDs), synthesized in aqueous medium, and functionalized with polyethylene glycol, were used as probes for the long-term imaging of glial cells. In vitro living healthy as well as cancer glial cells were labelled by direct insertion of a small volume of QDs contained in aqueous suspension into the culture wells. A long-term monitoring (over 7 days) of the cells was performed and no evidence of cell fixation and/or damage was observed. Two control groups, healthy and cancer glial cells, were used to compare cell viability. During the observation period, labelled and non labelled cells presented the same dynamics and no difference was observed regarding cell viability. To our knowledge, this is the first report of the viability of hydrophilic prepared quantum dots without any further surface treatment than the polyethylene-glycol coverage for the long-term imaging of living cells. Further, the study also permitted the observation of two distinct interaction mechanisms between cells and QDs. Healthy glial cells were mainly labelled at their surface, while non-healthy glial cells have shown a high rate in the uptake of QDs.

Long term imaging of living brain glial cancer cells

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Quantum dots pushing up in vitro diagnostics limits

Biopsies are conventionally performed in two dimensions. Histological slices in general present some micrometers in thickness, allowing that some molecular domains stay out of the resulting image. Thus the histopathological assay potentially is based on an incomplete set of information. The use of quantum dots as fluorescente probes allows the investigation of labelling pattern and biomarkers expression, along the three-dimensions of fresh histological slices, leading to more precise results. Present work show and discuss pattern and fluorescence intensity emission at the visible region obtained as a function of tissue thickness in histological (thickness(z)=7.6μm) breast cancer samples labeled with compact (7-10 nm) water soluble quantum dots. Series of 154 three-dimensional (3D) images were recorded from each tissue sample by laser scanning confocal microscopy, using 488 nm excitation.. In order to compare the results obtained, all the acquisition parameters were maintained constant. Results point to the possibility of more accurate histological diagnostics, once they clearly show distinct labeling patterns across sample thickness.