Sub-100 nm material processing and imaging with a sub-15 femtosecond laser scanning microscope

König, Karsten; Uchugonova, Aisada; Straub, Martin; Zhang, Huijing; Licht, Martin; Afshar, Maziar; Feili, Dara; Seidel, H.

doi:10.2351/1.4718858

Cited by 23 publications

(12 citation statements)

References 37 publications

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“…The instrumental setup is described elsewhere. 12 Briefly, it consists of a modified inverted microscope with a galvanometric scanner unit and a piezoelectric stage for z positioning of the objective. An 85-MHz mode-locked titanium-sapphire laser (Integral Pro 400; FemtoLasers Produktions GmbH, Vienna, Austria) generating~10-fs pulses with an M-shaped spectral profile (centered at 800 nm) and a bandwidth of~95 nm was used for sample excitation.…”

Section: Flim and Shg Imagingmentioning

confidence: 99%

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Batista

Breunig

König

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation.METHODS. Human corneas were imaged after different storage times: short-term (STS), mediumterm (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. RESULTS.Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a 1 /a 2 ) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a 1 /a 2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a 1 /a 2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 6 190 cells/ mm 2 was obtained for MTS samples based on TPI.CONCLUSIONS. TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

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Section: Flim and Shg Imagingmentioning

confidence: 99%

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Batista

Breunig

König

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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“…2PPis the only fabrication technique in which the scaffold geometry may be controlled at the cell scale (10 μm) and with a very high spatial resolution (less than 1 μm). Non-biodegradable "structural" materials used to fabricate scaffolds by 2PPinclude vinyls [12], epoxies [13], acrylates [14] and hybrid inorganic-organic materials [15], many of which have proven to be biocompatible in the cell culture environment [16]. In recent work, we have developed structural microscaffolds, or niches, of various complex 3D architectures, which were laser-written directly on the glass bottom of the cell culture chamber, using a photoresist consisting of a solgel-synthesized silicon-zirconium hybrid inorganic-organic [17].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Femtosecond Laser Polymerized Structural Niches to Control Mesenchymal Stromal Cell Fate in Culture

et al. 2014

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We applied two-photon polymerization to fabricate 3D synthetic niches arranged in complex patterns to study the effect of mechano-topological parameters on morphology, renewal and differentiation of rat mesenchymal stromal cells. Niches were formed in a photoresist with low auto-fluorescence, which enabled the clear visualization of the fluorescence emission of the markers used for biological diagnostics within the internal niche structure. The niches were structurally stable in culture up to three weeks. At three weeks of expansion in the niches, cell density increased by almost 10-fold and was 67% greater than in monolayer culture. Evidence of lineage commitment was observed in monolayer culture surrounding the structural niches, and within cell aggregates, but not inside the niches. Thus, structural niches were able not only to direct stem cell homing and colony formation, but also to guide aggregate formation, providing increased surface-to-volume ratios and space for stem cells to adhere and renew, respectively. OPEN ACCESSMicromachines 2014, 5 342

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“…The laser source, a Ti-sapphire oscillator (Integral Pro 400, FemtoLasers Produktions GmbH), supplied 10-fs pulses at a repetition frequency of 85 MHz. The emission spectrum ranged from 700 nm to 870 nm with two maxima at 770 nm and 827 nm [6]. The laser beam was coupled into an inverted optical microscope (AxioObserver.D1, ZEISS) equipped with a galvanometric x-y scanning unit and piezodriven focusing optics.…”

Section: Methodsmentioning

confidence: 99%

“…An ultra-short pulsed laser has been proven to be a useful tool for micro-and nanoprocessing of various materials because of its high precision compared to other light sources [5] and its high peak intensity at low average power [1,6]. In addition, two-or multi-photon absorption at the focal volume of the laser beam facilitates a real 3D structuring of transparent specimens [1].…”

mentioning

confidence: 99%

Submicron-dot chains at and beneath surfaces of glasses written by a picojoule 12-fs laser scanning microscope

et al. 2012

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A near-infrared 12-fs laser scanning microscope was employed for structuring glass surfaces. A Ti-sapphire laser operated at 85 MHz and emitted light pulses at wavelengths around 800 nm. A focused laser beam with a mean power of less than 27 mW, corresponding to a maximal pulse energy of 318 pJ, was applied for line scanning at and beneath the surfaces of cover slips as well as of a filter glass BG39. Periodic arrangements of dots along the processed lines were produced through digital control of the scanner. Depending on the pulse energy and the scan speed, the diameter of the dots ranged from 550 nm down to 100 nm. For the cover slips, the dots occur as cavities after wet chemical etching. For BG39, which exhibits strong near-infrared absorption, both chains of cavities and bumps can be generated without any etching process. The result shows that structures with a size down to 1/8λ can be generated, probably through nonlinear single-photon processes confined within the focal volume.

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Sub-100 nm material processing and imaging with a sub-15 femtosecond laser scanning microscope

Cited by 23 publications

References 37 publications

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Optimization of Femtosecond Laser Polymerized Structural Niches to Control Mesenchymal Stromal Cell Fate in Culture

Submicron-dot chains at and beneath surfaces of glasses written by a picojoule 12-fs laser scanning microscope

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