LIFTOSCOPE: development of an automated AI-based module for time-effective and contactless analysis and isolation of cells in microtiter plates.

BACKGROUND: The cultivation, analysis, and isolation of single cells or cell cultures are fundamental to modern biological and medical processes. The novel LIFTOSCOPE technology aims to integrate analysis and isolation into one versatile, fully automated device. METHODS: LIFTOSCOPE's three core technologies are high-speed microscopy for rapid full-surface imaging of cell culture vessels, AI-based semantic segmentation of microscope images for localization and evaluation of cells, and laser-induced forward transfer (LIFT) for contact-free isolation of cells and cell clusters. LIFT transfers cells from a standard microtiter plate (MTP) across an air gap to a receiver plate, from where they can be further cultivated. The LIFT laser is integrated into the optical path of an inverse microscope, allowing to switch quickly between microscopic observation and cell transfer. RESULTS: Tests of the individual process steps prove the feasibility of the concept. A prototype setup shows the compatibility of the microscope stage with the LIFT laser. A specifically designed MTP adapter to hold a receiver plate has been designed and successfully used for material transfers. A suitable AI algorithm has been found for cell selection. CONCLUSION: LIFTOSCOPE speeds up cell cultivation and analysis with a target process time of 10 minutes, which can be achieved if the cell transfer is sped up using a more efficient path-finding algorithm. Some challenges remain, like finding a suitable cell transfer medium. SIGNIFICANCE: The LIFTOSCOPE system can be used to extend existing cell cultivation systems and microscopes for fully automated biotechnological applications.

Increasing dental zirconia micro-retentive aspect through ultra-short pulsed laser microstructuring: study on flexural strength and crystal phase characterization.

OBJECTIVES: Although ultra-short pulsed laser (USPL) microstructuring has previously improved zirconia bond-strength, it is yet unclear how different laser-machined surface microstructures and patterns may influence the material's mechanical properties. Therefore, the aim of this study was to assess the flexural strength of zirconia after different USPL settings creating three different geometrical patterns with structures in micrometer scale. METHODS: One hundred sixty zirconia bars (3Y-TZP, 21 × 4 × 2.1 mm) were prepared and randomly divided into five groups (n = 32): no surface treatment (negative control-NC); sandblasting with Al2O3 (SB); and three laser groups irradiated with USPL (Nd:YVO4/1064 nm/2-34 J/cm2/12 ps): crossed-lines (LC), random-hatching (LR), and parallel-waves (LW). Bars were subjected to a four-point flexural test (1 mm/min) and crystal phase content changes were identified by X-ray diffraction. Surface roughness and topography were analyzed through 3D-laser-profilometry and SEM. Data were analyzed with parametric tests for roughness and Weibull for flexural strength (α = 5%). RESULTS: LR (Mean[95%CI]: 852.0 MPa, [809.2-894.7]) was the only group that did not show a significantly different flexural strength than NC (819.8 MPa, [796.6-842.9]), (p > 0.05). All laser groups exhibited higher Weibull moduli than NC and SB, indicating higher reliability and homogeneity of the strength data. An increase of monoclinic phase peak was only observed for SB. CONCLUSION: In conclusion, USPL created predictable, homogeneous, highly reproducible, and accurate surface microstructures on zirconia ceramic. The laser-settings of random-hatching (12 ps pulses) increased 3Y-TZP average surface roughness similarly to SB, while not causing deleterious crystal phase transformation or loss of flexural strength of the material. Furthermore, it has increased the Weibull modulus and consequently material's reliability. CLINICAL SIGNIFICANCE: Picosecond laser microstructuring (LR conditions) of 3Y-TZP ceramic does not decrease its flexural strength, while increasing materials realiability and creating highly reproducible and accurate microstructures. These features may be of interest both for improving clinical survival of zirconia restorations as well as enhancing longevity of zirconia implants.

In Vitro Feasibility Analysis of a New Sutureless Wound-Closure System Based on a Temperature-Regulated Laser and a Transparent Collagen Membrane for Laser Tissue Soldering (LTS).

For the post-surgical treatment of oral wounds and mucosal defects beyond a certain size, the gold standard is still an autologous skin or mucosal graft in combination with complex suturing techniques. A variety of techniques and biomaterials has been developed for sutureless wound closure including different tissue glues or collagen patches. However, no wound covering that enables for sutureless fixation has yet been introduced. Thus, a new system was developed that allows for sutureless wound covering including a transparent collagen membrane, which can be attached to the mucosa using a specially modified 2λ laser beam with integrated temperature sensors and serum albumin as bio-adhesive. The sutureless wound closure system was tested for its applicability and its cytocompatibility by an established in vitro model in the present study. The feasibility of the laser system was tested ex vivo on a porcine palate. The in vitro cytocompatibility tests excluded the potential release of toxic substances from the laser-irradiated collagen membrane and the bio-adhesive. The results of the ex vivo feasibility study using a porcine palate revealed satisfactory mean tensile strength of 1.2-1.5 N for the bonding of the membrane to the tissue fixed with laser of 980 nm. The results suggest that our newly developed laser-assisted wound closure system is a feasible approach and could be a first step on the way towards a laser based sutureless clinical application in tissue repair and oral surgery.

Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy.

Mammalian cells have become the predominant expression system for the production of biopharmaceuticals due to their capabilities in posttranslational modifications. In recent years, the efficacy of these production processes has increased significantly through technical improvements. However, the state of the art in the development of producer cell lines includes many manual steps and is as such very time and cost consuming. In this study we developed a process combination of Raman micro-spectroscopy, laser-induced forward transfer (LIFT) and surface-enhanced Raman spectroscopy (SERS) as an automated machine system for the identification, separation and characterization of single cell-clones for biopharmaceutical production. Raman spectra showed clear differences between individual antibody-producing and non-producing chinese hamster ovary (CHO) cells after their stable transfection with a plasmid coding for an immunoglobulin G (IgG) antibody. Spectra of producing CHO cells exhibited Raman signals characteristic for human IgG. Individual producing CHO cells were successfully separated and transferred into a multiwell plate via LIFT. Besides, changes in concentration of human IgG in solution were detected via SERS. SERS spectra showed the same peak patterns but differed in their peak intensity. Overall, our results show that identification of individual antibody-producing CHO cells via Raman micro-spectroscopy, cell separation via LIFT and determination of changes in concentrations of overexpressed protein via SERS are suitable and versatile tools for assembling a fully automated system for biopharmaceuticals manufacturing.

Influence of laser wavelength and beam profile on the coagulation depth in a soft tissue phantom model.

In laser tissue soldering (LTS), a protein solution is thermally denatured and cross-linked to obtain a strong bond between tissues or tissue and a wound dressing. However, if the extension of the heat-affected zone is too large, wound healing is impaired by thermal tissue injuries. Therefore, heat input and coagulation depth have to be limited. We investigate the influence of wavelength and beam profile on coagulation depth using a soft tissue phantom in case of weakly (980 nm) and strong (1540 nm) absorbed laser radiation. The soft tissue phantom is doped with polystyrene (PS) beads to obtain similar scattering properties as natural tissue. The propagation of the laser radiation in the phantom is simulated by Monte-Carlo method and the optical penetration (OPD) depth calculated from isophotes. The simulation results are compared with the experimental determination of the coagulation volume. The results reveal that scattering effect of tissues on laser radiation increases the losses of a Gaussian beam profile laterally leading to a half-sphere coagulation volume. The depth profile of the coagulation follows approximately the intensity distribution of the laser beams as long as scattering effects are weak. As scattering effects become significant, as for 980-nm radiation, the intensity distribution of the laser beam in the tissue deviates from the original one, leading to different profile of the coagulation depth.

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

New stereolithographic resin providing functional surfaces for biocompatible three-dimensional printing.

Stereolithography is one of the most promising technologies for the production of tailored implants. Within this study, we show the results of a new resin formulation for three-dimensional printing which is also useful for subsequent surface functionalization. The class of materials is based on monomers containing either thiol or alkene groups. By irradiation of the monomers at a wavelength of 266 nm, we demonstrated an initiator-free stereolithographic process based on thiol-ene click chemistry. Specimens made from this material have successfully been tested for biocompatibility. Using Fourier-transform infrared spectrometry and fluorescent staining, we are able to show that off-stoichiometric amounts of functional groups in the monomers allow us to produce scaffolds with functional surfaces. We established a new protocol to demonstrate the opportunity to functionalize the surface by copper-catalyzed azide-alkyne cycloaddition chemistry. Finally, we demonstrate a three-dimensional bioprinting concept for the production of potentially biocompatible polymers with thiol-functionalized surfaces usable for subsequent functionalization.

Surface Characterization and Short-term Adhesion to Zirconia after Ultra-short Pulsed Laser Irradiation.

PURPOSE: To evaluate the suitability of an ultra-short pulsed laser (USPL) to treat zirconia ceramic surfaces and increase their adhesion to dual-curing resin cement. MATERIALS AND METHODS: Twenty 10 × 10 × 5 mm³ blocks were prepared from a zirconia ceramic (Y-TZP). The specimens were polished and randomly assigned to four groups (n = 5) which received the following surface treatments: sandblasting (SB) with Al2O3 particles and silica coating (SC) with SiO2 particles as positive controls; two groups received USPL irradiation, one with 10 scan repetitions (L10) and the other with 20 (L20). Laser irradiation was performed at 1030 nm, 2.3 J/cm², 6 ps pulse duration. The ceramic blocks were duplicated in composite resin and cemented with a dual-curing resin cement. Half of the blocks were then stored in water (37°C) for 24 h and the other half for 1 month. At each time, 40 to 60 sticks per group were subjected to microtensile bond strength testing. Data were analyzed statistically using the Kruskal-Wallis test (α = 0.05). RESULTS: Laser-treated zirconia presented statistically significantly higher roughness than did SB and SC. After 24 h, the highest bond strength means (MPa) were achieved by L10 (42.3 ± 10.8) and L20 (37.9 ± 14.4), and both of them were statistically significantly higher than SB (22.0 ± 5.3) and SC (20.8 ± 7.1) (p < 0.05). After 1 month of storage, L10- and L20-treated zirconia still showed significantly higher bond strengths than did SB- and SC-treated zirconia (p < 0.05). CONCLUSION: USPL irradiation significantly increases bond strength of zirconia ceramic to dual-curing resin cement and might be an alternative for improving adhesion to this material.

Precise ablation of dental hard tissues with ultra-short pulsed lasers. Preliminary exploratory investigation on adequate laser parameters.

This study aimed to evaluate the possibility of introducing ultra-short pulsed lasers (USPL) in restorative dentistry by maintaining the well-known benefits of lasers for caries removal, but also overcoming disadvantages, such as thermal damage of irradiated substrate. USPL ablation of dental hard tissues was investigated in two phases. Phase 1--different wavelengths (355, 532, 1,045, and 1,064 nm), pulse durations (picoseconds and femtoseconds) and irradiation parameters (scanning speed, output power, and pulse repetition rate) were assessed for enamel and dentin. Ablation rate was determined, and the temperature increase measured in real time. Phase 2--the most favorable laser parameters were evaluated to correlate temperature increase to ablation rate and ablation efficiency. The influence of cooling methods (air, air-water spray) on ablation process was further analyzed. All parameters tested provided precise and selective tissue ablation. For all lasers, faster scanning speeds resulted in better interaction and reduced temperature increase. The most adequate results were observed for the 1064-nm ps-laser and the 1045-nm fs-laser. Forced cooling caused moderate changes in temperature increase, but reduced ablation, being considered unnecessary during irradiation with USPL. For dentin, the correlation between temperature increase and ablation efficiency was satisfactory for both pulse durations, while for enamel, the best correlation was observed for fs-laser, independently of the power used. USPL may be suitable for cavity preparation in dentin and enamel, since effective ablation and low temperature increase were observed. If adequate laser parameters are selected, this technique seems to be promising for promoting the laser-assisted, minimally invasive approach.

Laser-mediated fixation of collagen-based scaffolds to dermal wounds.

BACKGROUND AND OBJECTIVE: Collagen scaffolds are popular for the reconstitution of dermal equivalents. Usually, these scaffolds are fixed with sutures or staples and in many cases these devices have to be removed in a second procedure. Laser-mediated tissue welding in a wet environment is a potential alternative for collagen scaffold fixation and may be advantageous to suture, staple, and tissue glue fixation. MATERIALS AND METHODS: Welding was performed with a continuous-wave diode laser system emitting radiation at a wavelength of 968 nm. Tensile strength after fixation to porcine skin and laser parameters were determined in vitro. In vivo, 24 excisional deep partial thickness wounds were created on flanks of two Goettingen mini pigs and covered with collagen scaffolds. These were randomized and fixated with either (1) staples, (2) fibrin glue, or (3) laser-mediated welding. Tissue biopsies for histological analysis were periodically performed and analyzed for wound healing progression, epidermal thickness, and extracellular matrix formation. RESULTS: Biomechanical stability after laser welding was time dependent. A dwell time of up to 10 seconds led to a strong bonding with a tensile strength of more than 30 g. In vivo, the wound healing process was macroscopically comparable in all groups and showed no significant differences. Microscopic analysis determined a more progressed and quicker wound closure in both the laser and staples group compared to the fibrin glue fixated scaffold. Laser-mediated fixation led to a significantly reduced epidermal thickness when compared with stapling or fibrin glue (P < 0.05). CONCLUSIONS: Laser tissue welding is a feasible approach for temporary fixation of collagen scaffolds to the wound bed. It improves wound healing properties and may lead to faster wound healing and cosmetically better scarring. Laser tissue welding is thus a very interesting and promising alternative to currently established fixation methods in a single step, no touch procedure.

Magnetic resonance-guided angioplasty with delivery of contrast-media doped solutions to the vessel wall: an experimental study in swine.

OBJECTIVES: To assess the feasibility of magnetic resonance (MR)-guided delivery of a solution containing contrast-medium and immediate online monitoring of its distribution to the vessel wall during MR-guided angioplasty in peripheral arteries. MATERIALS AND METHODS: In 3 pigs, the feasibility of MR-guided atraumatic delivery of a solution containing contrast-medium and tissue dye (0.05 mmol/mL Gd-DTPA, 3% Evans blue dye) into the vessel wall of the iliac arteries was tested using a permeable balloon catheter (8 mm). Catheter placement was monitored using a steady-state free precession real-time imaging sequence.Additionally, in 5 pigs, surgically created bilateral stenoses in the external iliac artery were dilatated with the porous balloon. In these animals, contrast-enhanced MR angiography was performed before and after the interventions to assess the degree of the stenosis. In all animals, the vessel wall was delineated before and after dilatation using a T1-weighted gradient echo (GE) sequence. RESULTS: In the 3 animals without stenosis, contrast medium was successfully applied to the vessel wall. On the GE images, the normalized signal intensity of the vessel wall was 0.95 +/- 0.015 arbitrary units (a. u.) prior and 2.15 +/- 0.105 a. u. after the intervention (P < 0.01). In the animals with stenosis, MR angiography performed before and after the intervention demonstrated successful dilatation of 9 of the 10 stenoses. Before the intervention, 7 stenoses were severe (76%-99%) and 3 moderate (50%-75%), and after the intervention, 4 stenoses were completely removed and 5 mild (<50%). Also in these 5 animals, the solution was visible in the vessel wall of the arteries on the T1-weighted GE MR images (normalized signal intensity prior the intervention 1.33 +/- 0.16 a. u. and 2.97 +/- 0.23 after angioplasty; P < 0.05). Histology demonstrated the distribution of the Evan's blue dye within the vessel wall in all animals. CONCLUSIONS: MR-guided delivery of a contrast-medium containing solution and immediate online assessment of its distribution to the vessel wall during angioplasty in peripheral arteries is feasible.

Infrared picosecond laser for perforation of single plant cells.

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. In this report, we demonstrate the use of an infrared (1,064 nm) picosecond laser for the perforation of tobacco cell protoplasts. A single pulse was sufficient to perforate the plasma membrane enabling the uptake of dye from the surrounding medium into the cytosol. Moreover, the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

Transthoracic echocardiographic measurement of patent ductus arteriosus in dogs.

BACKGROUND: Patent ductus arteriosus (PDA) size and morphology influence the selection of the kind and the size of the embolization device used to effect shunt closure. HYPOTHESIS: That echocardiographic measurement of PDA in dogs is accurate. ANIMALS: Forty-five client-owned dogs with PDA. METHODS: Prospective observational study. Echocardiographic and angiographic data were compared. RESULTS: Measurement of the ductus in color Doppler echocardiography (CD-E) and 2-dimensional echocardiography (2D-E) was achieved from left parasternal views in 43 of 45 unsedated dogs (96%). In these 43 dogs, the angiographic minimal PDA diameter was 3.72 +/- 1.59 mm, and the diameter of the PDA ampulla was 8.46 +/- 3.01 mm. The CD-E minimal PDA diameter ranged from 2.3 to 9.5 mm (median, 4.0 mm). There was a significant mean difference to the angiographic measurements (1.15 +/- 0.95 mm; P < .0001). An agreement in a 1-mm range was found in 21 of 43 dogs (48%). The 2D-E minimal PDA diameter was 3.73 +/- 1.78 mm, and the mean difference to the angiographic measurements was not significant (0.00 +/- 0.72 mm; P = .98). An agreement in a 1-mm range was found in 31 of 43 dogs (72%). The 2D-E measurement of the PDA ampulla revealed a significant mean difference to the angiographic data (1.95 +/- 2.43 mm, P < .0001). An agreement in a 2-mm range was found in 21 of 43 dogs (49%). CONCLUSIONS AND CLINICAL IMPORTANCE: The 2D-E from the left cranial parasternal view is an excellent noninvasive method to estimate the PDA minimal diameter before doing catheter intervention.

Morphological effects of nanosecond- and femtosecond-pulsed laser ablation on human middle ear ossicles.

We evaluate the feasibility of nanosecond-pulsed and femtosecond-pulsed lasers for otologic surgery. The outcome parameters are cutting precision (in micrometers), ablation rate (in micrometers per second), scanning speed (in millimeters per second), and morphological effects on human middle ear ossicles. We examine single-spot ablations by a nanosecond-pulsed, frequency-tripled Nd:YAG laser (355 nm, beam diameter 10 microm, pulse rate 2 kHz, power 250 mW) on isolated human mallei. A similar system (355 nm, beam diameter 20 microm, pulse rate 10 kHz, power 160-1500 mW) and a femtosecond-pulsed CrLi:SAF-Laser (850 nm, pulse duration 100 fs, pulse energy 40 microJ, beam diameter 36 microm, pulse rate 1 kHz) are coupled to a scanner to perform bone surface ablation over a defined area. In our setups 1 and 2, marginal carbonization is visible in all single-spot ablations of 1-s exposures and longer: With an exposure time of 0.5 s, precise cutting margins without carbonization are observed. Cooling with saline solution result is in no carbonization at 1500 mW and a scan speed of 500 mms. Our third setup shows no carbonization but greater cutting precision, although the ablation volume is lower. Nanosecond- and femtosecond-pulsed laser systems bear the potential to increase cutting precision in otologic surgery.

Laser-induced crystallization of calcium phosphate coatings on polyethylene (PE).

Calcium phosphate (CaP) coatings are used for obtaining a desired biological response. Usually, CaP coatings on metallic substrates are crystallized by annealing at temperatures of at least 400-600 degrees C. For polymeric substrates, this annealing is not possible due to the low melting temperatures. In this work, we present a more suitable method for obtaining crystalline coatings on polymeric substrates, namely laser crystallization. We were successful in obtaining hydroxyapatite coatings on polyethylene. Because of the UV transmission characteristics of the CaP coatings, the use of a low wavelength (157 nm) F(2) laser was necessary for this. As a result of the laser treatment, the CaP coating broke up into islands. The cracks between the islands became larger and the surface became porous with increasing laser energy. The mechanism behind the formation of this morphology did not become clear. However, the fact that crystalline CaP coatings can be obtained on polymeric substrates in an easy way, possibly allows for the development of new products.

Percutaneous angiography of Patent Ductus Arteriosus in dogs: techniques, results and implications for intravascular occlusion.

OBJECTIVES: To evaluate the percutaneous angiography and the morphology of patent ductus arteriosus (PDA) in dogs. BACKGROUND: In contrast to surgical ligation the knowledge of angiographic morphology is necessary for intravascular PDA occlusion. METHODS: Forty-nine dogs with a left to right shunting PDA were included in a three-year, prospective study. In 43 dogs with a body weight >/= 3.0 kg a percutaneous approach to brachial artery was tested. In six miniature dogs (< 3.0 kg) placement of angiographic catheter from femoral vein antegrade through the PDA was studied. Angiographic morphology and dimensions of PDA were analyzed. RESULTS: Percutaneous approach to brachial artery was performed successfully in 41/43 (95%) dogs, in the remaining 2 dogs this approach was not possible and the femoral artery had to be used. Two dogs did not survive the procedure. Arterial bleeding was not a problem in any of the 39 cases in which percutaneous brachial artery catheterization was performed. Two dogs developed transient radial nerve paralysis that resolved 4 to 6 days later. The transvenous technique was successful in all 6 small dogs. In 39 dogs there was an elongated conical duct (type E). A conical duct (type A) was found in nine dogs and one dog had a PDA with two constrictions (type D). The mean PDA minimal diameter was 3.78 +/- 1.55 mm (range 1.5 to 6.9), in 20/49 dogs (41%) the minimal PDA diameter measured more than 4.0 mm. CONCLUSIONS: Percutaneous catheterization of the brachial artery using a vascular introducer is feasible and entails minimal risk of hemorrhage. Angiographic evaluation of the ductus arteriosus is easily performed with this technique, but coilimplantation is not possible by this approach. The frequency of PDA-types is different in dogs and humans and the PDA minimal diameter is larger in dogs.