A 2” × 2” (5.08 × 5.08 cm) positive 1951 USAF resolution target (58-198, Edmund Optics) was used to quantify the spatial resolution and DOF of our system. The carbon fiber sample was prepared by randomly distributing carbon fibers with a diameter of 6 µm on top of a glass slide. The fibers were mixed with UV-curing optical adhesive (NOA61, Thorlabs) and sealed with a cover glass. The optical adhesive was then cured by illumination of UV light from a light-emitting diode (LED). HeLa cells were placed on sterile glass slides and cultured in DMEM supplemented with 10% fetal bovine serum and a penicillin-streptomycin mixture (all from Invitrogen/Life Technologies) at 37 °C in a 5% CO2 air atmosphere. When cells were 70% confluent on the glass slides, we fixed them with an ice-cold mixture of ethanol and methanol (1:1 volume ratio). The glass slides placed in a 10-cm petri-dish were covered by the organic solvents and then incubated in a freezer (−20 °C) for 5–7 min. The organic solvents preserved the cells by removing lipids, dehydrating tissue, and denaturing and precipitating the proteins in the cells. After fixation, the glass slides were gently rinsed with phosphate-buffered saline to remove any fixation agent.
Noa61
Manufactured by Thorlabs
Sourced in Germany, United States
The NOA61 is a precision optical adhesive from Thorlabs. It is a single-component, UV-curable adhesive designed for bonding a variety of optical and electronic components.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin mixture, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Mlj150, by Thorlabs (1 mentions)
Ft200emt, by Thorlabs (1 mentions)
Pm200, by Thorlabs (1 mentions)
Irgacure 651, by Merck Group (1 mentions)
7 protocols using noa61
1
Quantifying Resolution and Depth-of-Field
2
Ultraflat Gold Surface on Glass
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A piece of solvent clean silicon is sputter coated (south bay technology) with 10 nm of gold. Optical UV curable epoxy (Thorlabs NOA 61) is used to glue the glass surface to the gold which is on the silicon. Once the silicon is stripped, the ultraflat gold surface on the glass cover slide has a roughness of 0.15 nm–0.35 nm.
3
Colloidal Probe Cantilever Preparation
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Tipless V-shaped Si3N4 cantilevers with a nominal spring constant of 0.06 N/m (NP-OW; Veeco Probes, Camarillo, CA) were treated for 30 min with UV/ozone (UV/Ozone ProCleaner; Bioforce Nanoscience, Ames, IA). Polystyrene microspheres were dispersed on a cleaned and UV/ozone-treated glass coverslip, and attached to the cantilever using a UV-cured adhesive (NOA 61; Thorlabs, Dachau, Germany). Positioning and attachment were aided by a micromanipulator (Patchstar; Scientifica, Uckfield, UK) and an optical microscope (Axio Observer; Zeiss, Oberkochen, Germany). The real cantilever spring constant was determined to be 0.076 ± 0.004 N/m, using the thermal noise method (34 ), from an average over three cantilevers with colloidal probes attached. Before use, the colloidal-probe AFM cantilever together with the chip and chip holder was immersed in BSA for 20 min, for surface passivation. Excess BSA was removed by washing with sample preparation buffer.
4
Depth Estimation Accuracy Validation
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Two phantoms were prepared to evaluate the depth estimation accuracy of the MI method under 660 nm excitation. A tilted microscope slide with a thin layer of Cy5.5 dye was raster scanned over 45 × 45 positions with the AOD. Another phantom was comprised of two single-channel microfluidic chips (inner diameter = 50 μm, Microfluidic ChipShop, Germany) overlaid in a crossing orientation and separated by approximately 260 μm along the axial (depth) direction. Similarly, the illumination pattern was raster scanned with 45 × 45 positions and the 3D compounded image was reconstructed as described in the image reconstruction section.
For validating the depth estimation by the SL method, orange fluorescence 1–5 μm diameter beads (460/594 nm, Cospheric, USA) were mixed with UV-curing glue (NOA61, Thorlabs, USA) and poured onto the microscope slide resulting in their effective distribution across different depths. The phantom was subsequently exposed to UV light for 10 min. SL image stack was collected by translating the phantom along the z axis with 10 μm step size using a motorized stage (MLJ150, Thorlabs, USA).
For validating the depth estimation by the SL method, orange fluorescence 1–5 μm diameter beads (460/594 nm, Cospheric, USA) were mixed with UV-curing glue (NOA61, Thorlabs, USA) and poured onto the microscope slide resulting in their effective distribution across different depths. The phantom was subsequently exposed to UV light for 10 min. SL image stack was collected by translating the phantom along the z axis with 10 μm step size using a motorized stage (MLJ150, Thorlabs, USA).
5
Cardiac Myocyte Contractility Assay
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Left ventricular tissue was mechanically homogenized in Isolation Buffer (8.91 mM KOH, 2 mmol/L EGTA, 7.11 mmol/L MgCl2, 10 mmol/L imidazole, 108.01 mmol/L KCl, 5.8 mmol/L adenosine triphosphate) containing 0.5% Triton X‐100 with 3 one‐second pulses at 7000 RPM and left on ice for 20 minutes to demembranate the myocytes. Myocytes were passed through a 70‐μm filter and pelleted by centrifugation at 120g and resuspended in Isolation Buffer without triton. Myocytes were attached by UV‐curing glue (NOA61, Thor Labs) to 2 pins, 1 attached to a calibrated force transducer (Kronex, AE801) and the other to a Piezo length controller. Calcium‐activated force measurements were performed at 6 different calcium concentrations (0.79, 0.97, 1.28, 2.41, 3.84, and 46.8 μmol/L), and the force produced was normalized to the myocyte cross‐sectional area, as previously described.
21 (link) Data were fitted to a Hill Curve to determine the myocyte maximum force and EC50 (calcium concentration required to elicit half‐maximal force). All experiments were performed at room temperature and a sarcomere length of 2.1 μm as measured by fast Fourier transform.
21 (link) Data were fitted to a Hill Curve to determine the myocyte maximum force and EC50 (calcium concentration required to elicit half‐maximal force). All experiments were performed at room temperature and a sarcomere length of 2.1 μm as measured by fast Fourier transform.
6
Fabrication of Optical Fiber Optoprobes
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Optical fiber was made as previously reported81 (link) A 0.39 NA, Ø200 μm core multimode optical fibers (FT200EMT, Thorlabs) were assembled with a stainless-steel ferrule terminals (SF230, Thorlabs) and then polished on one side; exposed the silica core on 1 cm, removed TECS cladding and shaped with hydrofluoric acid on the another side. Only optoprobes with ∼9 mW maximal current output power at the tip were used, as confirmed by a photodiode power sensor (S130C, Thorlabs) and a power meter (PM200, Thorlabs). After that, each optical fiber was glued to a single tungsten wire by a UV-curing optical adhesive (NOA61, Thorlabs), the tip of wire was 0.5 mm longer than the tip of optical fiber.
7
Two-Photon Photopolymerization of PEGDA Derivatives
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Norland optical adhesive NOA61 was from Thorlabs. It was used directly on bare glass coverslips for two-photon photopolymerization.
PEGDA (258, 575) derivatives were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and were mixed to 5% (w/v) of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651, Sigma-Aldrich) and protected from light before undergoing two-photon photopolymerization. PEGDA derivatives were used on silanized glass coverslips prepared as follows. Glass coverslips (Ø = 30 mm) were first cleaned for 2 h in piranha solution to remove organic contaminants (H 2 SO 4 / H 2 O 2 , 70:30 v/v-Warning: this is a highly corrosive solution). The substrates were then washed extensively with distilled water and dried under a filtered nitrogen stream. Cleaned wafers were exposed to monolayer deposition solutions prepared by mixing 100 μl of 3-acryloxypropyltrichlorosilane with 100 ml of dry 1,2-dichoroethane solvent at room temperature. The coating procedure was performed under an argon atmosphere. Samples were withdrawn from the silane solutions and washed several times with CHCl 3 and ethanol and then dried under a nitrogen stream.
PEGDA (258, 575) derivatives were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and were mixed to 5% (w/v) of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651, Sigma-Aldrich) and protected from light before undergoing two-photon photopolymerization. PEGDA derivatives were used on silanized glass coverslips prepared as follows. Glass coverslips (Ø = 30 mm) were first cleaned for 2 h in piranha solution to remove organic contaminants (H 2 SO 4 / H 2 O 2 , 70:30 v/v-Warning: this is a highly corrosive solution). The substrates were then washed extensively with distilled water and dried under a filtered nitrogen stream. Cleaned wafers were exposed to monolayer deposition solutions prepared by mixing 100 μl of 3-acryloxypropyltrichlorosilane with 100 ml of dry 1,2-dichoroethane solvent at room temperature. The coating procedure was performed under an argon atmosphere. Samples were withdrawn from the silane solutions and washed several times with CHCl 3 and ethanol and then dried under a nitrogen stream.
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