The optical imaging set-up consisted of a stereomicroscope (MZ16, Leica) associated with a fast CMOS camera (Orca Flash 2.0, Hamamatsu). Images of light diffusion into the brain tissues were acquired for continuous illumination at 100, 200, and 600 mW/mm2 using the same exposure time (20 ms) for the sake of comparison. Reflectance images showing the vasculature were obtained using wide field illumination in green (530 nm) light to check that no bleeding occurred following cranial window surgery.
Orcaflash2
Manufactured by Hamamatsu Photonics
Sourced in United Kingdom, Japan, France
The OrcaFlash2.8 is a scientific camera designed for low-light imaging applications. It features a large, back-illuminated CMOS sensor with high quantum efficiency and low read noise, allowing for sensitive and high-resolution image capture. The camera supports a range of image readout modes and bit depths to suit various experimental requirements.
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Lab products found in correlation
Uapo 20 0.75 objective lens, by Olympus (4 mentions)
Lambda dg 4 ultra high speed wavelength switcher, by Sutter Instruments (4 mentions)
Metafluor software, by Molecular Devices (4 mentions)
Nanoparticle free water, by Fresenius (3 mentions)
Dlp lightcrafter 4500, by Texas Instruments (3 mentions)
Cellsens version 11, by Olympus (3 mentions)
Bx61 microscope, by Hamamatsu Photonics (3 mentions)
Pclamp 10, by Molecular Devices (2 mentions)
Nanosight lm10 system, by Malvern Panalytical (2 mentions)
Hcimage system, by Hamamatsu Photonics (2 mentions)
Fura 2 am, by Dojindo Laboratories (2 mentions)
Zen 2012, by Zeiss (2 mentions)
Axio observer z1, by Hamamatsu Photonics (2 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Cellsens dimension software, by Olympus (2 mentions)
Eclipse ti e, by Nikon (1 mentions)
Multiclamp 700a, by Molecular Devices (1 mentions)
Pc 10, by Narishige (1 mentions)
Upright microscope, by Olympus (1 mentions)
Multiclamp 700a amplifier, by Molecular Devices (1 mentions)
65 protocols using orcaflash2
1
Optical Imaging of Brain Tissue
2
Calcium Signaling in C25 Cells
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C25 cells seeded on 25-mm glass coverslips were incubated with 5 μM Fluo-4 AM for 25 min at 37 °C.
Subsequently, Ca2+ signals induced with 10 µM ionomycin were obtained in an inverted microscope (Eclipse Ti-E, Nikon, Tokyo, Japan) using a 40× objective. Ionomycin was administered 20 s after the start of recording and was maintained throughout the acquisition. Images were acquired using a cooled digital camera (ORCA-FLASH 2.0; Hamamatsu Photonics, Hamamatsu City, Japan) and NIS-Element Advanced Research 4.3 software (Nikon, Tokyo, Japan). For cells transfected with the mCherry-tagged plasmid containing WT dynamin-2, the mutation p.A618T or the mutation p.S619L, experiments were performed 24 h after transfections. Data are presented as ∆F/F0, where F0 and F are the background-subtracted fluorescence intensities recorded immediately before and after the addition of the agonist, respectively.
Subsequently, Ca2+ signals induced with 10 µM ionomycin were obtained in an inverted microscope (Eclipse Ti-E, Nikon, Tokyo, Japan) using a 40× objective. Ionomycin was administered 20 s after the start of recording and was maintained throughout the acquisition. Images were acquired using a cooled digital camera (ORCA-FLASH 2.0; Hamamatsu Photonics, Hamamatsu City, Japan) and NIS-Element Advanced Research 4.3 software (Nikon, Tokyo, Japan). For cells transfected with the mCherry-tagged plasmid containing WT dynamin-2, the mutation p.A618T or the mutation p.S619L, experiments were performed 24 h after transfections. Data are presented as ∆F/F0, where F0 and F are the background-subtracted fluorescence intensities recorded immediately before and after the addition of the agonist, respectively.
3
Patch Clamp Recording of Cultured Inhibitory Neurons
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AP firing and spontaneous post synaptic current were recorded from cultured iN at DIV 22–32 days. The cover slip was placed in recording solution containing (in mM) 150 NaCl, 10 HEPES, 3 KCl, 2 CaCl2, 2 MgCl2, 5.5 glucose and 20 sucrose with pH 7.3. The recorded cell was visualized either directly via the microscope’s optics, or indirectly via a high-resolution CCD camera system (Orca Flash 2.1, Hamamatsu) that received the output of a CCD camera attached to the microscope’s video port. Whole-cell patch clamp recordings were obtained using borosilicate glass pipettes (resistance 4–8 MΩ) prepared using a 2-stage vertical pipette puller (Narishige PC-10). Voltage and current clamp recordings were acquired using Multiclamp 700A (Axon Instruments). Signals were filtered at 1–2 kHz. All recordings under these protocols were digitized at 5 kHz and analyzed using pCLAMP 10 software (Axon Instruments). Experiments with a holding current of more than −100 pA or in which there was a change in input resistance >30% of the control were rejected. Whole-cell recordings were carried out with K-gluconate-based internal solution containing (in mM) 140 K-gluconate, 7 NaCl, 10 HEPES, 4 MgATP and 0.3 Na3-GTP, in which the pH was adjusted to 7.2 with KOH (OSM = 289).
4
Electrophysiological Recordings of Cultured Neurons
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Neurons cultured on individual coverslips were transferred to the recording chamber mounted on the stage of an upright microscope (Olympus, Tokyo, Japan) fitted with a 40× water‐immersion objective lens. Cells were visualized either directly via the optical microscope or indirectly via a high‐resolution CCD camera system (Orca Flash 2.1, Hamamatsu photonics, Iwata City, Japan).
Recordings were obtained using a Multiclamp 700A amplifier (Molecular Devices, Sunnyvale, CA) and were filtered at 2 kHz. Current recordings under ramp protocol and step were digitized at 10 kHz with DigiDATA 1550B1 and analysed using pCLAMP 10 software (all from Molecular Devices).
During recordings, cells were superfused at 1‐3 mL/min with external solution contained 150 mmol/L NaCl, 10 mmol/L HEPES, 3 mmol/L KCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, 5.5 mmol/L glucose, 20 mmol/L sucrose, pH adjusted to pH 7.3, and micropipettes contained internal solution composed of 140 mmol/L potassium gluconate, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L EGTA, 40 mmol/L HEPES (pH 7.2 was adjusted with KOH). Series resistance was monitored throughout the experiments, and data were discarded if changes >20% were observed.
Recordings were obtained using a Multiclamp 700A amplifier (Molecular Devices, Sunnyvale, CA) and were filtered at 2 kHz. Current recordings under ramp protocol and step were digitized at 10 kHz with DigiDATA 1550B1 and analysed using pCLAMP 10 software (all from Molecular Devices).
During recordings, cells were superfused at 1‐3 mL/min with external solution contained 150 mmol/L NaCl, 10 mmol/L HEPES, 3 mmol/L KCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, 5.5 mmol/L glucose, 20 mmol/L sucrose, pH adjusted to pH 7.3, and micropipettes contained internal solution composed of 140 mmol/L potassium gluconate, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L EGTA, 40 mmol/L HEPES (pH 7.2 was adjusted with KOH). Series resistance was monitored throughout the experiments, and data were discarded if changes >20% were observed.
5
Multiparametric Analysis of Apoptosis
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Cell suspensions simultaneously stained with TMRE, Hoechst33342, CellEvent and annexin V-Alexa Fluor 647 were analyzed using a Nikon Ti Eclipse microscope under PlanApo 60×/1.4 objective. Images were recorded by Hamamatsu ORCA FLASH2 digital camera, using the following filter sets: Ex.450–490 nm/Em.510–540 nm; Ex.340–380 nm/Em.435–485 nm; Ex.530–560 nm/Em.573–640 nm and Ex.595–645 nm/Em.665–715 nm.
6
Nanoparticle Characterization by NTA
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A 15-μl aliquot of each fraction was taken and particle counts and particle size distribution were determined using the Nanosight LM10 system (NanoSight Ltd, Amesbury, UK) configured with a 405-nm laser and a high-sensitivity digital camera system (OrcaFlash2.8, Hamamatsu C11440, NanoSight Ltd, Amesbury, UK). 30-s videos were taken and analyzed using NTA software (version 2.3), with the minimal expected particle size set to automatic and camera sensitivity and detection thresholds set to 14 and 3, respectively, to reveal small particles. Each fraction was diluted in nanoparticle-free water (Fresenius Kabi, Runcorn, UK) to a concentration between 2 × 108 and 9 × 108 particles per milliliter within the linear range of the instrument. A mock gradient where no sample was added was also analyzed, revealing negligible counts for particles related to the sucrose gradient (not shown). This mock sample acted as a background control for the SOMAscanTM array.
7
Nanoparticle Analysis of Exosome Isolates
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Size distribution and the estimated concentration of nanoparticles in each purified exosome isolate were analysed using a light scattering technology via measurement of the rate of Brownian motion with the NanoSight™ LM10 system (NanoSight Ltd, Amesbury, UK) configured with a 488 nm laser and a high sensitivity digital camera (OrcaFlash2.8, Hamamatsu C11440, NanoSight Ltd).
All samples were diluted with nanoparticle-free water to obtain exosome concentration within the range of 5 × 107 to 5 × 109 particle/ml. Samples were administered and recorded under controlled flow (infusion rate of 100) using a NanoSight™ syringe pump and script control system. The ambient temperature was set at 25°C, with the camera sensitivity and detection threshold set between 9 to 12 for maximum particle detection. Five different videos of 60 seconds from 3 different replicates were collected and analysed using NTA-software (version 2.3) for each sample.
All samples were diluted with nanoparticle-free water to obtain exosome concentration within the range of 5 × 107 to 5 × 109 particle/ml. Samples were administered and recorded under controlled flow (infusion rate of 100) using a NanoSight™ syringe pump and script control system. The ambient temperature was set at 25°C, with the camera sensitivity and detection threshold set between 9 to 12 for maximum particle detection. Five different videos of 60 seconds from 3 different replicates were collected and analysed using NTA-software (version 2.3) for each sample.
8
Nanoparticle Tracking Analysis of Vesicles
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Concentration and size of vesicles present in conditioned medium were analysed by NTA using the NanoSight LM10 system (NanoSight, Malvern Instruments, Malvern, UK) configured with a 405 nm laser and a high-sensitivity sCMOS camera (OrcaFlash2.8, Hamamatsu C11440). Videos were collected and analysed using the NTA software (version 3.1), with the minimal expected particle size, minimum track length and blur setting, all set to automatic. Ambient temperature was recorded manually and did not exceed 25°C. Each sample was diluted 1:100 in particle-free PBS and had a final volume of 1.0 mL. For each sample, 5 videos of 60 seconds duration were recorded generating replicate histograms that were averaged.
9
Caffeine-Induced Calcium Transients
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SR Ca2+ content was assessed by rapid applying 10 mM caffeine and measuring the resulting Ca2+ transients in isolated cardiomyocytes. Fura-2 acetoxymethyl ester (Fura-2)-loaded myocytes were alternately excited at 340 and 380 nm using a Lambda DG-4 ultra-high-speed wavelength switcher (Sutter Instrument) coupled to an inverted microscope IX71 (Olympus) with a UApo 20×/0.75 objective lens (Olympus). The Fura-2 fluorescent signal was recorded by ORCA-Flash 2.8 (Hamamatsu Photonics) and analyzed using a ratiometric fluorescence method with MetaFluor software (version 7.7.5.0; Molecular Devices).
10
Nanoparticle Characterization by Tracking
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Particles present in samples were analysed by nanoparticle tracking, using the NanoSight LM10 system (NanoSight Ltd, Amesbury, UK), configured with a 405 nm laser and a high-sensitivity sCMOS camera (OrcaFlash2.8, Hamamatsu C11440, NanoSight Ltd). Videos were collected and analysed using the NTA software (version 2.3, build 0025), with the minimal expected particle size, minimum track length and blur setting, all set to automatic. Ambient temperature was recorded manually and did not exceed 25°C. Each sample was diluted in particle-free PBS and had a final volume of 0.4 ml. Samples were analysed within 15 min of the initial dilution with a delay of 10 seconds between sample introduction and the start of the measurement (16 ). Approximately 20–40 particles were in the field of view and the typical concentration was approximately 2×108–10×108 particles/ml for each measurement to keep within the linear range (14 (link)). For each sample, multiple videos of 60 seconds duration were recorded generating replicate histograms that were averaged. The typical number of completed and tracked events per measurements typically exceeded 2000, and 5 measurements taken per sample.
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