Protein fluorescence was detected by confocal laser-scanning microscopy using a Leica TCS-SP5 II setup consisting of an inverted confocal laser-scanning microscope DMI6000 (Leica) equipped with an × 63/1.2 water immersion lens (HCX PL APO lambda blue 63.0 × 1.20 Water UV). The following filters were used: mVenus, excitation 514 nm (Argon laser), scanning 525–550 nm; mCherry, excitation 561 nm (DPSS Laser), scanning 605–638 nm. Chlorophyll autofluorescence was detected at 690–740 nm (Supplementary Table 2 ). Image acquisition was performed using the Leica software (Leica Application Suite – Advanced Fluorescence 2.6.0.7266; Leica Microsystems). Images are shown in RGB mode and brightness and contrast were adjusted using Adobe Photoshop CS3 software.
Application suite advanced fluorescence 2
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Leica Application Suite – Advanced Fluorescence 2.6.0.7266 is a software package designed for image acquisition, analysis, and processing of fluorescence microscopy data. The software provides a comprehensive suite of tools for researchers to capture, visualize, and quantify fluorescence signals in their samples.
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6 protocols using application suite advanced fluorescence 2
1
Confocal Microscopy Analysis of Protein Fluorescence
2
Confocal Imaging of FRET Biosensors
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High-resolution imaging was performed as previously described [76 (link)], with a Leica DMI 6000B inverted microscope equipped with a Leica TCS SP5 laser scanning device and HDy, using the Leica confocal software (Leica Application Suite–Advanced Fluorescence 2.6.0.7266; Leica Microsystems, Wetzlar, Germany). For excitation, an argon laser with a 458-nm line was used. The CFP and FRET emissions were collected at 473–505 and 526–536 nm, respectively. Images were acquired with a 25x objective (HCW RAPO L 25.0 × 0.95 water). Image acquisition was conducted as follows: scanning speed (400 Hz), image dimension (512 × 512), pinhole (2–4 airy unit), and line average (4). YFP and CFP images were acquired as a time series in a 6-s interval. Offline calculation of the FRET ratio was performed using ImageJ RATIOPLUS plug-in.
3
CFSE-Based Cell Proliferation Assay
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Cells were seeded on a Petri dish as described earlier. Twenty-four hours after seeding, control and treated cells were stained with CFSE fluorescent dye (2 µM) within 3 to 4 h after adding DDC+B12b. Images were obtained using a TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) and analyzed in Leica Application Suite Advanced Fluorescence 2.1.0 software (Leica Microsystems).
4
Fluorescent Microscopy of Cultured Cells
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Cells were seeded on cover glasses inside a Petri dish as described earlier. Twenty-four hours after seeding, control cultures and cultures treated with DDC + B12b were stained with fluorescent dyes according to manufacturers’ recommendations. Images were obtained using a TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) and analyzed by the Leica Application Suite Advanced Fluorescence 2.1.0 software (Leica Microsystems).
5
Imaging Cerebral Microvascular Permeability
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Mice were anesthetized, operated on to construct a thin cranial window (3 mm in diameter), fixed on a custom-fabricated metal frame and placed under a two-photon laser scanning microscope (Leica, Germany)equipped with a water-immersion objective lens (25×). Data acquisition and laser scanning were performed using Leica Application Suite Advanced Fluorescence 2.5 software, at a wave length of 860 nm. To monitor the cerebral microvascular permeability using detection of leaked dyes, Rhodamine B isothiocyanate-dextran (1.4% in saline, 70 kDa molecular weight, Sigma-Aldrich) was injected intravenously to visualize the brain vasculature. We selected the red fluorescence channel for detection, and calculated the average fluorescence intensity in the extravascular compartment. Images of the XYZ stacks (512 × 512 pixels) were collected to a depth of 200 μm (2-μm step size) below the cortical surface, at 5and 10 min after the injection. We defined the vessels with a diameter of 20-40um as microvascular.
6
Thinned-Skull Imaging of BBB Permeability
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Age-matched mice (8-10 weeks) were exposed to the thinned-skull window preparation as described in previous studies [30] (link). In brief, mice were anesthetized and xed with a custom-fabricated metal frame by holding the head with a cyanoacrylate and dental cementon the stage of Leica DM6000 CFS (Lecia Germany) equipped with a water-immersion objective lens (25X). The skull over right somatosensory cortex was carefully thinned to ~ 20-30 µm within an area 3 mm in diameter. Imaging was performed within 30min of window construction. Data acquisition and laser scanning were performed using Leica Application Suite Advanced Fluorescence 2.5 software, at a wavelength of 860 nm. To monitor the BBB permeability using detection of leaked dyes, 100µL
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