Similar experiments were performed as in multiple-particle tracking studies, while movies were captured under STED microscopy (Leica TCS SP8 STED 3X, Leica Microsystems, Germany). Unlike the tracking studies, STED movies focus on a single particle to show its moving pattern. Movies were processed in Imaris.
Leica tcs sp8 sted 3x
Manufactured by Leica Microsystems
Sourced in Germany
The Leica TCS SP8 STED 3X is a super-resolution confocal laser scanning microscope capable of achieving resolutions up to 30 nm. It features a unique STED (Stimulated Emission Depletion) technology that allows for the visualization of nanoscale structures within living cells.
Automatically generated - may contain errors
Lab products found in correlation
Nicolet 6700, by Thermo Fisher Scientific (3 mentions)
Escalab 250xi, by Thermo Fisher Scientific (2 mentions)
Axioskop 2 plus, by Zeiss (2 mentions)
X pert pro diffractometer, by Bruker (1 mentions)
Ht7700, by Hitachi (1 mentions)
Fls1000, by Edinburgh Instruments (1 mentions)
F 7000, by Hitachi (1 mentions)
Uh4150, by Hitachi (1 mentions)
Alexa fluor 488 labeled goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
15 mm glass bottom cell culture dish, by NEST Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Live dead assay, by Thermo Fisher Scientific (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Hyd hybrid detector, by Leica camera (1 mentions)
Anti rabbitigg h l alexa fluor 555 conjugate, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
12 protocols using leica tcs sp8 sted 3x
1
STED Microscopy Particle Tracking
2
Fluorescent Labeling of Salmonella
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We labeled the PG of Salmonella isolates with HADA as previously described (22 (link)). Cells were grown to exponential phase and incubated with HADA (final concentration, 0.5 mM) for 3 hours at 37°C. Cells were then fixed in 70% ethanol for 10 min to prevent potential cell stress resulting from the washing steps. Cells were collected by centrifugation (5000 rpm, 5 min) and washed three times with PBS (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) to remove excess dye. Approximately 20 μl of the samples was fixed on glass slides and dried at room temperature in the dark. The stained cells were observed using superresolution confocal microscopy (Leica TCS SP8 STED 3X, Leica Microsystems, Wetzlar, Germany). The samples were excited with a laser at 405 nm, and the emission was detected through a 419- to 465-nm emission filter.
3
Comprehensive Characterization of ZnO Nanocrystals
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The X-ray diffraction (XRD) was recorded by X’ Pert Pro diffractometer with Cu Kα radiation (D8 Discover, Bruker, Germany). The morphology of the sample is characterized by transmission electron microscopy (TEM) (HT7700, Hitachi, Japan). The emission spectra of the ZnO nanocrystals were analyzed by a fluorescence spectrophotometer (F-7000, Hitachi, Japan; FLS-1000, Edinburgh Instruments, UK). The UV-Vis absorption of the ZnO nanocrystals was performed by a spectrophotometer (UH4150, Hitachi, Japan). The hydrodynamic diameter distribution of the sample was acquired by a malvern zetasizer nano series (ZEN5600, Malvern, UK). FTIR spectra of the nanocrystals were measured by a spectrometer (Nicolet 6700, Thermo Fisher Scientific, USA). The XPS measurements were obtained by a spectrometer (EscaLab 250Xi, Thermo Fisher Scientific, USA). The PL decay were determined by a time-corrected single photon counter system (FLS-1000, Edinburgh Instruments, UK). The PL QY of the sample was measured by a calibrated integrating sphere of a spectrometer (FLS-1000, Edinburgh Instruments, UK). The cellular and mice muscle tissue imaging pictures were acquired at a laser scanning confocal microscope (Leica TCS SP8 STED 3X, Leica Microsystems, Germany).
4
Immunofluorescence Staining of CD9 Protein
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Cells were grown on coverslips in media and were fixed with 4% paraformaldehyde for 10 min. Then, the cells were washed thrice with PBS, permeabilized with 0.1% Triton X-100 for 15–20 min, and again washed thrice. Cells were then treated with 1% BSA in PBS (IH solution) for 45 min followed by incubation with anti-CD9 rabbit monoclonal antibody (1 μg/mL) in IH solution at 4 °C overnight. Then, the samples were thoroughly washed thrice with PBS and exposed to Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Thermo Scientific, USA). Fluorescence was measured using a laser scanning confocal microscope (Leica TCS SP8 STED 3X, Leica Microsystems, Germany).
5
Visualizing Protein-Protein Interactions
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HEK293T cells were grown on a 15-mm glass-bottom cell culture dish (NEST). Cells were transfected with 1 μg plasmid and rinsed briefly with ice-cold PBS after 24 h of transfection, followed by fixing cells with 4% paraformaldehyde in PBS for 20 min and washing three times with PBS. Then, the cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min, blocked with 1% BSA in PBS for 30 min, incubated with corresponded antibody in PBST for 2 h, and washed three times with PBS, followed by incubating cells with the Alexa Fluor™ 488 or 568 secondary antibody in PBST for 1 h and washing three times with PBS. The last two steps are not required if the transfected protein carries a fluorescent label. Next, 1 μg/ml DAPI (Sigma) was used to stain nuclei for 1 min followed by rinse with PBS. Then, the cells were imaged by Leica TCS SP8 STED 3X (Leica Microsystems). The protein–protein co-localization coefficients were calculated by Leica microscope software. Cells from different fields of view were randomly extracted to assess co-localization coefficient.
6
Fluorescence Recovery After Photobleaching Analysis
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Confocal microscopy and FRAP analyses were conducted with the Leica TCS SP8 STED 3X (Leica microsystems) using a 100×, N.A. 1.4 oil-immersion apochromatic objective. Samples were applied to a 15-mm glass-bottom cell culture dish (NEST). A circular area of 1 μm radius or a speckle was bleached with the 65 mW argon laser and the PMT was used as detector. After completing the three steps of set up (setting parameters for pre- and post-bleach imaging), bleach (defining parameters for bleaching) and time course (defining numbers of pre-bleach, bleach, and post-bleach intervals), which depend on the sample and the purpose of the experiment, we obtained the displayed recovery showing all intensity values averaged over the regions of interest for all frames. The mean and standard deviation of fluorescence density were obtained from cells with different fields of view.
7
VEGF and PGE2 Spheroid Viability Assay
8
Multi-color STED Microscopy Protocol
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STED imaging was performed with a Leica TCS SP8 STED 3X (Leica Microsystems) equipped with a HCPLAPO100x/1.40 Oil STED WHITE objective. For 2-color STED imaging, the fluorophores Alexa Fluor 594 and Abberior STAR 635P were excited by a pulsed white light laser with lines at 598 nm and 653 nm, respectively. The pulsed STED laser at 775 nm was used for both imaging channels. A confocal channel excited at 488 nm was also used to outline the cellular structure by actin staining with Phalloidin labelled with Alexa Fluor 488. The channels were recorded sequentially with a pixel size of 25 nm at line scan speeds of 200–400 Hz. Three-color STED microscopy was applied with fluorophores Alexa Fluor 594, Abberior STAR 635P and TRITC, excited by pulsed white light lasers lines at 598 nm, 653 nm and 540 nm, respectively, and depleted with 775 nm laser light. STED imaging with both 2D vortex and 3D phase-ring pattern were applied to improve the resolution in the imaging plane and along the optical axis.
9
Multimodal Microscopy Imaging Protocol
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Cells were observed with a Zeiss Axioskop 2 plus equipped with epifluorescence. Images were acquired with a CoolSnap camera coupled with Metavue. Confocal acquisitions were made with a Leica SP8 equipped a UV diode (line 405), and three laser diodes (lines 488, 552 and 635) for excitation and two PMT detectors. Images stacks were processed with imageJ and Photoshop. STED imaging was performed using a Leica TCS SP8 STED 3X (Leica Microsystems CMS GmbH, Mannheim, Germany). The system was equipped with a WLL ranging from 470 to 670 nm for excitation and with 3D STED lasers at 592, 660, and 775 nm. A 100× 1,4 Oil STED white objective was used to acquire the images. GFP, AF568, and Cy5 were excited at 488, 561, and 643 nm, respectively. Detection ranges were 500–550, 575–625, and 660–700 nm, respectively. A pixel size of 25 nm was used. For deconvolution, SVI Huygens was used.
10
Multimodal Imaging of Cellular Structures
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Cells were observed with an epifluorescence Zeiss Axioskop 2. Images were acquired with a CoolSnap camera coupled with Metavue. Confocal acquisitions were made with a Leica SP8 equipped a UV diode (line 405), and three diodes laser (lines 488, 552 and 635) for excitation and two PMT detectors. Images stacks were processed with Fiji and Photoshop.
STED imaging was performed using a Leica TCS SP8 STED 3X (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with a 100×1,4 Oil STED white objective, a WLL ranging from 470 to 670 nm for excitation and with three STED lasers at 592 nm, 660 nm and 775 nm. GFP and AF568 were excited at 488 nm and 561 nm and detection range were 500-550 nm and 575-625 nm respectively. STED was performed at 592 nm for GFP and 775 nm for AF568. Signals were recorded on Leica HyD Hybrid Detectors using time gated detection of 1.5-6.0 ns and 0.5-6.0 ns for GFP and AF568 respectively. A pixel size of 25 nm was used. For deconvolution, SVI Huygens was used.
STED imaging was performed using a Leica TCS SP8 STED 3X (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with a 100×1,4 Oil STED white objective, a WLL ranging from 470 to 670 nm for excitation and with three STED lasers at 592 nm, 660 nm and 775 nm. GFP and AF568 were excited at 488 nm and 561 nm and detection range were 500-550 nm and 575-625 nm respectively. STED was performed at 592 nm for GFP and 775 nm for AF568. Signals were recorded on Leica HyD Hybrid Detectors using time gated detection of 1.5-6.0 ns and 0.5-6.0 ns for GFP and AF568 respectively. A pixel size of 25 nm was used. For deconvolution, SVI Huygens was used.
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