STED imaging was performed on an Abberior Instruments ‘Expert Line’ gated-STED coupled to a Nikon Ti microscope. DIV10 hippocampal neurons were imaged at a fixed distance of 80-100 μm from the cell body, with an oil-immersion 60x 1.4NA Plan-Apo objective (Nikon, Lambda Series) using confocal and STED modes. The system featured 40 MHz modulated excitation (405, 488, 560, and 640nm) and depletion (775nm) lasers. The microscope’s detectors were avalanche photodiode detectors. The 2D vortex STED images with lateral resolution enhancement were recorded with 20nm pixel size in xy, and the pinhole was set to 0.8 Airy units. To analyze ring periodicity, the maximum intensity of peaks was determined and the interpeak distance was measured.
Expert line gated sted
Manufactured by Nikon
The Expert Line gated-STED is a specialized lab equipment product from Nikon. It utilizes the stimulated emission depletion (STED) technique to enable super-resolution microscopy, allowing for imaging beyond the diffraction limit of light. The core function of this product is to provide high-resolution visualization and analysis of microscopic samples.
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Lab products found in correlation
Plan apo objective, by Nikon (7 mentions)
Ti microscope, by Nikon (4 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Axio imager z1, by Zeiss (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Lsm 710 confocal microscope, by Leica (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Huygens software, by Scientific Volume Imaging (1 mentions)
Star 635p, by Abberior (1 mentions)
Autoquant x software, by Media Cybernetics (1 mentions)
Zen 2008, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Plan apo, by Nikon (1 mentions)
8 protocols using expert line gated sted
1
Quantifying Neuronal Membrane Structure via STED
2
Three-Dimensional Imaging of Adult Ovary
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Adult ovaries were processed according to standard procedures (35 (link), 62 (link), 63 (link)). Images were acquired using a Zeiss LSM710 Confocal microscope and the Leica laser scanning confocal microscope TCS SP8, deconvolved using Huygens Software (Scientific Volume Imaging B.V.), projected using Fiji Software, and treated with Adobe Photoshop 2017 (Adobe Microsystems). Three-dimensional projections were generated with LAXs software from immunofluorescence images acquired using the Leica laser scanning confocal microscope TCS SP8. In Coherent-hybrid STED (CH-STED) Superresolution Imaging, 90% glycerol, 0.5% N-propyl gallate, and 20 mM Tris⋅HCl (pH 8) were used as mounting medium. The superresolution CH-STED images were acquired using Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope, an oil-immersion 60× 1.4 numerical aperture Plan-Apo objective (Nikon, Lambda Series), and a pinhole size of 0.8. The coherent-hybrid mode was previously described (66 (link)) and is set by imprinting a bivortex phase map (ρ = 0.92) onto a spatial light modulator (Hamamatsu LSH 0801392). Used antibodies are shown in SI Appendix, Table 1F .
3
Gated-STED Confocal Imaging Protocol
4
Multicolor Super-Resolution Microscopy
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For CH-STED microscopy, cells were grown as described above. Parental RPE-1 cells were seeded in the day before the experiment in coverslips coated with FBN. After fixation with 4% paraformaldehyde in cytoskeleton buffer, the cells were extracted in PBS with 0.5% Triton-X100 (Sigma-Aldrich). The coverslips were incubated with the primary antibodies (rabbit anti-TPR, 1:100, NB100-2867; and mouse anti-NUPs, 1:100, 24609; Abcam) in blocking solution overnight at 4°C. After washing with PBS-0.1% Triton-X, the coverslips were incubated with a 1:100 dilution of secondary antibodies (Abberior anti-rabbit STAR 580, cat. no. 2-0012-005-8, and Abberior anti-mouse STAR 635P, cat. no. 2-0002-007-5) at RT for 1 h. Later, coverslips were washed in PBS with 0.1% Triton-X100 and sealed on a glass slide using mounting medium (20 nM Tris pH 8, 0.5 N-propyl gallate, and 90% glycerol).
The images were acquired with an Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope. For all the acquisitions, we used an oil-immersion, 60× 1.4NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units. The CH-STED technique creates an orthogonal direction on the STED parametric space that enables the independent tuning of both resolution and contrast using only one depletion beam in a standard STED setup (circular polarization based).
The images were acquired with an Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope. For all the acquisitions, we used an oil-immersion, 60× 1.4NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units. The CH-STED technique creates an orthogonal direction on the STED parametric space that enables the independent tuning of both resolution and contrast using only one depletion beam in a standard STED setup (circular polarization based).
5
Super-Resolution Imaging Techniques for 3D
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3D wide-field images were acquired using an AxioImager Z1 (100× Plan-Apochromatic oil differential interference contrast objective lens, 1.46 NA, Carl Zeiss Microimaging Inc.) equipped with a CCD camera (ORCA-R2, Hamamatsu) operated by Zen software (Carl Zeiss, Inc.). Blind deconvolution of 3D image datasets was performed using Autoquant X software (Media Cybernetics). Confocal and super-resolution CH-STED images were acquired using Abberior Instruments “Expert Line” gated-STED coupled to a Nikon Ti microscope. An oil-immersion 60× 1.4 NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units were used in all confocal acquisitions. Super-resolution images were acquired using a CH-STED beam (Pereira et al., 2019 (link)). A second LSM800 confocal microscope (Carl Zeiss Microimaging Inc.) mounted on a Zeiss-Axio imager Z1 equipped with plan-apochromat 63×/1.40 oil differential interference contrast (DIC) M27 objective (Carl Zeiss, Inc.) was used and operated by Zen 2008 software (Carl Zeiss, Inc.).
6
Super-Resolution Imaging of Axon Diameters
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STED imaging was performed in an Abberior Instrument 'Expert Line' gated-STED coupled to a Nikon Ti microscope with an oil-immersion 60 × 1.4NA Plan-Apo objective (Nikon, Lambda Series) and a pinhole size set at 0.8 Airy units. The system features 40 MHz modulated excitation (405, 488, 560 and 640 nm) and depletion (775 nm) lasers. The microscope's detectors are avalanche photodiode detectors which were used to gate the detection between 700 ps and 8 ns. Typically, STED images were obtained near the entrance and exit of the microchannel patterns. After STED imaging, microchannel regions were fully imaged in a widefield microscope (20× objective) for mapping of the culture topology. This allowed for the precise measure of the STED image localization, in relation to the microchannel length. Axons were identified based on the Tau specific staining and the presence of periodic actin rings within the membrane periodic skeleton. The diameter of axons focused on the maximum wide plan was measured manually. Per axon, at least five measures were acquired perpendicularly to the longitudinal axon axis by connecting the brighter pixels (most often actin rings).
7
STED Imaging of Axonal Periodic Skeleton
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STED imaging was performed in an Abberior Instrument 'Expert Line' gated-STED coupled to a Nikon Ti microscope with an oil-immersion 60x 1.4NA Plan-Apo objective (Nikon, Lambda Series) and a pinhole size set at 0.8 Airy units. The system features 40 MHz modulated excitation (405, 488, 560 and 640nm) and depletion (775nm) lasers. The microscope's detectors are avalanche photodiode detectors (APDs) which were used to gate the detection between 700ps and 8ns.
Typically, STED images were obtained near the entrance and exit of the microchannel patterns.
After STED imaging, microchannel regions were fully imaged in a widefield microscope (20× objective) for mapping of the culture topology. This allowed for the precise measure of the STED image localization, in relation to the microchannel length. Axons were identified based on the Tau specific staining and the presence of periodic actin rings within the membrane periodic skeleton (MPS). The diameter of axons focused on the maximum wide plan was measured manually. Per axon, at least 5 measures were acquired perpendicularly to the longitudinal axon axis by connecting the brighter outer pixels (most often actin rings).
Typically, STED images were obtained near the entrance and exit of the microchannel patterns.
After STED imaging, microchannel regions were fully imaged in a widefield microscope (20× objective) for mapping of the culture topology. This allowed for the precise measure of the STED image localization, in relation to the microchannel length. Axons were identified based on the Tau specific staining and the presence of periodic actin rings within the membrane periodic skeleton (MPS). The diameter of axons focused on the maximum wide plan was measured manually. Per axon, at least 5 measures were acquired perpendicularly to the longitudinal axon axis by connecting the brighter outer pixels (most often actin rings).
8
Advanced CH-STED Microscopy Protocol
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For CH-STED microscopy, cells were grown as described above. Parental RPE-1 cells were seeded in the day before the experiment in coverslips coated with FBN. After fixation with 4% paraformaldehyde in cytoskeleton buffer the cells were extracted in PBS with 0.5% Triton-X100 (Sigma-Aldrich). The coverslips were incubated with the primary antibodies (rabbit anti-TPR, 1:100, NB100-2867; and mouse anti-NUPs, 1:100, Abcam 24609) in blocking solution overnight at 4º C. After washing with PBS-0.1%Tríton-X, the coverslips were incubated with the secondary antibodies (Abberior anti-rabbit STAR 580, 1:100, and STAR and Abberior anti-mouse STAR 635P, 1:100) at room temperature for 1h. Later, coverslips were washed in PBS with 0.1% Triton-X100 and sealed on a glass slide using mounting medium (20nM Tris pH 8, 0.5 N-propyl gallate, 90% glycerol).
The images were acquired with an Abberior Instruments "Expert Line" gated-STED coupled to a Nikon Ti microscope. For all the acquisitions, an oil-immersion 60x 1.4NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units were used. The CH-STED technique creates an orthogonal direction on the STED parametric space that enables the independent tuning of both resolution and contrast using only one depletion beam in a standard STED setup (circular polarization based).
The images were acquired with an Abberior Instruments "Expert Line" gated-STED coupled to a Nikon Ti microscope. For all the acquisitions, an oil-immersion 60x 1.4NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units were used. The CH-STED technique creates an orthogonal direction on the STED parametric space that enables the independent tuning of both resolution and contrast using only one depletion beam in a standard STED setup (circular polarization based).
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