3D-SIM was performed on a 3D-SIM Imaging System (GE DeltaVision OMX; Applied Precision Ltd.) equipped with a 60× 1.42 NA objective (Olympus). Raw data were reconstructed using Softworx (Applied Precision Ltd.; GE Healthcare) and 0.003 for the wiener filter constant. TIRF-SIM imaging was performed as described previously (Beach et al., 2014 (link); Li et al., 2015 (link)). In brief, images were acquired using an inverted microscope (Axio Observer; ZEISS) fitted with a 100× 1.49 NA objective (Olympus) and a spatial light modulator to provide phase grating for structured illumination (Li et al., 2015 (link)). Confocal imaging was performed on an IX81 with a 150× 1.4 NA objective (Olympus) fitted with a spinning disk confocal unit (Yokogawa Electric Corporation). Linear adjustments to images were made using ImageJ software (National Institutes of Health).
Spinning disk confocal unit
Manufactured by Yokogawa
The Spinning disk confocal unit is a specialized piece of laboratory equipment designed for high-speed, high-resolution imaging. It utilizes a rotating disk with microscopic lenses to rapidly scan a sample, providing optical sectioning and enhanced contrast for fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Sp8x confocal microscope, by Leica (2 mentions)
Axio observer 7, by Yokogawa (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
1.42 na objective, by Olympus (1 mentions)
Axio observer, by Zeiss (1 mentions)
Tsa indirect kit, by PerkinElmer (1 mentions)
Masson s trichrome staining, by StatLab (1 mentions)
Tie inverted research microscope, by Nikon (1 mentions)
Ixon du897 ultra, by Oxford Instruments (1 mentions)
6 protocols using spinning disk confocal unit
1
Super-resolution Microscopy Techniques
2
Immunohistochemical Profiling of Breast Tumors
3
Multimodal Imaging of Plant Growth
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Seedlings were imaged on a Leica SP8X confocal microscope. GFP was excited at 488 nm and acquired at 500 to 530 nm. mCHERRY was excited at 594 nm and acquired at 600 to 650 nm.
For meristem imaging, roots were stained with propidium iodide (PI) (Sigma-Aldrich). Images were taken by 20× or 40× objectives. Vertical imaging was done using a Carl Zeiss Axio Observer.7 armed with a Visiscope spinning disk confocal unit based on Yokogawa CSU-W1-T2 equipped with a VS-HOM1000 excitation light homogenizer and a PRIME-95B Back-Illuminated sCMOS camera, and a Plan-Apochromat 20x/0.8 M27 objective. For time-lapse microscopy experiments with plants expressing pDWF4:DWF4-GFP, pCPD:CPD-GFP, pROT3:ROT3-GFP and pBES1:BES1-GFP, 6-day-old seedlings were transferred to a 3Dprinted triple microscopy chamber (5 seedlings to each) on ½MS agar blocks, supplemented with propidium iodide (PI) and imaged overnight at 25-min intervals. Time series were assembled with stitching of tiled 3D microscopic image acquisitions 53 (link) and subsequently analyzed with Fiji or LAS X software.
For meristem imaging, roots were stained with propidium iodide (PI) (Sigma-Aldrich). Images were taken by 20× or 40× objectives. Vertical imaging was done using a Carl Zeiss Axio Observer.7 armed with a Visiscope spinning disk confocal unit based on Yokogawa CSU-W1-T2 equipped with a VS-HOM1000 excitation light homogenizer and a PRIME-95B Back-Illuminated sCMOS camera, and a Plan-Apochromat 20x/0.8 M27 objective. For time-lapse microscopy experiments with plants expressing pDWF4:DWF4-GFP, pCPD:CPD-GFP, pROT3:ROT3-GFP and pBES1:BES1-GFP, 6-day-old seedlings were transferred to a 3Dprinted triple microscopy chamber (5 seedlings to each) on ½MS agar blocks, supplemented with propidium iodide (PI) and imaged overnight at 25-min intervals. Time series were assembled with stitching of tiled 3D microscopic image acquisitions 53 (link) and subsequently analyzed with Fiji or LAS X software.
4
Multimodal Imaging of Plant Growth
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Seedlings were imaged on a Leica SP8X confocal microscope. GFP was excited at 488 nm and acquired at 500 to 530 nm. mCHERRY was excited at 594 nm and acquired at 600 to 650 nm.
For meristem imaging, roots were stained with propidium iodide (PI) (Sigma-Aldrich). Images were taken by 20× or 40× objectives. Vertical imaging was done using a Carl Zeiss Axio Observer.7 armed with a Visiscope spinning disk confocal unit based on Yokogawa CSU-W1-T2 equipped with a VS-HOM1000 excitation light homogenizer and a PRIME-95B Back-Illuminated sCMOS camera, and a Plan-Apochromat 20x/0.8 M27 objective. For time-lapse microscopy experiments with plants expressing pDWF4:DWF4-GFP, pCPD:CPD-GFP, pROT3:ROT3-GFP and pBES1:BES1-GFP, 6-day-old seedlings were transferred to a 3Dprinted triple microscopy chamber (5 seedlings to each) on ½MS agar blocks, supplemented with propidium iodide (PI) and imaged overnight at 25-min intervals. Time series were assembled with stitching of tiled 3D microscopic image acquisitions 53 (link) and subsequently analyzed with Fiji or LAS X software.
For meristem imaging, roots were stained with propidium iodide (PI) (Sigma-Aldrich). Images were taken by 20× or 40× objectives. Vertical imaging was done using a Carl Zeiss Axio Observer.7 armed with a Visiscope spinning disk confocal unit based on Yokogawa CSU-W1-T2 equipped with a VS-HOM1000 excitation light homogenizer and a PRIME-95B Back-Illuminated sCMOS camera, and a Plan-Apochromat 20x/0.8 M27 objective. For time-lapse microscopy experiments with plants expressing pDWF4:DWF4-GFP, pCPD:CPD-GFP, pROT3:ROT3-GFP and pBES1:BES1-GFP, 6-day-old seedlings were transferred to a 3Dprinted triple microscopy chamber (5 seedlings to each) on ½MS agar blocks, supplemented with propidium iodide (PI) and imaged overnight at 25-min intervals. Time series were assembled with stitching of tiled 3D microscopic image acquisitions 53 (link) and subsequently analyzed with Fiji or LAS X software.
5
Multicolor Immunofluorescence of FFPE Tissues
6
Visualizing Bead Internalization in GFP-N-WASP Cells
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RF/6A cells were transfected with GFP–N-WASP, and at 23 h posttransfection, fresh phenol red-free Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum was added, and cells were incubated with flash red beads (0.51-µm diameter; Bangs Laboratories) coated with rEtpE-C (11 (link)). Samples were moved to a live-cell imaging chamber at 37°C connected to a TI-E inverted research microscope (Nikon Instruments) controlled by Nikon Elements software and equipped with a spinning-disk confocal unit (Yokogawa Electric), a 100× objective lens (Plan Apochromat Lambda, NA 1.45; Nikon), and an electron multiplying charge-coupled device (EMCCD) camera (iXon DU897 Ultra; Andor Technology). Three-dimensional confocal image stacks were acquired at 1-min intervals for 2 h, with a step size of 0.1 µm along the z axis. The bead internalization movie (see Movie S1 in the supplemental material) was prepared with ImageJ software by creating a stack of confocal images acquired at the upper surface of an RF/6A cell.
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