Cells were imaged using a Nikon A1R HD confocal with a four-line (405 nm, 488 nm, 561 nm, and 640 nm) LUN-V laser engine and DU4 detector using bandpass and long-pass filters for each channel (450/50, 525/50, 595/50 and 700/75) mounted on a Nikon Ti2 using an Apo 100× 1.49 NA objective and operated using NIS Elements software. Image stacks were acquired in Galvano mode with unidirectional scanning with a 488 nm laser at 1.5% power with a frame size of 512×512 at scan zoom, 1 frame per second (fps), and 97.1 μm pinhole size. Small regions of interest (ROIs) for stimulation were drawn over the punctate structures and in the cytosol. The total FRAP series contained 3 images before bleaching (obtained at 2 s intervals), 2 cycles of ROI bleaching with the 488 nm laser at 100% laser power (5 frames at 1 fps), and 2 min of continuous acquisition to monitor fluorescence recovery.
Apo 100 1.49 na objective
Manufactured by Nikon
The Apo 100× 1.49 NA objective is a high-performance objective lens designed for advanced microscopy applications. It features a numerical aperture (NA) of 1.49, which allows for high-resolution imaging and excellent light-gathering capabilities. The 'Apo' designation indicates that it is an apochromatic lens, which means it has been designed to minimize chromatic aberrations for enhanced image quality.
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2 protocols using apo 100 1.49 na objective
1
Confocal Imaging and FRAP Analysis
2
Multicolor Confocal Imaging of Cells
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Cells were imaged using a Nikon A1R HD confocal microscope with a four-line (405 nm, 488 nm, 561 nm, and 640 nm) LUN-V laser engine and DU4 detector using bandpass and longpass filters for each channel (450/50, 525/50, 595/50, and 700/75), mounted on a Nikon Ti2 using an Apo 100× 1.49 NA objective. Image stacks were acquired in resonant mode with bidirectional scanning and 2× or 4× line averaging. The lasers used were 405 nm, 488 nm, 561 nm, and 640 nm. To avoid cross-talk between channels, z-stacks were acquired of the AlexaFluor 568 or tagRFP channel first, and the sfGFP or Alexa Fluor 488 and Alexa Fluor 647 channels were acquired subsequently. Illumination and image acquisition was controlled by NIS Elements Advanced Research software (Nikon Instruments).
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