Nikon Crest Spinning Disk Confocal Microscope
The microscope is configured for fast live cell imaging with added capabilities for ROI photo-activation, FRAP, FRET and TIRF. It is is specialized for fast imaging of live cells.
System specifications
- Nikon Ti2 inverted microscope with Perfect Focus 4 (PFS4) mechanism
- Motorized XY Stage, allowing large images and multi-point time-lapse imaging
- Crest X-Light V3 (15,00 rpm, 7 laser lines, ~800 mw/line)
- fast piezo Z stage (100 um travel)
- iLAS2 circular TIRF illuminator : 488/561/640 nm (60/50/40 mW)
- iLAS2 FRAP/Photostimulation : 405/473 nm (50/30 mW)
- WideField Imaging with large field of view (KINETIX 10MP CMOS 25 mm, 6.5 um pixel 3200x3200 pixels, 95% QE)
- Okolabs blackout enclosure w/ environmental control (temperature and CO2)
- High Content Screening and Throughput using the JOBS/HCS module in Elements; allowed to apply automatic screening of multi-wells plates / Tracking of object over time using the xy stage / Phenotypic screening etc.
Software
- NIS-Elements
Free NIS-Elements viewer:
https://www.microscope.healthcare.nikon.com/products/software/nis-elements/viewer
More information here:
Objectives
Name | Magnification | NA | Immersion | working distance |
PLAN APOCHROMAT/DIC | 4x | 0.20 | air | 20 mm |
PLAN APOCHROMAT/DIC | 10x | 0.45 | air | 4.0 mm |
PLAN APOCHROMAT/DIC | 20x | 0.8 | air | 0.80 mm |
PLAN APOCHROMAT/DIC | 40x | 0.95 | air | 0.17-0.25 mm |
PLAN APO TIRF/DIC | 60x | 1.49 | oil | 0.12 mm |
PLAN APO TIRF/DIC | 100x | 1.49 | oil | 0.12 mm |
Confocal lasers: 7-Line Solid-State Celesta Laser Unit
- 365 nm
- 440 nm
- 488 nm
- 514 nm
- 561 nm
- 640 nm
- 730 nm
Widefield illuminator
- Lumencor SPECTRA III (LED)
- 385 nm
- 475 nm
- 550 nm
- 621 nm
fluorescence emission filter | ||||
Description
| ||||
DAPI | 387/11 | |||
CFP | 430/24 | |||
Alexa488 | 490/20 | |||
YFP | 500/20 | |||
Alexa555 | 555/25 | |||
CY5 | 645/30 | |||
GFP/mCherry | "GFP" 470/40, "mCherry" 573/35 | |||
FURA2-C | 340/380 | |||
Cy7 | 809/81 |
Detection
- KINETIX 10MP CMOS 25 mm, 6.5 um pixel 3200x3200 pixels, 95% QE
Funding
This microscope was funded by Stanford University's c-ShARP initiative, David Lenzi PI / Jon Mulholland co-PI. Installation date: 01/2024
What to put in Material and Methods section (example for SDC)
Cells were cultivated in tissue culture dish with No. 1.5 cover glass bottom (FluoroDish) and mounted in an environmental chamber (in vivo scientific) for acquisition at 37°C and 5% CO2. DMEM media with 25mM Hepes (pH 7.2) and without phenol red was used during image acquisition, with a layer of mineral oil on top of the media to prevent evaporation. All image were collected with a Yokogawa spinning disk confocal on a Nikon Eclipse-TI inverted microscope (Nikon) equipped with a PLAN APO-TIRF 100X 1.49 N.A. oil immersion objective and the Perfect Focus System for continuous maintenance of focus. XX-EGFP fluorescence was excited with the 488nm line from a 50mW Cobolt Blues laser and collected with a quadruple band pass dichroic mirror (Semrock) and a 525/30 emission filter (Semrock). Images were acquired with a Photometrics Prime95B sCMOS camera controlled with NiS-Elements software. For timelapse experiments, images were collected every 1 min, using an exposure time of 500 ms and 2x2 binning, with illumination light shuttered between acquisitions. At each time point, 6 z-series optical sections were collected with a step size of 0.5 microns, using a piezo Z-axis stage (Mad City Labs). ZGamma, brightness, and contrast were adjusted on displayed images (identically for compared image sets) using NIS-Elements software.
Short instruction videos on how to use the microscope
Starting up the microscope
Cleaning the objective
Login into the workstation
Turning on CO2
Location