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Nikon Spinning Disk Confocal Microscope

RESERVE THIS MICROSCOPE


The microscope is configured for simultaneous, two channel fast imaging with added capabilities for ROI photo-activation, FRAP, FRET and TIRF. This imaging system is specialized for fast imaging of live cells.

 

System specifications:

 

  • Nikon TiE inverted microscope with Perfect Focus (PFS) mechanism
  • Motorized XY Stage, allowing large images and multi-point time-lapse imaging
  • Yokogawa CSU-X1 (1,800 rpm, 360 fps)/Borealis Upgrade/405/488/561/642 multichroic
  • fast piezo Z stage (100 um travel, 15 ms step response)
  • Nikon motorized TIRF illuminator
  • WideField Imaging with large field of view (2560x2160 pixels / 16.6x14.0 mm)
  • InVivo Scientific 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

 

NIS-Elements free viewer download page here:

http://www.nis-elements.com/resources-downloads.html

 

Objectives :

 

Name

Magnification

NA

Immersion

working distance

PLAN APO

4x

0.20

air

20 mm

PLAN APO

20x

0.75

air

1.00 mm

PLAN APO

40x

1.15

water

0.61 mm

PLAN APO IR

60x

1.27

water

0.17 mm

PLAN APO

60x

1.40

oil

0.13 mm

PLAN APO-TIRF

100x

1.49

oil

0.12 mm

 

Confocal lasers:

 

  • 405nm (100 mW)
  • 488nm (50 mW)
  • 561nm (50 mW)
  • 642nm (100 mW)

 

Widefield illuminator:

 

  • Lumencor SPECTRA III (LED)

 

  • 390/22nm (500 mW)
  • 440/20nm (500 mW)
  • 475/28nm (500 mW)
  • 510/25nm (400 mW)
  • 555/28nm (500 mW)
  • 575/25nm (500 mW)
  • 637/12nm (500mW)
  • 748/12nm (500mW)

 

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:

 

  • Photometrics Prime94B sCMOS camera for the Spinning Disk
  • Andor Neo sCMOS camera for the wide field and the TIRF

 

Funding:

 

This microscope was funded by jointly by the SOE and the Beckman Center (Dr. Alex Dunn and Jon Mulholland, PIs). Installation date: 09/2012

 

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


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