RPI Spinning Disk Confocal

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RPI Spinning Disk Confocal microscope
Hardware
  • Yokogawa CSU-22 spinning disk confocal scan head with Andor Borealis modification
  • Zeiss AxioVert 200M inverted microscope stand with DIC optics
  • 6 visible laser lines in SAR/Andor ILE:
    405nm, 445nm, 488nm, 515nm, 561nm,and 642nm
  • Hamamatsu Orca-ER cooled CCD camera
  • MetaMorph aquisition software

 

Filters:

Not sure which fluorophores will work best? Use an online spectra viewer (see Links) or contact Cassandra.

Widefield for viewing samples by eye:

  • DAPI
  • FITC/GFP/Alexa488
  • Rhodamine/Alexa 568
  • DIC available for 100x, 63x, 40x. See Wendy for instructions.

Confocal imaging:

Software setting Excitation laser Dichroic Emission
DAPI

405nm 100mW OPSL

 405/488/568/647 450/50nm
GFP 488nm 150mW OPSL 405/488/568/647 525/50nm
RFP 561nm 100mW OPSL 405/488/568/647 605/70nm
Cy5 642nm 110mW OPSL 405/488/568/647 700/75nm
CFP 445nm 75mW Diode 405/440/515/647 480/40nm
YFP 515nm 100mW OPSL 405/440/515/647 535/30nm
DIC Transmitted light 405/488/561/647 Analyzer

If you need alternate wavelength combinations, contact Wendy for assistance.

 

Objectives:

Mag. NA

Immersion Medium

 Corrections DIC prism **Scaling
(um/pixel)
100x* 1.4 Oil Plan Apochromat III 0.057
63x 1.4 Oil Plan Apochromat III 0.092
40x 1.3 Oil Plan Apochromat III 0.14
25x 0.8 Water, Glycerol, or Oil Plan NeoFluar II 0.23
10x 0.30 Air EC-NeoFluar N 0.57

*The pinhole size in the spinning disk is optimized for the 100x 1.4NA objective. Use of a differentobjective reduces the confocality of the image.
** These values work well for making scale bars. When performing careful distance measurements, it is best practice to confirm the calibration for the exact setup you use.

 

Sample holders:

Stage plates Compatible Sample holders
Adjustable Slides, 35mm dishes
Multipwell plate Any standard 96-well plate format, LabTek II chambers

 

Software:

To view your files:

  • With Volocity: Generate a library then drag the .nd file(s) into the image list.
  • With Fiji (ImageJ), using BioFormats Importer to open the .nd file. See our Links page to download.