Andor TIRF &
Spinning Disk Confocal

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Hardware:

  • Yokogawa CSU-X1 spinning disk confocal scan head
  • Nikon Ti-E invertedmicroscope stand with Perfect Focus System, motorized stage and DIC and phase optics
  • Prior Lumen200 metal halide light source
  • 4 laser lines in Andor ALC:
    405nm, 488nm, 561nm, and 642nm
  • 2 Andor iXon 897E back-illuminated EM-CCD CCD cameras
  • Andor Zyla 5.5 sCMOS camera
  • MetaMorph aquisition software
  • Andor iQ Live Cell Imaging software
  • Pathology Devices Live Cell stage-top incubations system
  • Mad City Labs stage-top piezo focus
  • Andor FRAPPA photamanipulation system

Filters:

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

TIRF imaging:

Software setting Excitation Dichroic Emission
TIRF 405 405nm 50mW Quad 450/50nm
TIRF 488 488nm 50mW    
TIRF 561 561nm 50mW    
TIRF 640 642nm 100mW    
Epi DIC Tungsten halogen   'scope Analyzer
Epi GFP      
Epi mCherry      
*Epi CFP1 ET436/20x T455lp ET480/40m
*Epi YFP5 ET500/20x T515lp ET535/30m
*Epi FRET6 ET436/20x T455lp ET535/30m

Confocal imaging:

Software setting Excitation laser Dichroic EM-CCD Emission sCMOS Emission
Conf 405

405nm 50mW

405/488/568/647

447/60nm
(dual w RFP)

447/60nm
Conf 488 488nm 50mW 405/488/568/647 525/40nm 525/50nm
Conf 561 561nm 50mW 405/488/568/647 607/36nm 617/73nm
Conf 640 642nm 100mW 405/488/568/647 685/40nm 685/40nm
*Conf CFP 405nm 50mW 405/488/568/647 483/35 482/35nm
DIC Tungsten Halogen   'scope Analyzer 'scope Analyzer

If you need alternate wavelength combinations, contact Wendy for assistance.
* This is not optimal for CFP but works.

Objectives:

Mag. NA

Immersion Medium

Corrections +DIC prism **TIRF Scaling
(um/pixel)
**SDC EMCCD
Scaling
(um/pixel)
**SDC sCMOS Scaling
(um/pixel)
10x 0.30 Air Plan Fluor Ph 1     0.692
40x 1.3 Oil Plan Apo H 0.390 0.345 0.168
60x 1.4 Oil Plan Apo H 0.268 0.235 0.114
60X TIRF 1.49 corr Oil Plan Apo TIRF IR 0.268 0.235 0.114
100x@ 1.4 Oil Plan Apo H 0.158 0.142

0.070

100x TIRF 1.49 corr Oil Plan Apo TIRF IR 0.158 0.142 0.070
100x 1.4 Oil Plan Apo Ph 3 0.158 0.142

0.070

*20X ELWD 0.45 corr Air Plan Fluor --      
*40x 0.95 Air Plan Apo H      

corr = objective has a correction collar that must be adjusted to prevent spherical aberration.
+ DIC prisms are installed as needed. See Wendy for instructions on how to install and uninstall.
@
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.
* Objectives with an asterisk are not installed by default. See Wendy if you require this lens.
** 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, LabTek II chambers, 35mm dishes, 60mm dishes
Multipwell plate Any standard 96-well plate format
Piezo stage Multiwell plate, slide, LabTek II, 35mm dishes

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.

 

 
 

 

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For website assistance please contact Nicki Watson