Andor TIRF & Spinning Disk Confocal
- 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 or contact Cassandra.
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 |
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 Cassandra 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 different objective 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.