Error Status code: 401
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Manufacturer: Andor
Model: Revolution WD
Year Installed: 2013
Camera 1: Andor Zyla 4.2 sCMOS
Camera 2: Andor iXon3 888 EMCCD 1024x1024
Camera aux: IDS Monochrome UI-3370CP-M-GL
Piezo Z Stage: PI 737.2SL - range: 220µm - Resolution: 1 nm
Keywords:
Spinning disk confocal microscopy is rapidly emerging as the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems are capable of providing high acquisition speeds with good contrast and minimal photobleaching at the low light levels available with this technique.
In this system, a spinning disk with multiple small holes is installed between the light source and specimen to excite point-like sources on specimen. The same hole serves as a detecting pinhole to remove out-of-focus light.
The Andor Spinning W1 system is a spinning disk laser confocal microscope with a sensitive EMCCD camera suitable for fast in vivo imaging. It's one of the fastest and most sensitive system at the institute. The system can also scan multiple positions in the sample, with multiple channels in timelapse mode. In order to stay in focus it also includes a perfect focus system to correct for vertical drift during long movies.
Confocal Z-series stacks were acquired on a Yokogawa CSU-W Spinning Disk confocal, using a 60x 1.49NA Oil immersion objective, using the 488nm and 561nm laser lines and a Andor Zyla 4.2 sCMOS camera where used to acquire images for the emission of the Alexa488 and Alexa 568 flurochromes.
We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).
Configuration |
Value |
Laser |
Excitation lines |
Maximum power |
Diode 405 |
405 nm |
100 mW |
DPSS 488 |
488 nm |
100 mW |
DPSS 561 |
561 nm |
50 mW |
Diode 640 |
640 nm |
100 mW |
Magnification |
Objective type |
NA1 |
Immersion |
Working distance (mm) |
Resolution3 @525nm in XY (µm) |
Pixel size (µm) |
Pixel size * 1.5x (µm) |
Nyquist Sampling @525nm in Z (µm) |
Link |
|||
Zyla |
iXon |
Zyla |
iXon |
|||||||||
10x ![]() |
PLAN DL |
0.25 |
- |
10.5 |
0.840 |
0.65 |
0.65 |
0.43 |
0.43 |
1.260 |
||
20x ![]() |
PLAN Fluor |
0.75 |
MImm2 |
0.35 |
0.280 |
0.33 |
0.33 |
0.22 |
0.22 |
0.420 |
||
40x |
Apo λ S LWD |
1.15 |
Water |
0.61-0.59 |
0.183 |
0.16 |
0.16 |
0.11 |
0.11 |
0.274 |
||
40x ![]() |
PLAN Fluor |
1.30 |
Oil |
0.2 |
0.162 |
0.16 |
0.16 |
0.11 |
0.11 |
0.242 |
||
60x ![]() |
Apo TIRF |
1.49 |
Oil |
0.12 |
0.141 |
0.11 |
0.11 |
0.07 |
0.07 |
0.211 |
||
60x ![]() |
PLAN APO VC |
1.20 |
Water |
0.31-0.28 |
0.175 |
0.11 |
0.11 |
0.07 |
0.07 |
0.263 |
Remember to choose the proper calibration if doing montages/size measurements.
The pixel size in microns of your picture is saved automatically (if you've chosen the correct calibration). Use LOCI to recognize it with ImageJ.
(1) NA - Numerical Aperture. For more information, follow this link.
(2) MImm: Multi Immersion (water,oil,glycerin). Speak with the UIC personnel if you need to change the immersion media.
(3) These values were calculated using the Resolution formula adapted to confocals as seen in the Olympus micro website: R = 0.4*λ/NA
The pixel size in microns of your picture is saved automatically when using µManager. Use Bio-formats to recognize it with ImageJ/Fiji (already installed in the Fiji package). When using Andor iQ, you have to input the values manually.
Name |
Transmission |
Description |
iQ number |
Link |
Quad Semrock |
440/521/607/700-25 |
Quadruple Filter |
1 |
|
DAPI Semrock |
447/60 |
DAPI filter |
2 |
|
GFP Semrock |
525/30 |
GFP or FITC filter |
3 |
|
RFP Semrock |
607/36 |
RPF or TRITC filter |
4 |
|
Cy5 Semrock |
685/40 | Cy5/Far red filter |
5 |
|
Empty |
- |
- |
6 |
- |
Cy5 LP Chroma |
655 LP |
Cy5 Long Pass (High efficiency) |
7 |
|
RFP Chroma |
595/50 |
RFP or TRITC filter (High efficiency) |
8 |
|
GFP Chroma |
525/50 |
GFP or FITC filter (High efficiency) |
9 |
|
DAPI Chroma |
450/50 |
DAPI filter (High efficiency) |
10 |
Turn on the cameras water cooling system (1 switches) and wait until it cools down to 15ºC. This should turn on both components of the water cooling system, if this does not happen, tell us as soon as possible!
Important:This should be done at least 10 to 15min before starting and you cannot start until it's at 15ºC.
Turn on the Main Cabinet key and switches.
Turn on the Power Bars.
Turn on the Spinning Disk scanhead by rotating the key.
Turn on the cameras. Only if the cooler is at 15ºC.
Press the button for the EMCCD and the switch for the sCMOS.
When you are finished and there is someone immediately after you, leave the entire system on. If you're the last or only person of the day, see the turn off procedures below.>
Save all you data and close all open windows.
Save all the files in the server using the shortcuts provided in the desktop.
Log off from Windows.
If you can't focus, make sure you haven't clicked the escape button. After you click escape, the focus doesn't move unless you click refocus.
Avoid recording your settings if looking through the eyepiece: If you click record while the microscope port is set to the eyepiece, the software will mix-up the ports. It will also remember whatever filter cube you are using which will affect your pictures.
If you have problems in iQ, make sure the items below are correct in your settings (Device setup window):
Acquiring images in a spinning disk confocal microscope is a complex procedure, first time users are required to contact the UIC staff for training. If you need assistance don’t forget to press “with assistance” at reservation time.
It is possible to acquire grids and tiles of big samples through Andor iQ. For a more detailed protocol, please check the following link or click in the image to the right.
Nikon has a hardware autofocus system called Perfect Focus System (PFS) which allows continuous and constant sample focus using the coverslip position as the reference, for a faster and less damaging focusing system compared to normal software ones. For more detailed information, follow the link or click the image to the right side.