IGC

Andor Spinning disk W1 | Reservations

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.

Spinning disk mechanism

The Andor Revolution XD system is a spinning disk laser confocal microscope with a sensitive EMCCD camera suitable for fast in vivo imaging. It's 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.


Suggestion for description in "Materials and Methods":

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.

Suggestion for "Acknowledgments":

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).

Maintenance log:

  • 17-03-2016 - Yokogawa CSU-W1 unit returned from the manufacturer for cleaning and dust inset precautions.
  • 30-06-2015 - Andor technician came to the IGC to clean Spinning disks, dichroics and to instal Dust Free kit in the scanner unit.
  • 15_16-01-2015 - Remote call with Andor. Minor tweaks to the piezo range and working manner.
  • 15-05-2014 - Computer backup made as a system hard drive image.
  • 29-04-2014 - New objective installed - Nikon 40x/1.3NA Oil Plan Fluor.
  • 10-04-2014 - New CMOS (IDS 1'' chip) installed in the right side port of the microscope.
  • 10-04-2014 - New Andor Zyla 4.2 replaced the malfuncioning one.
  • 18-03-2014 - Room structure changed and microscope moved to the back of the room.
  • 18-03-2014 - New Light Path prism installed at the microsocpe (R80/L20).
  • 17-03-2014 - New set of High Efficiency Emission filters (DAPI, GFP, RFP, Cy5) and Dichroic Mirror from Chroma installed at the Spinning Disk W1.

Booking Calendar details

Configuration
Value
Booking type
Pre-reservation with real-time tracking confirmation
Responsible
Unidade Imagiologia
Minimum booking time (in min)
30
Schedule tolerance (in days)
7
Detele tolerance (in hours)
1
Booking restriction status
Disabled

Laser Unit

Laser
Excitation lines
Maximum power
diode 405
405 nm
100 mW
DPSS 488
488 nm
100 mW
diode 561
561 nm
100 mW
diode 640
640 nm
50 mW

Available objectives

Place mouse over Magnification to see the objective.
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.

Emission Filterwheel

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

Spectra, lines & filters:

Installed Dichroic Mirrors:

Name
Transmission
Description
iQ number
Link
IR
685 IR
Infrared dichroic mirror
1
Quad (Semrock)
LD 405/488/561/640
Quadruple dichroic mirror
2
Quad (Chroma)
ZT 405/488/561/640
Quadruple dichroic mirror (High Efficiency)
3

Setup

Turn on procedures

  1. 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.

    Cooling system power button

  2. Turn on the computer.
  3. Log-In to the computer using your Agendo credentials (Username and Password).
  4. Turn on the Main Cabinet key and switches.

    main cabinet laser control

  5. Turn on the Power Bars.

    on/off button

  6. Turn on the Spinning Disk scanhead by rotating the key.

    CSU key

  7. Turn on the cameras. Only if the cooler is at 15ºC.

    Cameras

    Press the button for the EMCCD and the switch for the sCMOS.

    iXon EMCCD 1024x1024 Zyla sCMOS 2048x2048

  8. Start the iQ and input your iQ username and passowrd (it is different from the Agendo one!).
  9. If the software asks to initialize the stage, please do it BUT make sure the objective is lowered. You can click the escape button on the right side of the microscope and when it's done click refocus to return to the previous focus.
  10. Remember to choose the proper calibration if doing montages/size measurements.
  11. In iQ you need a different calibration for each objective and to check if wether you are using the 1,5x auxiliary Optovar magnification or not.

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.


Turn off procedures

  1. Save all you data and close all open windows.

    • Save all the files in the server using the shortcuts provided in the desktop.

  2. Log off from Windows.

    • Stop here if you are NOT the last user of the day.
  3. Shutdown the computer.
  4. Turn off both cameras.
  5. Turn off the Spinning Disk scanhead.
  6. Turn off the Power Bars.
  7. Turn off the Main Cabinet key and switches (see above).
  8. Turn off the camera cooling water system (1 switch in the power socket).
  9. Clean any immersion objectives you used.
  10. TAKE OUT EVERYTHING YOU BROUGHT! (pipettes, slides, etc...)

Common problems

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):

  1. On the Nikon Ti>Control tab, the filter changer is to be set to "Filter 3" (BF) and the port to "L100".
  2. On the Yokogawa tab, the shutter has to be open and the mode needs to be set to "Confocal"
  3. On the ALC tab, the shutter needs to be open and an intensity value has to be given for each laser to be used.
  4. Check that the filter on the Yokogawa tab (filter wheel) is correct.
  5. If you are making a montage and it doesn't look correct when finished, you need to make sure you selected the correct calibration on the protocol window. You need a different calibration for each objective and if wether you are using the 1,5x auxiliary Optovar magnification or not.

Acquisition

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.

  • Grid Colection / Multi-well / Mosaic

    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.

  • Tile Scan

  • Perfect Focus System (PFS)

    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.

  • Nikon PFS


Extra Info