IGC

WARNING

This is to remind you the basic rules of fluorescence lamps and gas lasers:

  • Never turn OFF the fluorescent lamp if it's been on for less than 30 min.
  • Never turn OFF the lasers if they've been on for less than an hour (1 Hour).
  • Never turn on the lamp if it's been off for less than 30 min (check to see if it's still warm).
  • Never turn on the lasers if they've been off for less than hour (1 hour).
  • Never turn off the lamp or lasers if someone else is using the microscope within the next 4 hours.
  • The Mercury lamp should always be first-on and last-off.
  • Not respecting these instructions may lead to instrument malfunction and complete loss of function! Both lasers and lamps are extremely expensive.

Andor Spinning disk | Reservations

  • Spinning disk Confocal
  • Location: Bartolomeu Dias wing, UIC Room 3

    Manufacturer: Andor

    Model: Revolution XD

    Camera: Andor iXon+ 512 EMCCD

    Operating: Full temperature atmosphere control (37oC + Humidity + CO2)

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 acceptable 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 ( manufacturer page / Guide ) 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-X Spinning Disk confocal, using a 100x 1.4NA Oil immersion objective, using the 488nm, 561nm and 640nm laser lines and a Andor iXon+ EMCCD camera where used to acquire images for the emission of the Alexa488, Alexa 568 and Cy5 flurochromes.

Maintenance log:

  • 03-03-2016 - New Piezo Z stage from MadCityLabs installed at the microscope, replacing old one.
  • 07-09-2015 - Piezo Z stage repaired and installed at the microscope. Has a 10% error while moving.
  • 26-05-2015 - Piezo Z stage replaced by static holder and sent to repair.
  • 05-03-2015 - Camera fan replaced by a new one similiar to the broken one.
  • 18-02-2015 - Liquid guide for the fluorescent lamp replaced by a new, shorter one. Lamp moved.
  • 12-01-2015 - Old camera fan replaced by a new temporary one. Back fan remained in place for camera cooling.
  • 19-11-2014 - Camera fan not working, causing overheating. Another stronger fan added to the back of the camera. Camera back remains open.
  • 22-09-2014 - Fluorescent lamp broke. Replaced by a new one.
  • 21_25-08-2014 - Frap unit realigned. Was missplaced and causing problems when using FRAPPA/Ablation.
  • 29-05-2014 - Comarine dye wahsed and renewed. MicroPoint calibrated and focused for the 20x dry objective.
  • 13-05-2014 - Laser Control Plugin updated for a 64bit system and installed at the microscope.
  • 06-05-2014 - Computer replaced for a new one. Clean installation of Andor iQ2.6 and MM softwares.
  • 17-04-2014 - MicroManager version updated to fix software crashes. Updated from 1.4.14 to 1.4.15.
  • 04-04-2014 - Transmitted light shutter working once again.
  • 04-02-2014 - Filter Wheel controller back on the microscope and working.
  • 24-02-2014 - Microscope working without the filter wheel, Quad filter in position for image acquisition of multiple channels.
  • 21-02-2014 - Sutter Filter Wheel controller out of order and sent to repair.
  • 20-02-2014 - Piezo Z stage was re-installed in the microscope.

Laser Unit

Laser
Excitation lines
Maximum power
405
405 nm
100 mW
OPSL CW 488
488 nm
50 mW
DPSS 561
561 nm
50 mW
DPSS 640
640 nm
40 mW

Available objectives

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Pixel size (µm)
Pixel size * 1.5x (µm)
Nyquist Sampling @525nm in Z (µm)
Link
10x
PLAN FLUOR
0.30
-
16
0.700
1.42
0.95
1.050
20x
PLAN APO VC
0.75
-
1.00
0.280
0.71
0.47
0.420
40x
PLAN Fluor
1.30
OIL
0.2
0.162
0.35
0.23
0.242
60x
PLAN APO VC
1.20
WATER
0.31-0.28
0.175
0.24
0.16
0.263
100x
PLAN APO
1.40
OIL
0.13
0.150
0.14
0.10
0.225

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) 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
DAPI
460/50
DAPI, CFP or Hoechst filter
460
GFP
525/50
GFP or FITC filter
525
RFP
595/50
RFP or TRITC
568
Cy5
685/40
Cy5/Far red Filter
685
DIC
-
Polarizer
1
488 LP
488LP
GFP or FITC (Longpass)
488
RFP LP
568LP
RFP or TRITC (Longpass)
565
Quad
400/521/607/700
Quadruple filter
4
Triple
525/600/640
Triple filter (GFP,RFP,Cy5)
3
BF
-
Empty - Full transmission
999

Spectra, lines & filters:

Setup

Turn on procedures

  1. Turn on the computer.
  2. Turn on the fluorescence lamp, should you need it.

    Fluorescence lamp

  3. Log-in to the computer using your Agendo credentials.
  4. Turn on the main cabinet switches.

    main cabinet

    laser control

    hardware control unit

  5. Turn on the Master Control Switch

    on/off button

  6. Start the Acquisition software.

    • If using Andor iQ2:
      • Open iQ with the Default configuration.
    • If using MicroManager (µManager):
      • Open iQ2 with FRAPPA configuration. Once it is open, you can close it.
      • Open MicroManager.
  7. 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.
  8. Remember to choose the proper calibration if doing montages/size measurements.

When you are finished and there is someone immediately after you, leave the entire system on (in particular, don't turn off the fluorescence lamp even if there is a large time difference between you and the next person). If you're the last or only person of the day, see the turn off procedures below.


Turn off procedures

  1. Close the software and save all your files
    • 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. Turn off the Master Control Switch.
  4. Turn off the Laser Control and the Hardware control (see above the main cabinet switches).
  5. Turn off the Fluorescence lamp.
  6. Clean any immersion objectives you used.
  7. 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.

If the camera doesn't see anything when using Micro-Manager, remember that you need to also start Andor iQ in FRAPPA configuration. You can close iQ after it has started.

If you don't see any fluorescence through the eyepiece, users of the ablation laser may have left the lamp blocked. Move the slider immediatly in front of the lamp housing.

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 6" 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 Sutter tab (filter wheel) is correct.
  5. If you don't get any laser light, on the ALC tab click on the "FRAPPA" checkbox and then back on the "CSU" checkbox.
  6. 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 µManager and 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