IGC

Leica Spinning Disk | Reservations

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

    Manufacturer: Andor

    Model: Revolution XD

    Microscope body: Leica DMI8

    Camera 1: Andor iXon Ultra 888 1024*1024 EMCCD

    Camera 2: Hamamatsu Flash Orca 4.0 v2.0 2048*2048 sCMOS

    Acquisition software: MetaMorph

    Additional Info: Confocal spinning disk with EMCCD camera or Widefiel with sCMOS, FRAP and Ablation systems.

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 on 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 Adaptive Focus Control ( spec sheet / guide ) to correct for vertical drift during long acquisitions.

Suggestion for description in "Materials and Methods":

Confocal Z-series stacks were acquired on a Yokogawa CSU-X Spinning Disk confocal, mounted on a Leica DMi8 microscope, with a 63x 1.3NA Glycerine immersion objective, using the 488nm and 561nm laser lines and a Andor iXon Ultra EMCCD 1024x1024 camera. The system was controlled with Metamorph software (Molecular Devices).
Photoablation was performed using Andor's MicroPoint system with a 350nm pulsed laser and 432 dye to adjust wavelenght for effeciency.
Photoconverion/FRAP experiments were performed unsing Andor's Mosaic system with a 470nm laser.

Maintenance log:


Laser Unit

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

Available objectives

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Pixel size (µm)
Nyquist Sampling @525nm in Z (µm)
Link
Confocal
Widefield
Confocal
Widefield
Confocal
Widefield
20x
HC PL APO
0.8
-
0.4
0.263
0.400
0.468
0.325
0.394
0.600
63x
PLAN APO
1.30
Gly
0.30
0.162
0.246
0.148
0.103
0.242
0.370
100x
PLAN APO
1.40
Oil
0.13
0.150
0.229
0.094
0.065
0.225
0.343

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 Spinning Disk

Name
Emission λ
Most common fluorophores
CSU 405 BP
460/50
DAPI or Hoechst
CSU 488 BP
525/50
GFP, FITC or Alexa488
CSU 561 BP
595/50
RFP, TRITC or A568
CSU 640 BP
685/40
Cy5, A633 or A647
DIC
-
Bright field with DIC optics
Quad
450/521/607/700
Quadruple channel

Widefield Filterset and Cubes

Filter Cube
Excitation filter
Dichroic
EMission filter
Code
Filter position
DAPI
BP 360/40
400
BP 470/40
A5
1
GFP
BP 470/40
500
BP 525/50
L5
2
Texas Red
BP 560/40
595
BP 645/75
TXR
3
Cy5
BP 620/60
660
BP 700/75
Y5
4
FRAP/MicroPoint
-
470
-
DAPI+TL
5
Confocal
-
empty
-
DAPI+TL
6

Spectra, lines & filters:


Setup

Turn on procedures

  1. Turn on the computer.
  2. Turn the power bar in the left side of the table On by pressing the red button, a green light should turn on.

    Power Bar 1

  3. Turn the power bar on the right side of the table On.

    This power bar in on top of the table behind the computer screen.

    Power Bar 2

  4. Turn on the Fluorescent lamp if you need it.
    • There are 2 buttons in the lamp: power supply (1) [left side] and LED remote shutter(2) [right side].

      Lamp

    • Only turn On the power supply button (1).

      Lamp PSU

  5. Turn on the CSU unit (Spinning Disk) by rotating the key to the On position.

    CSU unit

  6. Turn on the Camera or Cameras you are going to use. If you are going to use the spinning disk, turn on the EMCCD (left side picture), if you are going to use the widefield only, turn on the sCMOS (right side picture).

    EMCCD camera sCMOS camera

  7. Open MetaMorph software depending on the configuration you want to use: Confocal or Widefield.

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. Close the software and save all your files.
    • Save all the files in the server using the shortcuts provided in the desktop.
  2. Turn off the Cameras.
  3. Turn off the CSU scanhead (Spinning disk) to the Off position.
  4. Turn of the Fluorescent lamp by turning Off the left side button (1), check pictures above in case of doubt.
  5. Turn off both power bars (left and right side), check images above in case of doubt.
  6. Clean any immersion objectives you used.
  7. TAKE OUT EVERYTHING YOU BROUGHT! (pipettes, slides, etc...)-

Mosaic Setup

  1. Turn on the Andor HPL (High Power Laser) by rotating the key to the on position.
  2. HPL key

  3. Turn on the Mosaic.
  4. Mosaic On button

  5. Turn on the HPL laser control software - NovaLum.
  6. NovaLum laser control

  7. Adjust all beam splitters so that the laser goes freely into the microsocpe.
    These mirrors are in the back of the microscope and need to be fitted into their respective position.
  8. Beam splitters

  9. Start MetaMoprh in the CSU configuration.
  10. If you need to do Frap experiments, please speak with the UIC personnel before hand.

Acquisition

  • Grid Colection / TileScan

    It is possible to acquire grids and tiles of big samples through MetaMorph software. For a more detailed information, please check the following link or click in the image to the right.

  • Tile Scan

  • Adaptive Focus Control (AFC)

    AFC is Leica's version of Autofocus through hardware. This funcionality allows continuous focus of the sample through coverslip position, keeping the sample focused during the whole acquisition with the main objective of preventing drift in Z over long periods of time. For more detailed information, please visit the following link.

  • Leica AFC


Extra Info