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3i Marianas Spinning disk | Reservations

  • 3i Marianas

  • Location: Bartolomeu Dias Wing

    Manufacturer: Intelligent Imaging Innovations

    Model: Marianas

    Year Installed: 2021

    Camera: Photometrics Prime 95B Scientific CMOS

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

    Keywords:

Spinning disk confocal microscopy is the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems provide 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 3i Marianas system is a spinning disk laser confocal microscope with a back-illuminated sCMOS 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 ( manufacturer page / Guide ) to correct for vertical drift during long movies. The spinning disk unit comes coupled with a uniformizer, which uniformizes the illumination for the whole field of view. Also has a laser ablation system with a mSwitcher to quickly switch between ablation and imaging.

Suggestion for description in "Materials and Methods":

Confocal Z-series stacks were acquired on 3i Marianas with a Yokogawa CSU-W1 Spinning Disk confocal, using a 100x 1.45NA Oil immersion objective, using the 488nm, 561nm and 637nm laser lines and a Prime 95b sCMOS camera and appropriate emission filters were used to acquire images for the emission of the Alexa488, Alexa 568 and Cy5 fluorochromes.

Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility, funded by FCT (Portugal) via grant #PPBI-POCI-01-0145-FEDER-022122, and the project LISBOA-01-0246-FEDER-000037_SingleCell.

LaserStack

All lasers are solid state.

Laser wavelength
Common fluorophores
405 nm
DAPI
445 nm
CFP
488 nm
GFP
561 nm
RFP
594 nm
Texas Red
637 nm
Cy5

The system also has an Ablation laser (355nm).

Available objectives

Place mouse over Magnification to see the objective.
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm) / Nyquist
Pixel size (µm)
Pixel size with 2x magnifying lens (µm)
Nyquist Sampling @525nm in Z (µm)
Ablate! compatible
Link
10x
Plan apochromat lambda
0.45
-
4
0.583 / 0.254
1.1
0.55
TO CHECK ON SYSTEM
NO
20x multi-immersion
Plan Fluor
0.75
Water, Silicone, Glycerol and Oil
0.51-0.33
0.350 / 0.152
0.55
0.275
TO CHECK ON SYSTEM
YES
40x Dry
Plan apochromat
0.95
-
0.25-0.17
0.276 / 0.12
0.275
0.138
0.36
NO
40xSil
Plan apochromat
1.25
Silicone
0.3
0.21 / 0.091
0.275
0.138
TO CHECK ON SYSTEM
NO
60xWater
Plan apochromat
1.20
WATER
0.31-0.28
0.219 / 0.095
0.183
0.092
TO CHECK ON SYSTEM
YES
100x
Plan apochromat
1.45
OIL
0.13
0.181 / 0.079
0.11
0.055
0.23
NO

(1) NA - Numerical Aperture. For more information, follow this link.

(2) These values were calculated using the Resolution formula seen in Nikon's microscopyu website: R = 0.5*λ/NA

The pixel size in microns of your picture is saved automatically when using slidebook. Use Bio-formats to recognize it with ImageJ/Fiji (already installed in the Fiji package).

Emission Filterwheel

Name
Transmission
Description
Slidebook filter Number (TO CHECK ON SYSTEM)
Link
Quad (c###q)
440/521/607/700
DAPI, GFP, RFP and Cy5
1
lnk
c405s
445/45
DAPI, CFP or Hoechst filter
2
lnk
c488s
525/30
GFP or FITC filter
3
lnk
c561s
617/73
RFP or TRITC
4
lnk
c637s
692/40
Cy5/Far red Filter
6
lnk
Dual (c###d)
 525/642
GFP and RFP
7
lnk
c445s
482/25
CFP
8
lnk
c591s
642/80
Texas Red
5
lnk
Not setup
542/27
YFP
9
lnk
cBF
-
Brightfield
0
N/A

Setup

Turn on procedures

  1. Turn On the Master power bar (table's left side).

    3i Main switch

  2. Turn the laser key to the On position.

    Laser key image

    Here, laser key is on the OFF position

  3. If your using temperature and/or CO2, turn on OkoLab power bar (under the table)

    CO2 and temp power bar

  4. Turn On PC. This has to be the last step

    marianas PC

  5. Open the Slidebook pretended for the configuration you need: Phasor (photomanipulation) or Ablate! (Photoablation). Use Phasor configuration for normal Imaging.

    Phasor has 2 versions, 100x and 60x. If you want to use the 60x to do FRAP, use Phasor 60x.

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.
    • Stop here if you are NOT the last user of the day.
  2. Turn off the Master power bar.
  3. Turn laser key to Off position.
  4. If used, turn off the okolab (temperature) power bar.
  5. Clean any immersion objectives you used.
  6. TAKE OUT EVERYTHING YOU BROUGHT! (pipettes, slides, etc...)

Tips and Tricks

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 it again.

If the software does not start, it could be that it's not recognizing the camera. If that's the case, restart the computer.

If you don't see any image on the camera, the laser key might have not be correctly turned on. Switch off and on the laser key again.

Camera chip size needs to be changed on the capture window. Apply it by pressing “Live” on the Capture window.

Extra Info

funding