Error Status code: 401
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.
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.
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.
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.
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).
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).
Name |
Transmission |
Description |
Slidebook filter Number (TO CHECK ON SYSTEM) |
Link |
Quad (c###q) |
440/521/607/700 |
DAPI, GFP, RFP and Cy5 |
1 |
|
c405s |
445/45 |
DAPI, CFP or Hoechst filter |
2 |
|
c488s |
525/30 |
GFP or FITC filter |
3 |
|
c561s |
617/73 |
RFP or TRITC |
4 |
|
c637s |
692/40 |
Cy5/Far red Filter |
6 |
|
Dual (c###d) |
525/642 | GFP and RFP |
7 |
|
c445s |
482/25 |
CFP |
8 |
|
c591s |
642/80 |
Texas Red |
5 |
|
Not setup |
542/27 |
YFP |
9 |
|
cBF |
- |
Brightfield |
0 |
N/A |
Turn On the Master power bar (table's left side).
Turn the laser key to the On position.
If your using temperature and/or CO2, turn on OkoLab power bar (under the table)
Turn On PC. This has to be the last step
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.
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.