Table of Contents
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Location: [Oeiras] Room 0B05 |
Andor Dragonfly SDC Quality Control
Andor Dragonfly Usage StatisticsMicroscope overview
Spinning disk confocal microscopy (SDC) is a technique well suited for observation of cells and organoids. Compared to conventional widefield microscopy, SDC provides instant optical sectioning, allowing the imaging of cells and shallow tissues (typically a maximum of a few tens of micrometres deep) in 3D. Compared to laser scanning confocal microscopes (such as Leica Stellaris, Zeiss Airyscan), the Dragonfly SDC provides much faster acquisition (up to 400 fps, with limited ROI (2048×128), 40 fps full frame), with lower phototoxicity and photobleaching. A spinning disk with multiple small pinholes is installed between the light source and specimen to generate point-like illumination covering the whole area “simultaneously”. Each small aperture serves as a detecting pinhole to remove out-of-focus fluorescence. This system was built to be versatile, and allow imaging of different sample sizes. It includes both high and low magnification objectives (which require different pinhole sizes, 25 or 40um). The scanhead allows 1x or 2x magnification (for extra magnification and to allow Nyquist sampling and deconvolution - see table below).
System components
Lasers
| Laser lines | Maximum power | Measured at objective @100% (mW) |
|---|---|---|
| 405 nm | 100 mW | 7 |
| 488 nm | 100 mW | 17 |
| 561 nm | 100 mW | 20 |
| 640 nm | 100 mW | 17 |
Emission Filterwheel Spinning Disk
| Filter “Name” | Transmission (nm) | Description (use for): | CSU dichroic |
|---|---|---|---|
| Quad (pos0) | 445/521/594/698 | (faster switching) | 405/488/561/640 |
| 405 (pos1) | 445/46 nm | DAPI, Hoechst | 405/488/561/640 |
| 488 (pos2) | 521/38 nm | GFP, FITC, Alexa488 | 405/488/561/640 |
| 561 (pos3) | 594/43 nm | A568 | 405/488/561/640 |
| 637 (pos4) | 698/77 nm | Cy5, A633, A647 | 405/488/561/640 |
| 561 extra (pos8) | 606/64 nm | mCherry | 405/488/561/640 |
| Pol (pos5) | All (50%) | TR-POLR-DIC-FWL | N/A- |
Objectives
| Objectives | NA | Imm. | WD (mm) | Max FoV (1600x1350px) | ideal CSU pinhole | Theor. Resol. um | Pixel size in μm (1x / 2x zoom) | PFS Compatible |
|---|---|---|---|---|---|---|---|---|
| 10x | 0.3 | Dry | 16 | 1.3mm | 25um | 1.07/0.43 | 1.020 / 0.510 | Yes |
| 20x | 0.8 | Dry | 0.8 | 0.68mm | 40um | 0.40/0.16 | 0.510 / 0.255 | Yes |
| 40x | 0.95 | Dry | 0.25-0.17 | 0.34mm | 40um | 0.34/0.13 | 0.255 / 0.108* | Yes |
| 60x | 1.2 | Water or Sil | 0.31-0.28 | 0.23mm | 40um | 0.27/0.11 | 0.170 / 0.085* | Yes |
| 20x* | 0.75 | Multi | 0.51-0.33 | 0.68mm | 25um | 0.43/0.17 | 0.510 / 0.255 | No |
| 40x* | 1.15 | Water/Sil | 0.59-0.61 | 0.34mm | 40um | 0.28/0.11 | 0.255 / 0.108* | Yes |
| 100x* | 1.45 | Oil | 0.13 | 0.13mm | 40um | 0.22/0.09 | 0.102 / 0.050* | Yes |
*Optional
Turn On Procedure
1. Turn on the Computer
2. Turn on power bar #1 and then power bar #2. These power bars are behind the microscope table, on a rail just above the floor.
3. Turn on laser key
4. (Optional) Turn on incubator powerbar if you are planning to use the incubator and/or the CO2
5. (Optional) Turn on the valves for CO2 and Nitrogen mixture, if you planning to use CO2 on your experiment
6. Login in Agendo and open the Fusion software.
7. Please ask assistance in advance, if you are going to use either the micropump system or/and the incubation setup.
Turn Off Procedure
1.Close the software and save all your files to the “files1” server (shortcut in desktop). DO NOT CONNECT external disks/USB flash drives to the workstation! Check if you are the last user in Agendo and if you’re not, proceed to step 5.
2. Turn the laser key to the Off position
3. Turn off both the power bars in the back of the microscope table
4. Clean any immersion objectives used. Use ONLY lens paper provided by staff, NEVER use other types of paper (eg lab towels) or cotton swabs. Apply a few drops of cleaning solution (EtOH) to the lens paper and gently swipe the tip of the objective in a linear motion without pressure (no circular motion). Repeat with a different part of the paper to remove all oil residue.
5. Take your belongings (slides, pipettes, etc…); there's a glass disposal bin in the lab!
6. Log off your computer session.
