Table of Contents
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Location: [Oeiras] Room 0B02 |
Deltavision OMX-SIM Usage StatisticsMicroscope overview
SIM microscopy is one of the imaging techniques that can overcome the limits of resolution of widefield fluorescence microscopes (~200nm), ie, it allows “sub-diffraction” imaging. By using patterned (“structured”) illumination, higher order spatial information is obtained that can then be extracted by computational algorithms in Fourier space. A typical DM OMX-SIM image is a reconstruction of 15 images, where the illumination pattern changes in angle (3x) and phase (5x). SIM works best with shallow samples, such as cells attached to glass, and is less reccomended for tissue imaging. Optimal optical conditions are important to avoid reconstructions artifacts. When successful, SIM can provide valuable information about organelles, cytoskeleton and other fine intracellular details. The speed of acquisition of the DV OMX system also allows live-cell imaging.
Suggestion for description in “Materials and Methods”: “Images were acquired on an Deltavision OMX system, equipped with 2 PCO Edge 5.5 sCMOS cameras, using the a 60x 1.42NA Oil immersion objetive, GFP + CY5 fluorescence filtersets and DIC optics. Images were deconvolved/reconstructed with Applied Precision's softWorx software.
System components
Lasers
| Excitation | Wavelength | Power |
|---|---|---|
| Violet | 405 nm | 100mW |
| Blue | 488 nm | 100mW |
| Green | 568 nm | 100mW |
| Red | 640 nm | 100mW |
Objectives
| Magnification | Immersion | NA | WD (mm) | Image Pixel Size | Reference |
|---|---|---|---|---|---|
| 20x UPlan SAPO | - | 0.75 | 0.6 | 0.240 | |
| 60x Plan APO N | Oil | 1.42 | 0.15 | 0.080* |
* after SIM reconstruction this system is capable of achieving resolutions of 100-150nm!
Emission filters/cameras
| Channel | Emission wavelength | Dyes | Camera |
|---|---|---|---|
| DAPI | 435/31 | DAPI, CFP, Hoescht… | 1 |
| GFP | 528/48 | GFP, Alx488… | 1 |
| RFP | 609/37 | RFP, Alx568… | 2 |
| Cy5 | 683/40 | GCy5, Alx633… | 2 |
| DIC* | - | Brighfield | 1/2 |
* though possible, DIC imaging is not recommended as it may introduce artifacts in the fluorescence reconstructions
Cameras (1 and 2)
2x PCO Edge 5.5 sCMOS 2560×2160 (note that each camera uses only 1024×1024 pixels to avoid field illumination problems and to allow simultaneous acquisition of 2 channels per camera)
Operating Procedures
For more detailed information consult the full SOP/Guide here: (under construction)
Turning ON
- Turn on the main power switch at the top module
- Turn on the laser chassis module (key switch)
- If necessary turn on temp controller
- Log on the OMX SR sworkstation (user: “worx” Password: “system serial number”
- Start the AcquireSR software, and in the acquistion software click “Instruments|Status”
- In the Instrument status dialog box, select “Restart Hardware” and wait.
- Once hardware restart is finished, select “Reset All Cameras”, and wait until the camera SNs are reported in the log box.
- Mount sample and start imaging.
Turning OFF
If you are NOT the last user for the day:
- Transfer your data to the institutional server. Data will be deleted every month!
- Close SoftWorx
- Clean the objective and remove all your materials
Else:
- Turn “OFF” the laser control and Hardware control.
- Log off from the workstation
