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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), by using patterned (“structured”) illumination. The illumination patterns produce high order spatial information that can be extracted by computational algorithms in Fourier space. A typical SIM image is a reconstruction of several patterned images, where the illumination pattern changes in both angle or phase in order to extract almost twice the original resolution. In the DV OMX system, a total of 15 images are acquired for each image (5x phases x 3 angles). SIM works best with shallow samples, such as attached cells, and is less employed for tissue imaging. Optimal optical conditions are important to avoid reconstructions artifacts. When successful, SIM can provide valuable information about organelles, cytoskeleton and fine intra-cellular 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 |
"Channels" & respective emission filters/cameras
| Position | 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 |
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)
Procedures
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
