deltavision
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deltavision [2025/01/11 19:00] bioimaging |
deltavision [2025/01/11 19:13] (current) bioimaging |
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| ===== Microscope overview ===== | ===== Microscope 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. | + | 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":** | **Suggestion for description in "Materials and Methods":** | ||
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| ^ Magnification ^ Immersion ^ NA ^ WD (mm) ^ Image Pixel Size ^ Reference ^ | ^ Magnification ^ Immersion ^ NA ^ WD (mm) ^ Image Pixel Size ^ Reference ^ | ||
| | 20x UPlan SAPO | - | 0.75 | 0.6 | 0.240 | | | 20x UPlan SAPO | - | 0.75 | 0.6 | 0.240 | | ||
| - | | 60x Plan APO N | Oil | 1.42 | 0.15 | 0.080 | | + | | 60x Plan APO N | Oil | 1.42 | 0.15 | 0.080* | |
| - | ==== "Channels" & respective emission filters/cameras ==== | + | * after SIM reconstruction this system is capable of achieving resolutions of 100-150nm! |
| - | ^ Position ^ Emission wavelength ^ Dyes ^ Camera ^ | + | |
| + | ==== Emission filters/cameras ==== | ||
| + | ^ Channel ^ Emission wavelength ^ Dyes ^ Camera ^ | ||
| | DAPI | 435/31 | DAPI, CFP, Hoescht... | 1 | | | DAPI | 435/31 | DAPI, CFP, Hoescht... | 1 | | ||
| | GFP | 528/48 | GFP, Alx488... | 1 | | | GFP | 528/48 | GFP, Alx488... | 1 | | ||
| | RFP | 609/37 | RFP, Alx568... | 2 | | | RFP | 609/37 | RFP, Alx568... | 2 | | ||
| | Cy5 | 683/40 | GCy5, Alx633... | 2 | | | Cy5 | 683/40 | GCy5, Alx633... | 2 | | ||
| - | | DIC| - | Brighfield | 1/2 | | + | | DIC* | - | Brighfield | 1/2 | |
| + | |||
| + | * though possible, DIC imaging is not recommended as it may introduce artifacts in the fluorescence reconstructions | ||
| ==== Cameras (1 and 2) ==== | ==== Cameras (1 and 2) ==== | ||
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| 2x PCO Edge 5.5 sCMOS 2560x2160 (note that each camera uses only 1024x1024 pixels to avoid field illumination problems and to allow simultaneous acquisition of 2 channels per camera) | 2x PCO Edge 5.5 sCMOS 2560x2160 (note that each camera uses only 1024x1024 pixels to avoid field illumination problems and to allow simultaneous acquisition of 2 channels per camera) | ||
| - | ===== Procedures ===== | + | ===== Operating Procedures ===== |
| + | For more detailed information consult the full SOP/Guide here: | ||
| + | (under construction) | ||
| ==== Turning ON ==== | ==== Turning ON ==== | ||
deltavision.1736618400.txt.gz · Last modified: 2025/01/11 19:00 by bioimaging
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