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Leica SP8 Confocal
- Location: Room B273
- Serial No: 8100001591
- Motorised XY Scanning Stage Operation with software extension for tile scanning
- Epi fluorescence (CCD Camera) Operation
- Laser Set: Super Continuum “White Light Laser” (470-670nm) + 405 & 442nm CW
- Linear Unmixing (Available during acquisition – Real time fluorescence unmixing
- Configurable USB Control Panel
- Three detectors: 2x HyD and 1x PMT. The HyDs can be used for photon counting.
- Deconvolution option via Huygens.
The SP8 has a white light laser and tunable bandpass filters. This means it can tune to create whatever excitation wavelength is needed and also catch whatever emission of light you need. There are no set laser wavelengths or filter sets to work around, like on the Zeiss Imager and Axioscan.
| Magnification | Numerical Aperture | Field of View | Working distance | Resolution at 1024x1024(/pixel) | Link for more info |
|---|---|---|---|---|---|
| x10 | 0.4 | 1.11mm x 1.11mm | 2.74 mm | 1.08um | 506407 |
| x10 water immersion | 0.4 | 1.11mm x 1.11mm | 360um | 506293 | |
| x25 water immersion | 0.95 | 442um x 442um | 2.4 mm | 432nm | 506375 |
| x40 oil immersion | 1.3 | 276um x 276um | 240 um | 270nm | 506358 |
| x63 oil immersion | 1.4 | 175um x 175um | 140 um | 171nm | 506350 |
| x20 Clearing Obj (BABB) | 0.95 | 1.95 mm | 507702 | ||
| x25 Motor Correction Collar (CLARITY) | 1 | 6 mm | 507703 |
- Move the sample stage/arm out of the way from objectives both before/after your session.
- Use the correct immersion medium for the objective
- Turn off the HXP lamp if there will be no user within 2 hours.
- Contact Peter for training or troubleshooting
The LAS AF software is comprised of four domains.
- Image Acquisition & Control Settings: these functions control the static acquisition settings for image collection, such as Frame Size, Scan Speed, Averaging, Zoom, etc.
- Beam Path Configuration: where to set the beam path configuration and control the detector settings to optimize the fluorescence signal.
- Image Display: this area is where the image will be displayed. Settings here allow the user to control how the image is viewed on screen.
- Scan Action Functions: these functions start/stop scanning and initiate our experiment acquisition.
Once the system has been powered on, we can mount our specimen on the stage below the microscope objective.
- Raise stage to appropriate height and mount sample.*need picture about home and focus bottons
- Switch microscope TFT to ‘FLUO’ to activate the incident path from default ‘CS’ in Combi.
- Choose filter.
- Open/Close reflected light shutter with ‘IL-Shutter’.
- Via the microscope eyepieces, visualize your sample and adjust the focus (Z) and stage position (X,Y) accordingly
- When sample is in focus and positioned correctly – switch microscope back to CS – Combi to begin imaging with the confocal.
- Open the dye assistant
- Choose your fluorochromes from the dropdown list.
- Define which type of detector you need.
- For the least bleedthrough, choose the line sequential setting.
- Be patient for the hardware to initialize!
- The SEQ will indicate that you are scanning sequentially.
- Click between the different sequences (without scanning) and check that emission bands are not moving between sequences.
The Acquisition Mode parameters located on the left-hand side of the software display contain all the settings available for image optimization and collection.
- From the dropdown, choose the Acquisition Mode. For example, if you want to collect 3d image stacks, select ‘xyz’ from the dropdown
- The remainder of the settings in the acquisition mode control dialog can adjusted, depending on sample morphology and signal quality.
The default image size in pixels is 512x512, however this can be optimized based on zoom and objective settings. For "ideal" Nyquist sampling you can quickly select with ‘Optimize XY Format’. However, this is not always necessary and is use-case dependent. Ask us if unsure.
The default pixel depth is 8 bits If you want to change that, see the "Configuration" tab, "Hardware" submenu, resolution drop down list.
- Scan speed is measured in Hz (lines per second). It refers to the frequency of two galvanometers which drive scanning mirrors, shading laser light across the specimen.
- The slower the speed, the higher singal-to-noise ratio (SNR). You can select between 10-1800 H, but 600H is recommended value.
- Bidirectional scanning will double the speed as pixels are recorded in both scan directions.
- Sometimes when bidirectional is selected, mismatch in the phase might occur, you can correct this with the control panel (phase correction)
To improve your image quality, you might choose to apply averaging. You can choose between line averaging, frame averaging or a mixture of both. Averaging takes the sum of pixels from the specified number of scan and uses arithmetic mean as the final value of the image. Averaging preserved those persistent pixel values that are mostly specific signal while divide away along times those fluctuated values that are mostly noise.
If you see the message "Safety interlock activated - no laser light. Please check position of safety interlock" you should turn the key on the box under the microscope.