- High spatial resolution: The Serial Sectioning Microscope (SSM) acquires multiphoton fluorescence contrast and achieves near-diffraction limited resolution (about 300 nm x 300 nm x 800 nm, depending on optical configuration).
- Large imaging volume: Total imaging volumes can reach cm3. For acquisition, a sample is mounted on a glass slide and immersed in buffer. After the top-most portion of the sample has been imaged, the integrated vibratome mechanically sections off the imaged portion and imaging of underlying layers proceeds. Hence, imaging depth is not limited. Typical lateral fields of view are hundreds of µm, but we typically acquire mosaics for post-acquisition stitching.
- Image acquisition, mosaicing, and mechanical sectioning are automated.
- Excitation: Multiphoton laser (Coherent Chameleon; 690 nm to 1080 nm)
- Detection: Simultaneous dual-color acquisition
- Section thickness depends on the achievable imaging depth in the sample, but typically is about 100 µm (the sliced off section can be collected for further use). The working distance of the objective limits the thickness to less than 1 mm.
- Spatial resolution: Standard multiphoton resolution (about 300 nm x 300 nm x 800 nm, depending on optical configuration).
- Imaging time: Depends on the imaging volume, sampling density, signal-to-noise, etc. A single-field-of-view image typically requires a few hundred ms, volume blocks several minutes. A volumetric image of an entire section can take hours, an entire cm3 serial sectioned volume many hours to about a day.
- For mechanical sectioning with the vibratome the sample needs to be embedded in agarose for mechanical stability. Note that this is embedding, NOT optical clearing. For more details on the sample mounting, see http://dx.doi.org/10.1038/nmeth.1854.
- ALIS staff can provide embedding protocols for mouse brains, but embedding other tissue types will likely involve trial & error.
Data analysis, visualization, and storage
- Data from the SSM is in the form of TIFF stacks (one stack per imaging block). For mosaics, blocks need to be stitched for full volume visualization. Because of the high resolution and sampling density, datasets can be on the order of 1 TB.
- Our workstation is available for simple analysis and visualization with commercial (Imaris, Amira) or open source packages (ImageJ, napari).
- While we provide temporary storage space for processing and visualization, the long-term storage of the acquired data is the responsibility of the user. We will retain raw data for up to 3 months after acquisition or until we reach capacity of our 100 TB storage array (whichever occurs first).
- SSM operation: $100.00 fixed setup fee (billed as 2 hr setup time) plus $50.00/hour experiment time. The setup fee is required for the start of each experiment run.
- Consulting & Training services: $100.00/hour
- Image analysis: $50.00/hour
- ALIS staff will align, calibrate, and operate the SSM.
- Sample embedding is performed by the user (with assistance from staff).
- ALIS staff is available for microscope operation, but the user should be present for imaging guidance. Repeat users can be trained to operate the microscope by themselves.