A Scanning Electron Microscopy-Compatible Optical Imaging Method for Mesoscopic All-Cell Brain Mapping

Our research focuses on connectomics. We develop high throughput optical or electron image acquisition methodologies, enabling the analysis of a neuro circuitry and a synaptic connectivity features to decipher the fundamental connection logic of neuro circuits. Original high revolution connectomics is limited to small brain volumes due to costly data acquisition and precisely, whereas rapid microscopic analysis and the targeted microscopic study offering potential for whole brain connectomics.

Compared to other microscopic approaches, such as various Larsens microscopes, OMLIT captures images of all neurons with dendrite and axon worry information. Besides its compatibility with electron microscope, there is further nanoscale resolution imaging under EF.OMLIT helps build comprehensive brain models, mapping all long range projections, including their direction, strength, and type alongside the local micro circuits in combination with serial theme. It offers insight into the structured architecture and information flow across the whole brain.

We are developing automated OMLIT imaging and EM data acquisition guided by OMLIT defined RIs, enabling efficient connectomic data collection and analysis across large or whole brain volumes. To begin, select either capton or D50 film with a thickness of 50 micrometers and mounted onto the motorized winding system. Using a double head magnetron sputtering system, deposit a uniform thin film of chromium or other metals such as aluminum, silver, or copper onto the surface of the tape.

Position the tape at a distance of 80 millimeters from the sputtering target. Perform the process under direct current power, and at a pressure of one pascal regulated by 99.99%argon gas. Now, set the tape winding speed to 0.6 millimeters per second to achieve a metal coating thickness of 50 nanometers.

After deposition, take out the tape from the system once it has cooled down to room temperature. Evaluate the thickness and uniformity of the coating, using a stylus profiler, atomic force microscopy, or scanning electron microscopy. For low reflectivity strategy, clean and render the tape hydrophilic using a plasma cleaner at 80 watt power while moving the tape at seven millimeters per second.

After treatment, place water droplets on the tape surface to confirm they rapidly spread into a thin film. Using a small grinder or similar cutting tool, rough trim the resin on the sample side to remove surrounding blank resin and expose the sample area. Place the resin embedded block in the sample holder and tighten it to secure the block firmly.

Mount the sample holder on the microtome's movable arm. Install a glass knife or diamond trimming knife at a 45 degree angle on the knife holder. Under the microscope, trim the sample surface into a pyramid shape and smooth it, then trim and smooth the four sides of the sample block to remove excess resin.

Rotate the knob to align the front and rear edges of the block in a horizontal position. Remove the trimming knife and replace it with a diamond knife, set at a 45 degree angle. Set the tilt angle of the microtome base to six degrees and move the knife holder slowly using the knob until the front edge of the knife is one to two millimeters from the sample surface.

Observe the bright band between the sample surface and the knife edge, and adjust the tilt angle so the band is even from top to bottom and side to side. Now, inject distilled water into the groove of the diamond knife to wet the blade. Remove some water with a syringe until the liquid level dips and the reflection appears silvery.

Next, set the section thickness cutting speed and cutting window in the control unit. Adjust the sectioning speed to 0.6 millimeters per second and the section thickness to 60 nanometers, depending on the sample quality. Begin sectioning with the microtome.

Once stable and uniform sections are produced, pause the process. Then use a fine brush to remove cut sections and debris. Now install both the coated tape reel and an empty takeup reel on the automatic, ultra thin section collecting system.

Secure the locking mechanism and perform a trial run to confirm smooth tape movement at constant speed and proper collection onto the takeup reel. Immerse the collection head of the taped collection device in the diamond knife's water bath and adjust the head to be parallel to the knife edge at 1.5 times the sample slice length. Secure the collection device and resume sectioning, while simultaneously running the tape collection device.

After collecting sufficient continuous sections, pause sectioning. Cut the tape in an area without sections. Run the tape collection device until all tape is wounded onto the spool.

Next, remove the spool and place it in an electronic drying oven. Clean the tape collection device and the microtome and return all accessories to their proper places. For mounting on a silicon wafer, peel off the transparent protective film from the double-sided conductive tape.

Apply the tape parallel to the double-sided conductive tape, placing up to three segments of tape on each piece. Place the silicon wafer on the stage of the optical microscope, and secure it with non-residue adhesive tape. Use a five X objective lens to obtain an overview image of the silicon wafer, the tape, and the sample.

On the overview image, outline each section and sort them, then add focus and exposure points to perform automatic imaging at 20 or 50 X magnification across the entire wafer. After imaging, save the images and check their quality, refocusing and re-imaging if any are out of focus or poor quality. Import the TIFF image stack into VAST 22 by selecting import, followed by import image volume from images to VSV file.

Connect an external tablet and use the brush tool and draw segment mode to manually segment and trace structures. Use the shortcut keys A and Z to navigate between image slices. Now, visualize the segmented structure in three dimensions by selecting window, followed by 3D viewer, view and update.

Finally, save the segmentation results after selecting file and save segmentation. In the high reflectivity images, the cytoplasmic and vascular lumen regions showed higher intensity compared to the surrounding areas, while in the low reflectivity images, the cytoplasmic and vascular lumen regions exhibited lower intensity. Quantified results demonstrated that the high reflectivity and low reflectivity strategies.

Each had advantages in terms of contrast and information entropy. OMLIT optical microscopy imaging allowed identification of axons, blood vessels, cell bodies, and dendrites. Manual segmentation of omelet images, using VAST, showed numerous tightly arranged cell bodies, dendrites, and axons.

The segmented results were combined with the original image to produce a three dimensional visualization Magnified OMLIT images showed insufficient resolution to reveal finer structures. Electron microscopy images of the same region revealed synapses, mitochondria, cell nuclei, and vesicles.