The image is for the visualization of various types of cells in the nervous system for histological purposes. The final product is a photograph or drawing. The methods have been replicated to the closest to the original processes that Cajal performed, using the materials which were available to me. Nearly all components of the method I used have been around for at least 50 years. The primary sources used are Cajal’s classical books, images, and texts, most notably the one cited.1 The tools necessary for sample preparation and imaging are 50 mg of potassium dichromate, 50 mg of lead nitrate, alcohol, acetone, a hot plate, glass slides, 5 Beral pipettes, an optical microscope, and a camera. Technological changes, such as digital cameras, allow for direct capture of the images. Likewise, the image is being submitted in digital format, which can be reshared by the recipients an unlimited number of times without loss of resolution. The process was tedious. Is there a faster way to do sample prep and staining, without the need of a human? With lower resolution microscopes, like what Cajal had, it is very hard to see fine details of these neurons. I wonder what type of personality is required to do this hundreds of times for a bunch of animals, and then record everything by hand, because it is backbreaking meticulous work. It took around an hour to prepare everything and stain one specimen, which needs to be reoptimized for every sample to get a good slide.
References:
1. Ramón y Cajal, S., Histology of the nervous system of man and vertebrates. Oxford University Press: New York, 1995.
FIELD NOTE 1
I would have done the procedure on a sparrow or a rat, but most critters with good brains that Cajal liked to use are difficult to acquire around these parts of the world because you aren’t allowed to kill them in the way Cajal did 100 years ago. Times have changed. Instead, I bought a live catfish from the supermarket. At first, I almost resorted to ballistic methods to extract the brain. However, I decided that I wanted the morphology of the specimen to be intact. So, I cracked open its head with a knife and a pair of pliers. During the process, I got spiked deep by the catfish because the bottom feeder was slippery. I almost got an infection, but I washed the wound with iodine solution to kill off the microbes, and therefore, I survived to eat the fish.
Figure 1. The catfish.
FIELD NOTE 2
I do not have a cryomicrotome, so frozen section method is out. Neither do I have formalin solution and paraffin, so those methods are also out. Also, slicing leads to poor visualization of neurons unless if done along the length of neurons (which is difficult to do for fish, as their brains are not well-structured), because all that will be visible if the tissue is sliced perpendicular to the neurons is a bunch of circles. Thus, I did another common method that has been around for at least half a century, to make the sample sufficiently thin to visualize by optical microscopy. I took a piece of its brain and performed a squash smear. As seen below, a small piece of tissue is placed on the slide. I chose a few pieces of the brain. The slides are pressed together, and then slid perpendicular to each other.
Figure 2. (a) A small piece of brain tissue is placed onto the slide. (b) The slide is pressed against another slide, and subsequently, the two slides are smeared perpendicularly with respect to each other to leave a thin film.
The resulting specimen retains its microstructure, but the macrostructure may be distorted. The squash smear procedure allows for decrease of thickness of sample down to the single-cell level.
The specimens are dehydrated with a 70% alcohol rinse, and excess lipids are removed with a 50/50 alcohol/acetone mixture. Subsequently, they are placed on the hot plate and dried for 15 minutes at 50 °C to adhere the tissue to the slide. 50 °C is a good choice, because it is compatible with many staining procedures, if additional staining procedures are to be used. Thus, the tissue is heat fixed to retain microstructural morphology, and the drying prevents the tissue from delaminating during the staining procedure.
FIELD NOTE 3
Finally, the specimens were stained. We chose a lead dichromate precipitation stain. This stain substitutes out the silver nitrate that Cajal and Golgi used, with lead nitrate. Lead nitrate is much cheaper, being less than 0.2 cents per gram versus 1-2 dollars per gram. In chromate and dichromate stains, the color primarily originates from the hexavalent chromium atom center, with very minor electronic shifts that can be approximated as spherically symmetric fields attributed to the precipitated cation combined with solvation effects. Like Cajal and Golgi, the hexavalent chromium source is potassium dichromate, which is less than 0.3 cents per gram. NO3-, and Na+ are colorless counterions. More importantly, Pb2+ and Ag+ are both colorless as well. Likewise, the electron density distributions surrounding Pb2+ and Ag+ are very similar, meaning that the color of the precipitate does not deviate substantially. Both precipitated chromate salts will give good optical microscopy and electron microscopy contrast.
Unlike Cajal, I also did not use osmic acid to increase contrast (osmic acid selectively stains unsaturated lipids, which neuronal structures are full of), because osmic acid is also expensive, and is around 100 dollars per gram. However, for low resolution optical microscopy, it is okay to not use osmic acid, because the hexavalent chromate and dichromate ions have some affinity for unsaturated sites, and as negatively charged species, strong affinity for cationic sites (choline heads of lipids, positively charged amino acids, positively charged sites of neurofilaments, etc). The staining procedure is as follows.
- 0.5 wt% solutions of Pb(NO3)2 and K2(Cr2O7) were prepared, in 25 wt% alcohol/water mixture. The alcohol increases penetration rate and improves staining.
- Note that the concentration used is quite high (more than 10x the necessary, to increase precipitation rate and to get bigger precipitated crystals that will allow for increased visibility of large structures at low magnification), as very high resolution is not necessary for optical microscopy. If I were to do electron microscopy, I would use a much lower concentrations (<0.05 wt%) as well as organic solvent mixtures (xylene/alcohol), which would lead to more difficult staining procedures (it would require pre-staining sample prep that increases selectivity for neurofilaments and nuclei, osmic acid treatment to allow for catalytic site-specific nucleation, along with multiple cycles of staining, rinsing, and clearing), but far better resolution due to smaller crystals that nucleate more selectively.
2) The sample is first treated with the dichromate solution for around 15 seconds. Then, it is rinsed with 70% ethanol to remove excess extracellular dichromate that may lead to background precipitation (which cause crystalline artifacts). This removal of extracellular dichromate ensures that the majority of the precipitate is intracellular.
3) Subsequently, the sample is treated with lead nitrate. A yellow precipitate is observed to form around the tissue.
4) Finally, the sample is rinsed off with 70% ethanol, and dried on the hotplate at 50 °C for 5 min.
I did not use a cover slip, because I did not have glue to mount the slip. The final product is shown below. Note that the precipitate forms where there is tissue, or where there is some residual lipid. Although the whole slide was immersed, only certain regions are stained because of the alcohol rinse between the staining steps.
Figure 3. The final stained slides before imaging with optical microscope. Note that only the portions with tissue or with residual lipid are stained.
Note that the lead chromate is highly opaque. For observation with the naked eye, silver chromate is slightly more reddish brown. However, under optical microscopy and electron microscopy, they are both opaque and appear black. Hence, the “black reaction” of Cajal and Golgi does not refer to the color of the precipitate by naked eye, but rather, the opacity of conferred to the specimen that is observed with an optical microscope.
Final Images
Figure 4. A long multipolar neuron that extends several hundred microns. The axon and telodendria are visible. As expected, the neuron is “black” under optical microscopy. Note the oligodendrocyte.
Figure 5. Ink drawing of Figure 4.
Figure 6. A bunch of neurons entangled together. A variety of axon diameters and bundles of neurons are observed. A mess of dendrites and telodendria.
Figure 7. This is NOT a neuron! It is some type of glial cell, although the cell is too badly damaged (which is unsurprising because the specimen was stored at 4 °C for a day) for us to see clear details which may indicate if it is a microglia cell (which is most likely), or a protoplasmic astrocyte.