This procedure begins with defining the reference space. A stereotaxic atlas may be helpful in determining the boundaries of the area of interest. Next, organize the slides and determine the section sampling frequency.
Insert the first section to be sampled into the microscope and fill out the sampling parameters. In theologist system, a grid will be placed over the reference space and at each intersection, an optical dissector will be sampled. Once all optical dissectors across each section are sampled, the stereo system will provide the results for the study.
Hi, I'm Dr.Mark Burke from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hi, I'm Dr.Shaheen Zur, also from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hello, I'm Dr.Morris Petto, director Of the Visual Neuroscience Laboratory at the School of Optometry, university of Montreal.
Today we'll show you a procedure for performing unbiased radiology in the non-human primate. We use this procedure in our laboratory to estimate total neuronal populations in various brain areas. So let's get started.
Start this protocol with a brain, which you have previously perfused. Well, with a fixative such as para formaldehyde, the brain should be blocked in stereotaxic space, cryo protected, and frozen. To see these protocols in detail consult our recent JoVE article, dissecting the non-human primate brain.
In Stereotaxic space, the brain should be systematically sectioned to contain all of the reference space. To see this protocol in detail, consult our recent JoVE article, brain Banking, making the most of your research specimens. In this experiment, we use the frontal lobe of the vervet brain.
The series is set to one out of 10 sections throughout the cortex and sectioned at 50 micrometers. To calculate the estimated cell numbers N, we use the following equation where SSF is the section sampling fraction. A SF is the area sampling fraction.
TSF is the thickness sampling fraction, where the measured thickness of the tissue is divided by the dissector height, and a cube is the total number of objects of interest counter within the dissector. To determine the section sampling fraction, the entire reference space must be well-defined from anterior to posterior and dorsal to ventral. Here we are interested in the frontal lobe, which we define as the region from the tip of the frontal pole to the central sulcus and the lateral sulcus.
Excluding the insular for the frontal lobe of this subject, a total of 760 sections were systematically collected with 76 stained for crest violet. Since the target was 10 sections throughout the reference space, one outta six crestal violet stain sections were sampled. This leads to a section sample fraction or SSF of about one outta 60.
We randomly start with one of the first crestal violet stain sections and systematically sample the rest of the one outta six. Thereafter, a total of 13 sections are sampled for the selected subject. To avoid bias from recognition errors, it is imperative that a standard definition is followed for the particular biological feature of interest.
Here we were interested in counting neurons and defined a neuron as having a visible, centrally located nucleus and a clearly defined cytoplasm. For this study, we use stereo, which is a computerized theology system. The system prompts the user to fill out the necessary information in a step-by-step fashion.
In the study information section, the parameters of the study are established. The volume parameter should be selected to define a reference space for the study. The object volume may also be selected here to estimate the number weighted volume for the population of objects of interest.
In this case, only the volume of the reference space was selected. For each object, select the number and define the feature of interest in this case neurons. To establish the sampling information, enter the slab sampling interval.
If separate slabs of tissue is sliced exhaustively, and each section is placed in sequential order, then enter one here. Next, enter the total number of sections taken through the reference and then enter the section sampling interval. The system will then calculate the number of sections to be sampled.
To define the grid and dissect a size, use a low magnification of 2.5 to 10 x. To define the grid spacing under the edit menu for volume, the object magnification should be performed at 100 x. The frame area is the size of the dissector.
In this case, we used 50%screen. The frame height is the thickness of the dissector. Here it is set at 10 micrometers and will determine the tissue sampling fraction.
The frame spacing is the size of the grid. For smaller areas, a smaller grid size should be used for this subject, and within the frontal lobe, a grid size of 2, 500 micrometers yields an average of 254 dissectors. The size of the dissector should yield between zero to five counted objects.
In this case, neurons, both the dissector size and grid size are used to calculate the area sampling fraction. Once the study parameters have been established, sampling through the tissue can begin under low magnification. The program will prompt the user to trace the reference space on the section.
The system will then place a grid over the section based on the pro parameters. This grid is used to calculate volume. Next, the user will verify that the points fall within the reference space.
If a point is not within the reference space, simply click on the point and it will not be calculated into the volume. Afterwards, the system will place a new grid over the reference space based on the frame spacing parameters. The intersections on this grid determine where each dissector will be placed.
Now, verify that the intersections fall within the reference space. The system will prompt the user to switch to the higher magnification objective and move the stage to the first dissector. At this point, the system prompts the user to define the top and bottom of the section.
This is done to determine TSF, the thickness sampling fraction and will be repeated at each dissector location. To determine the measured thickness focus through the Z plane until the first cell comes into focus. Then back up slightly to the top of the section until the last object appears, just outta focus.
Then to find the bottom of the tissue, focus through the Z plane until the cells are barely outta focus while focusing through the Z plane. Note that the cells should be stained at every depth. If not, this may indicate an incomplete penetration of the stain or non-uniform dehydration of the tissue, in which case the sections should be restrained.
The default dissector height for this study is 10 micrometers. The difference between the dissector height and the measured section thickness is the guard height, which is the volume of tissue where no biological features are counted. The system then sets the sampling space in the Z axis.
The user will then click on each object that falls within the dissector through the Z plane. Objects that touch the red lines or the bottom of the dissector are not counted. Once each object is counted, click next.
This is used to calculate the total number of objects. The stage will move to the next dissector and repeat the previous steps. Once all of the dissectors for the section have been counted, the system will prompt the user to insert the next sequential section to be probed.
The steps will be repeated until all of the sequential sections have been sampled. Once finished, the system will then provide the calculations for area sampling, fraction section sampling, fraction tissue sampling fraction, and the total number of objects. Based on these parameters, the system will generate an estimated n reference volume and coefficient of errors for both the estimated number and volume.
It'll also give recommendations to become more efficient or to reduce the coefficient of errors. We've just shown you how to perform unbiased theology in a biological specimen. When doing this procedure, it is important to remember to systematically sample throughout the entire reference space, carefully measure tissue thickness at each optical dissector, and have a well-defined object of interest.
So that's it. Thanks for watching and good luck with your experiments.
