Human Spinal Cord

Human Spinal Cord

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The spinal cord is a long, thin, tubular bundle of nervous tissue and support cellsthat extends from the medulla oblongata in the brainstem to the lumbar region of thevertebral column. The brain and spinal cord together make up the central nervous system (CNS).

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Posterior Gray Horn

the three membranes that envelop the brain and spinal cord with the primary function of the meninges is to protect the central nervous system.

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Meninges

the three membranes that envelop the brain and spinal cord with the primary function of the meninges is to protect the central nervous system.

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Posterior Gray Commisure

The gray commissure is a thin strip of gray matter that surrounds the central canalof the spinal cord and, along with the anterior white commissure, connects the two halves of the cord. It comprises lamina X in the Rexed classification.

Human Scalp

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The structure of the scalp can be broken down into five layers. The human scalp is covered with one of the thickest sections of skin on the body. Its largest thickness is located at the back of the head and as it reaches the front it becomes thinner. The skin of the head contains numerous sebaceous gland and hair follicles; they all produce oil.Beneath the surface is a thin connective tissue layer called the superficial fascia. The connective tissue is a dense layer of fats and fibrous tissue that contain the nerves and vessels of the scalp. Next is the epicranial aponeurosis which is a tough layer of dense fibrous tissue. Following is a loose areolar connective tissue layer that provides a separation between the upper three layers and the paricranium. The paricranium is the membrane that covers the outer surface of the bone and provides the nutrients needed for the bone as well as for repair.

Epidermis Layer
Sebaceous Gland

The Bigger Picture

Wide field imagery in Microscopy takes the smallest of objects and presents them in a grandiose way. This methods purpose is to be able to display the entirety of a sample for reasoning’s of research or demonstrations.

The process of making one of these images usually takes a lot more than just one picture. For me it was generally in the 200 range. This is because the images have to be at a high enough magnification to delineate small structures within a sample, such as a nucleus within a cell. By doing that, however, you cannot get the entire subject within the photo. This process for me usually is an hour long session slouched over a camera making small adjustments after each photo.

After the initial photographing of the images comes a session of   staring at a computer screen, to stitch all the photos. This part can sometimes be fairly tedious. To begin this section of the process you need to load all your images into a stitching software, which there are several. Next you wait anxiously to see if the software will actually stich the photos. Depending on well you shot this can go either way. When the software finally spits out a correctly stitched image of your sample and not a cubist representation of it you can sit back in your chair and cheer a little.

To finish your image you need to add an appropriate bar scale to show the size of you subject. Then if you’d like you can process it a bit to make it look pretty. Yeah, that’s essentially it in a nutshell. If you follow most of these steps you should hopefully have a presentable wide field photomicrograph.

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Structures of the Human Body

The lens can be seen in the bottom right of the image of the eye of the human fetus. The lens, along with the cornea, allows light to be focused on the retina. The retina is located at the back of the eye. The suspensory ligament can be seen left of the lens. This structure helps to keep the lens in place, and is located on both sides of it. The middle picture is a bone marrow sample. Bone marrow is the spongy material inside bones. Bone marrow has stem cells, which produce blood cells. Red blood cells, platelets, and white blood cells are all examples of blood cells that are located throughout the body. The bottom picture is a portion of the heart wall. The heart is the strongest muscle in the human body. Veins carry oxygenated blood towards the heart, while arteries carry deoxygenated blood away from the heart.

Wide Field Photomicrography

To create wide field images there are simple steps to follow. First, set the camera to raw format in the settings. Next, go pick out a slide or two and attach the camera to the microscope. Find the specimen and focus the image for the camera. Start on one side of the slide and work across, leaving about ¼ of the previous image in the next. Continue to work across and down until the end of the specimen. Take the SD card out of the camera and put it into the computer. Pull the photos into Photoshop and automatically Adobe Camera Raw will pop up. Here, make some minor changes throughout the images and save the images as JPEGs. Next, go into Adobe Bridge and select the best images of each section of the specimen that can be combined. Choose about 5-10 images, hover over the tools drop down menu and hover over Photoshop and click photomerge. Automatically the program will go into Photoshop and then select reposition for the images chosen. Hit done and the section selected will be made. Do the same thing for the rest of the images. By the end there should be multiple sections about 5-6 depending on how many primary images. Next, flatten the layers in all the selection photographs and save them as sets of JPEGs. Now go back to Adobe Bridge and select the section images and repeat what was done to make the section images. Make a bar scale of 100 microns. Lastly, give the image a title, the name of the slide, and save the whole image as a TIFF file.

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Figure 1
Figure 2
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Figure 3
Figure 3

The Effects of the Aperture Diaphragm on an Image

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The Aperture Diaphragm is one of the most
important controls found on the
microscope.
It controls contrast, resolution, brightness,
and depth of field. Proper adjustment and
focusing of the substage condenser are essential
in order to maximize the effectiveness of the objective.
Most subjects benefit from having the aperture diaphragm
from halfway, to one-third open.
The following figures demonstrate the effects
of the aperture diaphragm on the image of a particular subject.

Figure 1-(Aperture Diaphragm fully open)(Top left)
High Brightness, High Contrast, Low Detail,
Shallow Depth of Field

Figure 2-(Aperture Diaphragm 1/2 closed)(Top right)
Acceptable Brightness, Acceptable Contrast,
Medium Detail, Acceptable Depth of Field

Figure 3-(Aperture Diaphragm 3/4 closed)(Bottom left)
Ideal Brightness, Ideal contrast, Ideal Detail,
Ideal Depth of Field

Figure 4-(Aperture Diaphragm completely closed)(Bottom right)
Low Brightness, Low Contrast
Too much Detail, Too much Depth of Field.

The Effects of Aperture Diaphragm in Photomicrography

Closed Aperture Diaphragm
Figure 1
Part Closed Aperture Diaphragm
Figure 2
Part Open Aperture Diaphragm
Figure 3

The purpose of the aperture diaphragm is to control image contrast and resolution by regulating the amount of light that is concentrated on the specimen. The aperture diaphragm in an upright light microscope is located in the sub-stage condenser. It can be seen at the exit pupil of the objective. The image characteristics that are dictated by the aperture diaphragm are resolution, contrast, brightness, and depth of field. Microscopists must balance resolution and contrast in order to create successful scientific microscopic images. This series of aperture diaphragm images is composed of frog skin imaged using a 40x objective with a Canon 5D MkIII on a Carl Zeiss Axio Lab A1 upright light microscope.

Open Aperture Diaphragm
Figure 4
In Figure 1 the aperture diaphragm is completely closed down resulting in a high contrast and large depth of field image at the expense of lower resolution and a darker image.
In Figure 2 the aperture diaphragm is partially closed giving us a decent amount of contrast and depth of field, but still losing some resolution and brightness.
Figure 3 has arguably the most successful use of aperture diaphragm, where there is a good amount of resolution and brightness, while retaining contrast and depth of field.
Figure 4 has an aperture diaphragm that is wide- open, resulting in an image that is soft because of its lack of contrast and depth of field.

Aperture Diaphragm

The aperture diaphragm is one of the most important parts of the microscope used in photomicrography. It is located within the substage condenser, which allows for changes in working distance and therefore focus.

The aperture diaphragm controls four characteristics of the image. It controls intensity (brightness), contrast, resolution (fine detail), and depth of field (range of focus). On the front is a dial in which to control these settings. Turning the dial toward the left opens the aperture diaphragm up (toward 0.25). This increases brightness, increases resolution, lowers contrast, and lowers depth of field (see image A). Turning the dial toward the right closes the aperture diaphragm down (toward 0.10). This decreases brightness, decreases resolution, increases contrast, and increases depth of field (see image D). Changing these settings may also affect the focus, so be sure to adjust the focus accordingly.

So, what is the best setting? It depends on the sample. Some samples require more contrast to show the important details. Others need more resolution to serve their purposes. The optimal setting is different for every subject. In personal experience, finding a middle ground between more contrast or more resolution is my best technique.

Image A

Image A

Image B

Image B

Image C

Image C

Image D

Image D

Umbilical Cord

The umbilical cord is a canal between a developing fetus and the placenta in a mammal. It contains one vein, located at the bottom, and two arteries, which are both above the vein, within a gelatinous substance in the cord. This is called Wharton’s jelly and it protects the blood vessels inside the cord. The umbilical vein provides the fetus with nutrient-filled and oxygenated blood from the placenta. It is directly attached to the placenta, which allows the fetus to draw nutrients from the mother without directly mixing their blood. The arteries each contain two layers: an outer layer of smooth muscle and an inner layer of loosely arranged cells.

Close up of umbilical vein
Close up of umbilical vein
Close up of umbilical cord cross section
Close up of umbilical cord cross section
Figure 1: Wide field image of umbilical cord cross section
Figure 1: Wide field image of umbilical cord cross section

Fluorescence Photomicrography

Fluorescence photomicrography is a special technique for imaging microscopic subjects involving sample excitation. Using a beam of short wavelength energy, the electrons in the sample become excited and rise in energy level. When they fall back down to their ground state they emit longer wavelength energy, which is the bright fluorescence that becomes visible. This technique is often used to track proteins through a process called tagging, where the protein has a fluorescent dye attached to it, so that when it is excited later the protein becomes highly visible. Some subjects – like the sambucus root seen bellow – exhibit autofluorescence, meaning that everything within it fluoresces on its own to some degree, no chemical tags needed.
In order to ensure that full subject detail remains visible and any unnecessary fluorescence does not ruin the image, three filters are used: excitation, emission, and dichroic filters. These filters cut down on the amount of light that passes through, restricting it to very specific bands of color. The excitation filter selects the wavelength band that is going to be used to excite the sample, emission filters cut back on excess fluorescence by absorbing unwanted wavelengths, and the dichroic filter acts as a beam splitter to send the desired excitation frequency to the sample but allow rejection of unwanted frequencies. Since the light is restricted this much exposure times can be a few seconds or longer, sometimes leading to boosting the ISO of the sensor to compensate.

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Normal Brightfield Illumination
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Fluorescence with 500-600nm (green) light. Useful for preserving internal structures and keeping high contrast.
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Fluorescence with 400-500nm (blue) light. Useful for achieving an overall structural view of the subject, though some internal detail is lost.