Microscopy

Object details at a larger scale

Microscopy is a technique for producing visible images of structures or details too small to otherwise be seen by the human eye. To improve the resolution within various light source can be used, for example:

The demonstration of details is low with visible light but very high with electron beams as used in electron microscopy (EM).

 

Visible Light

The workhorse of histologists was the light microscope. It was invented already in the 16. Century: Using various arrangements of glass lenses it was possible to obtain detailed cellular differences.
Modern light microscopes allow a 1000-times enlargement in tissue sections of as low as 0.5 to 1 µm. For this purpose, brain tissue is perfused with formaldehyde, embedded in paraffin, and cut with a microtome into 1 – 30 µm sections. These tissue slices are transferred to an object holder and stained with adequate dye procedures.
This procedure still remains as the classic tool to estimate the cellular integrity within the various tissues.


Picture 1:
Picture 2:
Picture 3:

Picture 1:
A classic light microscope of the 60’s.

Picture 2:
Embedding automate for formaldehyde fixated brains

Picture 3:
Stained and native paraffin sections

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Fluorescence microscope: A UV light source (black box at left side) illuminates the tissue section from below. With adequate filter arrangements fluorophore-labeled antibodies can be visualized.

UV light

Fluorescence microscopy uses UV light for the illumination of an object instead if visible light. Fluorophores can then be visualized with adequate filter arrangements. Fluorescence microscopy is preferentially employed with immunohistochemical procedures because the various antibodies can be labeled different classes of fluorophores.
Alternatively, one can inject fluorescent dyes intravenously to demonstrate perfused blood in brain tissue. Under normal conditions labeled blood will remain within the space of all vessels providing evidence of the intactness of the so-called “blood-brain-barrier”. Under pathophysiological conditions, the BBB can become leaky and generate vasogenic brain edema, a severe complication for survival after stroke or brain trauma.

Ex-vivo fluorescence: the red-fluorescent dye (Evans-Blue) is only visible within the smallest units of brain vessels, the so-called capillaries.
Ex-vivo fluorescence: Following a BBB-damage of brain tissue, for example after a disturbance of brain perfusion, large confluent areas of labeled blood-borne fluorophores (Evans-Blue) are visible. This process can lead to massive brain swelling (brain edema).

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Microtome for the cutting of paraffin embedded brain tissue into very this sections
(less than 1 μm)