Atlas of plant and animal histology

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Histological techniques


Fine features of tissues and cells are visualized with microscopes. However, only small thickness samples can be readily observed with microscopes, otherwise there would be diffusion and poor penetration of light in light microscopy, or electrons in transmission electron microscopy. For these reasons, it is necessary to obtain sections from tissues we want to study. Section thickness may rank from hundreds of micrometers to a few nanometers. Some tissues, like blood or cell cultures, can be observed without sectioning after they are extended onto a slide or because they are a few cells thick.

Microtomes are the laboratory apparatus for obtaining histological sections. There is a wide range of microtomes designed for different purposes: obtaining sections with different thickness, cutting soft or hard embedding media, or tissues not embedded at all, and to obtain sections from frozen samples.

Most common microtomes:

Paraffin microtome. It is designed to obtain sections from paraffin embedded samples. The section thickness may range from 5 to 20 µm, and they are intended for light microscopy studies. This type of microtome is present in every histology lab.

Hand microtome. It is a very simple and easy-to-use apparatus consisting of a sample holder that can be raised manually. The microtome is held with a hand and sections are obtained with a blade handled with the other hand. It is useful for plant samples, which have hard tissues and can be cut without embedding. Sections thickness are usually above 100 µm, intended to be observed at light microscopy.

Vibratome. It can cut not-embedded material (relatively soft samples) and produces sections ranging from 30-40 µm to hundreds µm in thickness. These sections are studied at light microscopy.

Freezing microtome. With this microtome, 30 µm to 100 or 200 µm thick sections can be obtained from frozen samples. The sections are for light microscopy studies.

Cryostat. From frozen samples, 10 to 40 µm thick sections can be obatined and be studied at light microscopy.

Ultramicrotome. This apparatus is able to cut samples embedded in resin and obtain sections ranging from dozens of nanometers (semithin sections) to 1 µm in thickness. Thicker sections (0.5 µm or higher) are studied at light microscopy and those thinner than 100 nanometers are intended for transmission electron microscopy.

Ultracryotome. It is not a widely used apparatus in histology labs, but it is needed when ultrathin sections (nanometers in thickness) have to be obtained from non-embedded material. These samples are first frozen, therefore hardened, and then ultrathin frozen sections can be obtained for transmission electron microscopy studies.

Traditionally, the most used microtomes for the study of the general features of tissues are the paraffin microtome for light microscopy and the ultramicrotome for transmission electron microscopy. Nowadays, the cryostat is gaining popularity because it saves time (all the embedding process) and provides a general better molecular preservation; It can even cut non-fixated material.

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