The Guide To Specimen Preparation For Microscopes

For microscopy techniques, specimen formation is fundamental.  Because of the ideal biological specimen, the microscopy technique can be easily used. There are many good reasons to examine the biological cells and tissues under an inverted microscope or simple microscope.  The medical and biological research is underpinned by the internal structural details of the cells and tissues.  It is seen that in the normal healthy state, all cells and other tissue elements are in good regular shape and recognizable patterns. But in the diseased forms, all such patterns get irregular due to the presence of chemical and physical influences of other elements. This is diagnosed with the help of microscopic techniques like phase contrast microscopy, inverted microscopy, darkfield microscopy, brightfield microscopy, etc.  

The detection of diseases becomes easy with an ideal specimen formation. Identification of all these ailments is based on the histopathology and cytopathology of the cells and tissues.  This marks the ways for the specializations of modern medicine.   In this method, microscopy techniques play an important role in pathological as well as biological research.  The study of parasites like viruses, bacteria, etc is detected by the microscopy techniques like phase-contrast microscopy.   For the ideal microscopic detection, biological specimens are required. With the help of skilled persons, the formation of biological specimens is done which is fundamental to the microscopy technique.

Microscopy Technique

All types of biological specimens are visualized under the cell culture microscope, culture microscope, and tissue culture microscope.  In the scientific world, many types of microscopes are used to observe cells and tissues.   The most employed form of microscopy technique is  as follows :

• Brightfield Microscopy
• Phase Contrast Microscopy
• Darkfield Microscopy
• Electron Microscopy

These microscopy techniques are used to illuminate the biological specimens with a beam of light rays that pass through a microscope trinocular head. The same happens with the electron microscope,  but with a beam of electrons. 

In the following ways, the cells and  the elements are preserved for the microscopic analysis :

• All the cells and other related elements in the specimen are preserved in a “life-like” state or active state. This is called fixation.
• The biological specimen is transparent rather than opaque. So,  the light rays from the light source can penetrate the specimen’s plane.
• The biological specimen should be thin and flat. This is important for the subtle visualization of cells. The single layer of cells is analyzed under a trinocular compound microscope.
• In biological specimens, some of the components are differentially colored or stained. This marks the presence of infected cells and tissues. Moreover, it also helps in clearly analyzing the samples.  The stained part helps in the powerful bioimaging of the cells and tissues. 

Steps In Biological Specimen Formation For Microscopy Technique

For analyzing the various biological specimens, one requires the ideal biological specimen and that requires the good procedure of biological specimen.

Following are the steps are required for the formation of biological specimens :

1. The Section Preparation

It is seen that all fresh tissues are very delicate and easily distorted and can get easily damaged.  Due to this fragility of tissues, they can be cut into thin sections of slices. For transforming the cut section into biological specimens, the fixatives are used which helps in the process of preservation of cells and tissues. For fixation, it is  broadly divided into the following types :

•  In this category, the tissue is rapidly frozen and kept frozen until its sections are cut down. This cutting down of tissues is done with a cryostat microtome which is used in the freezing chamber.  The resultant of this process is called the frozen sections of tissues of cells. The advantage of forming the frozen sections is that they can be prepared quickly without taking so much time.  For the observations of frozen sections, phase contrast microscopy is used because of its great illumination process.
• In this second category of formation of biological samples, the specimens are infiltrated with the help of a liquid agent which helps in converting into the form of solids.  It is done to back up the cutting down of the specimens.  Moreover, it allows the solids to be cut down into thin sections.  For infiltration and supporting process, agents like epoxy and methacrylate resins and paraffin waxes. The produced specimens are called paraffin sections.   Here, the rotary microtome is used.  The rotary microtome is here for cutting down the solids into sections. In the pathological research labs, the paraffin waxes are routinely prepared from almost every biological sample. These are used in the diagnosis process.

2. Specimen Reception

It is seen that many different biological specimens come from several different sources.  This ranges from very large specimens to tiny sources. The fragments are cut down to their cellular or tissue level.  Following are the sources of biological specimens used in histopathology systems or biological research :

• The excision specimens like surgical biopsies, where whole organs or affected areas are removed at operation for treatment.
• In an incisional biopsy specimen, the tissue is removed for diagnosis from within an affected area of the diseased body. The section is further cut down to be analyzed in an inverted phase contrast microscope.
• In the case of punch biopsies, the punches are used to remove a small piece of suspicious tissue or cell for examination under an inverted phase microscope.
• The case of Shave biopsies consists of small fragments of tissue that are shaved from a skin surface.
• In Curettings specimen type, the tissue is removed in small pieces from the lining of the uterus or cervix from the female body.
• For the case of Core biopsies, a small tissue sample is removed using a special needle from any part of the body or skin. 

3. Fixation Of Biological Specimen

Fixation of a biological specimen is a crucial step in the preparation of specimens for the microscopy technique. The main objective of fixation is to prevent the cells and tissues from decaying. It helps to preserve the cells and tissues in a ‘life-like state.  This is done by stopping the enzyme killing and microorganisms killing and hardening of sections of cells and tissues. All this is done by maintaining the molecular structure intact within cells and tissues. 

As soon as fixation is initiated, the separation of the specimen from its blood supply is done.  This ensures a good result for the biological specimens.  For the fixation process, the most common fixative agent is formaldehyde that is used in the form of a phosphate-buffered solution, called formalin.  Usually, all biological specimens are used for analyses under an inverted microscope or simple microscope, the samples are immersed in formalin for 6 to 12 hours, before the process.

4. Grossing Of Biological Specimen

Grossing refers to a cut-up of the cells and tissues which involves careful examination and description of the biological specimen. This also includes appearance, dimensions, and several pieces.  In the case of larger biological specimens,  it requires further dissection to produce representative pieces from the concerned areas. On the contrary, in the case of small biological specimens,  no further cutting is required.  They can be further processed.

Conclusion

The best trinocular microscope is used to examine the biological specimens for better examination. For better observational study and biological research, specimen formation is mandatory which should be done carefully.