Phase contrast microscopy is an indispensable diagnostic procedure in gynecology that greatly facilitates the examination of vital cells in cervical mucus (cervical mucus) and vaginal secretions (vaginal fluid). In addition, this procedure also allows for the evaluation of sperm (semen cells).
Indications (areas of application)
- Diagnostics of vaginal flora – when examining the vaginal flora (vaginal flora), the following examinations are possible by phase contrast microscopy: for example, the diagnosis of a lack of bacterial colonization or a normal vaginal flora (lactic acid bacteria) with or without cytolysis (cell destruction) can be made with the help of this diagnostic procedure. Furthermore, it is possible to identify bacteria in the preparation and to classify them into the groups of immobile and motile bacteria. In addition, flagellates such as Trichomonas vaginalis, which belong to the group of parasites, can usually be reliably detected by the phase contrast microscope. Furthermore, mycoses (fungal diseases) can be reliably detected in phase contrast microscopy, so that the diagnosis of thrush mycosis can be made with the aid of phase contrast microscopy. Based on these diagnostic capabilities, the detection of an infectious cervical cause of sterility is usually possible.
- Inflammatory markers – by phase contrast microscopy, the number and type of leukocytes (leukocyte morphology) can be determined.
- Cycle phase determination – vaginal cytology is used to determine the cycle phases of the menstrual cycle. In this examination step, superficial (superficial) cells of the vagina (vagina) are examined microscopically. The cell assessment provides information about the current menstrual cycle phase of the woman.The female cycle is divided into two phases, the first phase is called the follicular phase (egg maturation phase) and the second cycle phase is called the corpus luteum phase (corpus luteum phase). However, it should be noted that the cycle phase determination by means of phase contrast microscopy has now been largely replaced by hormone diagnostics, which ensures a high degree of diagnostic certainty.
- Sims-Huhner test – this test procedure, which is also called the post-coital test, is used to determine whether sperm (sperm cells) can penetrate the cervical mucus (cervical mucus) to reach the site of fertilization.
- Assessment of sperm – in addition to assessing the ability of sperm to penetrate (pass through) the cervical mucus, phase contrast microscopy can further examine the number of sperm, their motility and morphology (appearance).
- Smear examination – in case of suspicion, for example, mycosis (fungal disease), there is the possibility of a smear of the vagina (vagina) or vulva (the totality of the external primary sexual organs of a woman. It consists of the labia and clitoris) or possibly from the anal region to take and evaluate microscopically.
Preparation of phase contrast microspie using the example of the evaluation of the vaginal secretion present:
- At the beginning of the assessment of vaginal secretions by a phase contrast microscope, the preparation of two suitable native preparations (fresh, unfixed, usually unstained preparation of body fluids, tissues or organ contents for microscopic detection of live pathogens) must be made.
- The addition of potassium hydroxide solution (KOH) and table salt to the vaginal secretion is performed. After mixing is complete, the combined secretion is applied to a slide and covered with a coverslip. Subsequently, the phase-contrast microscopic assessment can be made.
The procedure
The principle of phase contrast microscopy is based on the interference of light rays in the beam path of the microscope, which can achieve a high-contrast image of unstained cells. Structure and function of the phase contrast microscope:
- The annular aperture, condenser, annular phase plate, and objective are particularly important to the function of the phase contrast microscope. By inserting the annular aperture into the condenser and the phase plate into the objective, a separation of non-diffracted and diffracted light beams is caused.The resulting delay in the wavelength of the diffracted light rays causes a phase difference that optically makes the structural details of the microscopic object to be examined appear darker than the surrounding medium. Physically, the phase platelets convert the phase or path differences into amplitude differences. As a result of these amplitude differences, the image of the object appears in the microscope by mutual cancellation or amplification of the direct light rays passing through the object and diffracted at the object.
- Thus, a disadvantage of this principle is the fact that ideal phase preparations are not the rule, since amplitude effects are usually superimposed on the phase effects.
- Based on the principle of imaging the structures by amplitude differences, the existing cell structures are represented as graded shades of gray depending on their own optical density. To improve the reproduction of the structures, the use of thin preparations and phase platelets is indicated. Depending on the nature of the various phase platelets, both positive and negative phase contrasts can be achieved, resulting in the objects of interest being rendered either darker or lighter than their surroundings.
- In phase contrast microscopy, it is also important to note that necrosis (death of cells) can occur relatively quickly due to exposure to light and additional damage to the vital cells. As a result of this rapid necrosis development, the assessment of the slides must be performed within one to two hours in order to maintain the validity of the diagnostic measure. Thus, this time limitation on the evaluation of vital specimens usually requires documentation by microphotography or, if necessary, by performing microkinematography. A photomicrograph is a photograph of a microscopic image taken with a camera attached to a microscope. Through microkinematography, there is the possibility to capture objects under microscopic observation in moving images.
Advantages of phase contrast microscopy:
- Immediate diagnostics – time-saving and inexpensive to perform.
- Relatively easy to perform and therefore quickly teachable and learnable.
- Depending on the experience of the investigator with relatively good sensitivity (percentage of diseased patients in whom the disease is detected by the application of the procedure, i.e., a positive finding occurs) and specificity (probability that actually healthy people who do not suffer from the disease in question are also detected as healthy by the procedure) afflicted
- The diagnosis is feasible without outside assistance (i.e., without a third-party laboratory).
