APPLICATION OF MICROSCOPY IN PHARMACOGNOSY.docx

**APPLICATION OF MICROSCOPY IN PHARMACOGNOSY** Microscopy is a useful tool in Pharmacognosy for the study of the internal structure, constitution, and inclusions of plant and animal cells or other objects in detail. It is necessary for the detection of adulterants and contaminants of herbal prepara...

**APPLICATION OF MICROSCOPY IN PHARMACOGNOSY** Microscopy is a useful tool in Pharmacognosy for the study of the internal structure, constitution, and inclusions of plant and animal cells or other objects in detail. It is necessary for the detection of adulterants and contaminants of herbal preparations and thus provides a means for assessing the authenticity and quality of herbal drugs. Microscopy is also useful for the detection of physical characteristics such as size, shape, and relative position of different cells and tissues as well as the chemical nature of the cell walls, and the form and nature of cell contents during microscopic analysis of crude drugs. Different kinds of microscopes are available for microscopical analysis. They include; **Optical microscopy**: This is the most common type of microscopy and uses visible light to illuminate the sample. It includes bright-field, dark-field, phase-contrast, and fluorescence microscopy. Depending on the number of eyepieces or ocular lenses, a microscope may be mono-, bi-, and trinocular, bright-field, dark-field, phase-contrast, fluorescence microscope, etc. The optical microscope can be simple or compound depending on the number of lens systems. Hence it is possible to have; **Simple microscope**: here the microscope uses a single lens for the magnification of the sample. The working principle is on the fact that when a sample is placed within the focus of the microscope, a virtual, erect, and magnified image is formed at the least distance of distinct vision from the eye that is held at the lens. **Compound microscope**: the lens system is more than one. It uses a combination of lenses and two optical parts known as the objective lens and an eye piece or ocular lens to magnify the given object. The principle is that the combination of lenses enhances the magnification of the object. The sample is first viewed as a primary object in the tube and viewed again in the eyepiece as a magnified erect image. **Electron microscope**: this is a type of microscope that uses a beam of electrons as the light source to illuminate the sample. It is regarded as a special type of microscope with the ability to magnify images in nanometers. Various types include; transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning transmission electron microscopy (STEM). The principle of the electron microscope is based on the excitation of Tungsten (the metal used in the microscope) on the application of a high-voltage current which results in the formation of a continuous stream of electrons that is used as a beam of light. The lenses used are magnetic coils that can focus the electron beam on the sample such that the sample gets illuminated. **Stereomicroscope**: this type of microscope provides images on a three-dimensional plane. It is also known as the dissecting microscope. In a stereo microscope, there are separate objective and eyepiece lenses such that there are two separate optical paths for each eye. **Scanning probe microscope**: this type of microscope finds application in industries where the examination of the specimen is done at the nanoscale. The study of a specimen's properties, its reaction time, and its behavior when stimulated can be done with the help of a scanning probe microscope. Generally, the botanical microscopic uses the characteristics of botanically authenticated multiple samples that have been compared and cross-checked against other microscopic characterizations for consistency and completeness. Microscopical evaluation of crude drugs may be qualitative or quantitative or both. Qualitative microscopy includes studies of the transverse sections of leaf, and root bark, as well as the longitudinal section of root bark under photomicrograph with or without staining. In the case of powder microscopy, different staining reagents such as iodine for the detection of starch grains and calcium oxalate crystals while phloroglucinol for the detection of lignified components are used. Quantitative microscopy of some pharmacognostic parameters like vein-islet number, vein termination number, stomatal number, stomatal index, and palisade ratio are used for identification, purity determination, and evaluation of crude leafy drugs. Drawing of morphological and histological structures of plant and animal organs and various other minute structures (e.g., trichomes, glands, stomata, calcium oxalate crystals) is also used for quantitative microanalysis of admixed or adulterated powdered drugs. Plant sections or powders of the drug are mounted in water or dilute glycerol for light microscopic examination. Color and clearing, bleaching, and defatting reagents are used to stain and clear before microscopic examinations. Tissues are macerated by using chemicals to disintegrate the middle lamella and isolation of tissues for study. Micrometry and camera lucida drawing to scale of tissues, cells, cellular elements, cell inclusions, and other minute structures are of significant value in the examination of crude drugs for quality assessment in the presence of adulterants. With the worldwide increase in popularity and acceptance of herbal medicines, classical tools like microscopy are urgently needed for the assessment and quality control of plant products such as crude herbal drugs, registered herbal medicinal products, over-the-counter herbal products, or health foods. Modern pharmacopeias offer new monographs on herbal drugs, including their microscopy. **Principles of Microscopy** For a microscope to achieve its intended goal of use, it must achieve three fundamental goals: First it must create **magnification**; ostensibly, this is the motivation of microscopy and is simple and easy to produce. Magnification, however, must be accompanied by both **resolution** and **contrast** to be useful. 1. **Magnification**: Magnification is the enlargement of the appearance of an object. It can also be considered as the ability of lenses to enlarge an object visually. If the magnifying power of the lens is 10X, it means that the given lens can enlarge the object up to 10 times. In a compound microscope, the magnification is the product of the magnifying power of both lenses. The magnifying power of the lens depends on the **focal length**. The lower the focal length, The higher the magnifying power of the lens. The focal length is a measure of how strongly the system converges or diverges light. It is the inverse of the focal power. 2. **Resolution**: The resolving power is the ability to distinguish two closely placed points. The resolution power of the lens allows us to observe the details of an object. The resolution of the microscope can be out by using Abbe's equation. https://cdn-fgnif.nitrocdn.com/DXTfpExRWnFuRKBvfNFPNrfazQrUitPW/assets/desktop/optimized/rev-fed9070/wp-content/fd77af52f8e3e7adddc3fa7336f834ab.webpc-passthru.php Where, d -- distance between two closely distant points λ -- wavelength of light n sin θ -- numerical aperture The microscope with higher magnification has a small d value. λ is the wavelength of light, shorter is the wavelength; the higher is the resolution. The wavelength of visible light is from 300 to 700 nm. The best resolution for a light microscope is obtained in the range of 450 to 500 nm. 'n' is the refractive index of the medium. The refractive index is the ability of the medium to bend the light. The angle of the cone of light is affected by the refractive index of the medium. The refractive index of air is 1. 'θ' is the half of the angle of the cone of light that enters the microscope. The value of 'Sin θ' cannot be more than 1 because the angle of entering the cone of light cannot be more than 90° and the value of sin 90 is 1. d= 0.5 x 450 nm               1 d= 225 nm 0r 0.2 μm Hence, the resolution limit of light microscope is 0.2 μm. **BASIC QUALITY PARAMETERS OF MICROSCOPIC IMAGES** The microscopic images should have four basic quality parameters, through which the microscopes can be graded. 1. **Focus:** It refers whether the image is well defined or blurry (out of focus). The focus can be adjusted through course and fine adjustment knobs of the microscope which will adjust the focal length to get clear image. The thickness of specimen, slide and coverslip also decide the focus of the image. (Thin specimens will have good focus). 2. Brightness: It refers how light or the dark the image is. 2. **Brightnes**s: of the image depends on the illumination system and can be adjusted by changing the voltage of the lamp and by the condenser diaphragm. 3. **Contrast**: It refers to how best the specimen is differentiated from the background or the adjacent area of the microscopic field. More contrast will give good images. It depends on the brightness of illumination and color of the specimen. The contrast can be achieved by adjusting illumination and diaphragm and by adding color to the specimen. The phase contrast microscopes are designed in such a way that the contrast can be achieved without coloring the specimen. 4. 4\. Resolution: It refers to the ability to distinguish two objects close to each other. The resolution depends on the resolving power, which refers minimum distance between the two objects that can be distinguishable **MAGNIFICATION AND RESOLUTION** The total magnification of the compound microscope is the product of the magnifications of the objective lens and eyepiece. Magnification of about 1500x is the upper limit of compound microscopes. This limit is set because of the resolution. Resolution refers to the ability of microscopes to distinguish two objects close to each other, it depends on resolving power, which refers to the minimum distance. Ex: Man has a resolving power of 0.2 mm (meaning that he can distinguish two objects with a distance of 0.2 mm close to each other) If he wants to see beyond the limit of his resolving power, further magnification is necessary. µ Resolving power = \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- n (sin ᶿ ) where µ is the wavelength of the light source and n (sin ᶿ ) is the numerical aperture (NA). For compound microscopes, the resolving power is µ/2NA. The resolving power of a microscope can be improved either by reducing the wavelength of light or by increasing the n(sin ᶿ) value. The numerical aperture (n sinᶿ) measures how much the light cone spreads out between the condenser and specimen. More spread of light gives less resolving power means better resolution. The numerical aperture depends on the objective lens of the microscope. There are two types of objective lenses available in any compound microscope. 8.2.4. THE LIMIT OF RESOLUTION The limit of resolution refers the smallest distance by which two objects can be separated and still be distinguishable or visible as two separate objects **NOTE**: In case of microscopes with oil immersion, when light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends. The refractive index of air is 1.0, which is less than that glass slide (1.56). So, when light passes from glass (dense medium) to air (lighter medium), the rays get refracted, which led to loss of resolution of image. Light of different wavelengths bends at different angles, so that as objects are magnified the images become less and less distinct. This loss of resolution becomes very apparent at magnifications of above 400x or so. Even at 400x the images of very small objects are badly distorted. Placing a drop of oil (Cedar wood oil) with the same refractive index (1.51) as glass between the cover slip and objective lens eliminates two refractive surfaces and considerably enhances resolution, so that magnifications of 1000x or greater can be achieved. Oil immersion is essential for viewing individual bacterial cell. A disadvantage of oil immersion viewing is that the oil must stay in contact, and the oil should be viscous. 8.3.1.2. DARK-FIELD MICROSCOPE In dark-field microscopy, the specimen is brightly illuminated against a dark background **Challenges that can arise when using microscopes:** 1. [**Low contrast and glare**: Samples with low contrast can be difficult to observe, and some areas on a sample image can be too bright or too dark due to the sample material, texture, or color, which can make it difficult to clearly observe surface conditions ](https://www.qualitymag.com/articles/92779-common-microscope-challenges) 2. [**Difficulty in viewing large samples at high resolution**: As magnification is increased, the observation field (field of view) becomes narrower, and it becomes very hard to obtain an overall image of the sample. ](https://www.qualitymag.com/articles/92779-common-microscope-challenges) 3. [**Contamination**: Contamination of samples can pose major obstacles to measurements. ](https://academic.oup.com/mam/advance-article-abstract/doi/10.1093/micmic/ozad090/7252196) 4. [**Crystallization**: Crystallization of samples can pose major obstacles to measurements. ](https://academic.oup.com/mam/advance-article-abstract/doi/10.1093/micmic/ozad090/7252196) 5. [**Macroscopic bending of the samples**: Macroscopic bending of the samples can pose major obstacles to measurements. ](https://academic.oup.com/mam/advance-article-abstract/doi/10.1093/micmic/ozad090/7252196) **Some common Terms in Microscopy** **The field of view: This** in a microscope is the maximum area visible when looking through the microscope eyepiece and is usually given as a diameter measurement. It is determined by the field number or the diameter of the diaphragm, and the magnification of the lens. The field of view for a microscope is the extent of the observable area in units of distance. To calculate the field of view, magnification, and field number of the microscope's lens currently in use required. The field number is divided by the magnification number to determine the diameter of the microscope's field of view. Whenever the microscopes is changed, or the eyepieces or objective lenses are switched, the FOV calculations with the new field number and magnifications should be repeated. **ASSIGNMENT (Note that this is not to be submitted, but to enhance your understanding)** Using mathematical expression to show/establish the relation between; 1. Focal length and Magnification 2. Magnifying power and focal power 3. Magnification and resolution 4. Resolving power and Magnification 5. Numerical aperture and focal lenght