[Music] hello everyone welcome back to my channel today we'll start with a new topic that is microscopy a microscope is a scientific instrument that enables the observation of objects or specimens that are too small to be seen with the naked eye it uses a combination of lenses and light to magnify and enhance the details of microscopic structures microscopes have played a pivotal role in advancing scientific understanding by revealing the Hidden World of tiny organisms cells and intricate structures time lapse Imaging is a technique in microscopy that involves capturing a series of images of a sample at regular intervals over a specified period of time this approach allows researchers to observe Dynamic processes changes and interactions occurring in life cells or other biological samples microscopes are used to evaluate the Integrity of samples during an experiment to map the fine details of the spatial distribution of macromolecules within cells to directly measure biochemical events within living tissues for Imaging whole animals in meters through tissues and embryos measured in micrometers and down to cells proteins and DNA in nanometers the study of living cells may require time resolution from days for example when Imaging neuronal development or disease processes now let us understand the difference between magnification and resolution magnification is not the best measure of a microscope rather resolution that is the ability to distinguish between two closely spaced points in a specimen is a much more reliable estimate of microscopes utility the shorter the wavelength of Illuminating Light the higher the resolving power of the microscope according to Rayleigh Criterion which defines the minimum resolvable distance between two point sources of light the angular resolution denoted by Theta is given by the following formula that is Theta is equal to 1.22 multiplied by the wavelength of The Illuminating Light divided by the diameter of the objective lens aperture in Optics an aperture is a hole or an opening through which light travels shorter wavelength of light such as blue or violet light result in smaller values for wavelength in the Rayleigh Criterion equation a smaller wavelength leads to a smaller angular resolution which means that the microscope can distinguish smaller details and closer points as separate objects standard light microscopes have a lateral resolution limit of about 0.5 micrometer for routine analysis the limit of resolution for a microscope that uses visible light is about 300 nanometer with a dry lens that is in air and 200 nanometer with an immersion lens the next line shows the different types of microscopes that we'll study first category is light microscopy that includes bright field microscopy which is further divided into three subtypes that are simple microscopes compound microscopes which can be upright as well as inverted and then stereo microscopes second type of light microscopy is dark field microscopy then we'll discuss fluorescence microscopes and then we'll discuss electron microscopes which are further categorized as scanning electron microscopes and then transmission electron microscopes now let's discuss all these microscopes one by one in detail light microscopy it is one of the most common and basic techniques in Optical microscopy used to observe an image stained or naturally colored specimen here its principle include light source a bright field microscope uses a light source typically a halogen lamp to provide a uniform and steady illumination of the specimen condenser the condenser is a lens system located beneath the microscope stage it focuses and concentrates the light in onto the specimen creating a well illuminated field of view specimen the specimen is placed on the microscope slide and positioned on the stage the specimen can be stained with dies to enhance contrast as many biological specimens are transparent and lack natural coloration objective lens the objective lens is located directly above the specimen it collects the light that has passed through the specimen and magnifies the image formed by the transmitted line eyepiece the eyepiece or ocular lens further magnifies the image formed by the objective lens allowing The Observer to view the specimen with greater detail image formation as light passes through the specimen it interacts with different structures densities and refractive indices within the specimen some region of the specimen May absorb more light and appear darker While others may allow more light to pass through and appear lighter contrast enhancement contrast is generated by difference in light absorption or scattering within the specimen so basically this absorption is a principle of your right field microscopy and scattering is for dark field microscopy stain structures or regions with varying Optical properties produce varying levels of contrast against the background image observation the magnified and contrast enhanced image of the specimen is observed through the eyepiece the final image is a result of the interplay between the absorbed scattered and transmitted light creating a visible representation of the specimens internal structure now coming to the first type of light microscopy that is bright field microscopy the principle of bright field microscopy involves a transmission of white light through a specimen which interacts with the sample to produce contrast and form a visible image some regions of the specimen it may absorb more light and appear darker While others may allow more light to pass through and appear lighter resulting in Darker areas against a brighter background stain structures often create absorption contrast contrast in bright field microscopy primarily depends on variation in the refractive index and thickness of different structures within the specimen this technique works well for samples with inherent contrast now what kind of samples can be viewed by bright field microscopy bright field microscopy is suitable for observing a wide range of specimen from cells and tissues to microorganisms both living and dead specimens and simple materials it is more suitable for specimens with intrinsic contrast staining enhances the contrast however it is limited by its inability to effectively observe transparent or low contrast specimens phase contrast microscopy and differential interface microscopy are Advanced Techniques developed to address this limitation by enhancing the contrast of transparent specimens types of bright field microscope they can further be categorized as simple microscopes that use single lens magnifiers compound microscopes those with multiple lenses and objective lenses they are available in two models as upright microscope and inverted microscope and then stereo microscopes for 3D imaging compound microscopes are more capable of providing higher magnifications and better resolution compared to simple or stereo microscopes now let us discuss simple microscopes first a simple microscope typically has a single convex Glass Lens often referred to as magnifying glass mounted in a metal frame this lens is used to magnify objects and bring them into Focus for observation here the specimen requires very little preparation and is usually held close to the eye in the hand focusing of the region of interest is achieved by moving the lens and the specimen relative to one another the source of light is usually the Sun or ambient indoor light the detector is the human eye the recording device is a hand drawing applications the magnification power of a simple microscope is limited compared to more complex microscopes but it still provides a useful level of magnification for various everyday applications such as reading small texts examining small objects or performing simple visual inspections compound microscopes a compound microscope specifically refers to a type of light microscope that uses multiple lenses to magnify a specimen the major components are the condenser lens the objective lens and the eyepiece lens each of these components is in turn made up of combination of lenses which are necessary to produce magnified images illumination compound microscope use built-in light source typically located at the base of the microscope to illuminate the specimen light passes through the condenser and the specimen to reach the objective lens diaphragm the diaphragm is an adjustable aperture located below the stage that controls the amount of light passing through the specimen adjusting the diaphragm helps optimize image contrast and quality condenser the condenser is a lens system located beneath the stage that focuses and concentrates light onto the specimen it helps improve the illumination of the specimen and enhances image clarity stage the stage is a flat platform where the specimen slide is placed for observation it often includes mechanical controls for moving the slide in horizontal and vertical directions enabling precise positioning for observation objective lenses compound microscopes have multiple objective lenses with varying magnification example 4X 10x 40x and 100x these lenses are positioned on a rotating turate or nose piece and are used to capture the initial magnification of the specimen nose piece or rotating to it the nose piece holds and allows for easy switching between different objective lenses this feature eliminates the need to manually change lenses for different magnifications eyepiece or ocular lens the eyepiece is the lens at the top of the microscope that you look through it further magnifies the image produced by the objective lens typically compound microscopes have eyepieces with 10x magnification the image is either viewed directly by I in the eyepiece or it is most often projected onto a detector for example photographic film or more likely a digital camera the images are displayed on the screen of a computer Imaging system stored in a digital format and reproduced using digital methods course and fine focus knobs these knobs are used to adjust the focus of the microscope by moving the stage vertically the course Focus Norm is used for rough focusing while the fine focus knob provides precise focusing so this diagram on the right is very important where you can see the source of light focusing the light wire diaphragm to the condenser lens which further condenses the light to the specimen and from the specimen the light enters the objective lens which actually magnifies the images and from there the light passes to the eyepiece or ocular lens which further magnifies the images and finally the image can be viewed with the eye now let us discuss in more detail the different parts of this compound microscope light source the correct illumination of the specimen is critical for achieving high quality images and photomicrographs this is achieved using a light microscope typically light sources are halogen lamps Mercury lamps Xenon Lambs laser or laser emitting diodes light from the light source passes into the condenser lens which is mounted beneath the microscope stage in an upright microscope and above the stage is an inverted microscope the condenser focuses light from the light source and illuminates the specimen with parallel beams of light a correctly positioned condenser lens produces illumination that is uniformly bright and free from glaze across the viewing area of the specimen also termed as color illumination as you can see here in the diagram cooler illumination is a technique used in microscopy to achieve optimal illumination of a specimen it involves adjusting the light source and Optical components to ensure even and uniform illumination of the specimen while maximizing resolution and contrast this technique is widely used in bright field microscopy and is named after its developer August Kohler condenser misalignment and an improperly adjusted condenser aperture diaphragm that is responsible for controlling the angle of The Illuminating Light cone are major sources of poor images in the light microscope so this diagram makes it very clear that how the diaphragm should be open for uniform illumination of the entire field area the specimen or sample preparation specimen preparation for bright field microscopy involves several critical steps to ensure that the specimen is appropriately mounted stained if necessary and prepared to achieve clear and detailed images first step is specimen collection and fixation the specimen sometimes called the sample can be the entire organism or a dissected organ hole mount and ellicotte collected during a biochemical protocol for a quick check of the preparation or a small part of an organism that is biopsy or smear of blood or spermatozoa in order to collect images from it the specimen must be in a form that is compatible with the microscope if the specimen is living it may need to be fixed prior to sectioning to preserve its structure and prevent Decay or degradation it holds biological activity and immobilizes structures and maintains a structural Integrity specimens morphology for observation under a microscope popular fixatives include formaldehyde or formalin that cross-links proteins preserving cellular structures glutaldehyde similar to formaldehyde used for electron microscopy ethanol or methanol used for preserving DNA often in cytogenetics buoyance fluid used for preserving soft tissues and cellular details after fixation the specimen is usually permeabilized in order to allow a stain to infiltrate the entire tissue the amount of permeabilization time and severity depends upon several factors for example the size of the stain or the density of the tissue sample mounting a relatively thin and transparent piece of specimen is mounted on the piece of glass line add a drop of mounting medium or suitable liquid such as water saline tissue culture medium glycerol or immersion oil to the specimen with a thin square of glass mounted on top known as coverslip the mounting medium helps prevent drying and provides a medium for light transmission cover slips cover slips are graded by the thickness the thinnest one are labeled as number one which corresponds to a thickness of approximately 0.17 mm the cover slip side of the specimen is always placed closest to the objective lens it is essential to use a cover slip that is optimally matched to the objective lens in order to achieve optimal resolution this is critical for high magnification Imaging because if the cover slip is too thick it will be impossible to achieve an image microtome many specimens are too thick to be mounted directly onto a slide and these are cut into thin sections using a device called a microtome as you can see in the first diagram the tissue is usually mounted in a block of wax and cut with the knife of the micro Dome Into Thin sections between 100 millimeter to 500 millimeter in thickness the sections are then placed onto a glass slide stained and sealed when mounting medium with a cover slip cryostat some samples are frozen and cut on a cryostat which is basically a microtome that keep a specimen in the Frozen State and produce Frozen sections more suitable for immunolabling a biochemical process that enables detection and localization of an antigen to a site within a cell organ or tissue contrast and light microscope most cells and tissues are colorless and almost transparent and lack contrast when viewed in a light microscope therefore to visualize any details of cellular components it is necessary to introduce contrast into the specimen this is achieved either by Optical means using a specific configuration of microscope components or by staining the specimen with a Dye or more usually using a combination of optical and staining methods staining staining can enhance contrast and highlight specific features within the specimen different regions of the cell can be stained selectively with different stains choose an appropriate staining method based on the specimen type and structures you want to visualize types of stain basic dyes these dies are positively charged and bind to negatively charged cellular components acidic dyes these dyes are negatively charged and bind to positively charged cellular components common stains include hematozylene and eosin a standard stain used in histology to highlight nuclei by hematozylene and cytoplasm by eosin gym sustain used to stain blood smears for identifying blood cells and malaria parasites Crystal wallet stains bacterial cells for visualization in microbiology acridine orange stays nucleic acids for fluorescence microscopy oil Red O stains lipids in cells Optical contrast contrast is achieved optically by introducing various elements into the light path of the microscope and using lenses and filters that change the pattern of light passing through the specimen and the optical system this can be as simple as adding a piece of colored glass or a neutral density filter Into The Illuminating Light path by changing the light intensity or by adjusting the diameter of a condenser aperture usually all of these operations are adjusted until an acceptable level of contrast is achieved for Imaging specimen stage the specimen stage is a mechanical device that is finely engineered to hold the specimen firmly in place any movement or vibration will be detrimental to the final image the stage enables the specimen to be moved and positioned in fine and smooth increments both horizontally and transversely for locating a region of Interest objective lens after placing the prepared slide on the microscope stage select the appropriate objective lens and adjust the focus to bring the specimen into view the objective lens is responsible for producing the magnified image and can be the most expensive component of the light microscope objectives are available in many different varieties and there is a wealth of information inscribed on each one this may include manufacturer magnification such as Forex 10x 20x 40x 60x and 100x immersion requirements like oil water or glycerol cover slip thickness usually 0.1 mm and often more specialized Optical properties of the lens objective lenses can either be dry that is glass air and cover slip or immersion lenses that includes glass oil or water and cover slip as a thumb rule most objectives are 10x and 40x are air objectives and those above 40x are immersion usually 100x the lens will be marked to indicate the use of other immersion media like glycerol and water hanging drop method is a laboratory technique used to observe the behavior and interactions of microorganisms cells or small organisms in a controlled environment for example demonstration of the mobility of flagellated bacteria as you can see here in the diagram below dipping lens are specially designed to work without a cover slip and dipped directly into the water or tissue culture medium these are used for physiological experiments as you can see here in the diagram the dry dipping as well as immersion lenses numerical aperture numerical aperture is a key parameter that defines or measures the light Gathering and resolving capabilities of a microscope's objective lens from the specimen lenses with a low numerical aperture collects less light than those with a high numerical aperture higher numerical aperture objectives yield the best resolution the numerical aperture is always marked on the lens this is a number usually between 0.04 and 1.4 numerical aperture takes into account the refractive index of the medium between the objective lens and the specimen and the half angle of the cone of light that can enter the lens image forming light waves pass through the specimen and into the objective in an inverted cone as Illustrated in figure the angle Nu is one half the angular aperture denoted by a the maximum angle of light that can be accepted by the objective lens and is related to the numerical aperture through the following equation that is numerical aperture is equal to n sine Nu where n is the refractive index of the Imaging medium between the front lens of the objective and the specimen cover glass a value that ranges from 1 for air to 1.51 for specialized immersion oils the points on the right side shows on what parameters numerical aperture depends the refractive index is a measure of how much a material can bend light different media such as air oil or water have different refractive indices and using a medium with a higher refractive index can increase the numerical aperture and thus the resolution cone of light the half angle new represents the angle at which light rays are accepted by the objective lens the larger the angle the more oblique rays of light can be gathered allowing for greater resolution resolution numerical aperture is directly related to the resolution of the microscope higher numerical aperture objectives yield the best resolution a higher numerical aperture allows the microscope to resolve smaller details and distinguish closely spaced structures objective lens selection microscope objectives with higher numerical aperture are capable of capturing more light and finer details resulting in better image quality different objectives may have different numerical apertures based on their designs and applications numerical aperture and wavelength numerical aperture is inversely proportional to the wavelength of light used shorter wavelength example blue light result in higher numerical apertures leading to improved resolution this relationship is important in fluorescence microscopy eyepiece or ocular lens the eyepiece Works in combination with the objective lens to further magnify the image and allows it to be detected by eye or more usually to project the image into a digital camera for recording purposes eyepieces usually magnify by 10x since an eyepiece of higher magnification merely enlarges the image with no improvement in resolution any magnification above this point is often called empty magnification the best way to improve magnification is to use a higher magnification and higher numerical aperture objective lens in addition to the human eye and photographic film there are two types of electronic detectors employed on Modern light microscopes these are area detectors that usually form an image directly for example video cameras and digital imaging systems that is charged couple devices types of compound like microscope stand the part of the microscope that holds all the components firmly in position is called the stand there are two basic types of compound like microscope stand and upright microscope the light microscope is below the condenser lens and the upright microscope and the objective lenses are above the specimen stage as you can see in the first diagram this is the most commonly used format for viewing specimens and inverted microscope is engineered so that the light source and the condenser lens are above the specimen stage and the objective lenses are beneath it moreover the condenser and light sources are often be swung out of the light path this allows additional room for manipulating the specimen directly on the stage for example for the micro injection of macromolecules into tissue culture cells for in vitro fertilization of eggs or for viewing developing embryos over time applications of compound microscope biology and Life Sciences cellular observation bright fill microscope is used to observe and study the morphology structure and behavior of cells tissues and organelles in histology it is employed in the examination of stained tissue sections for diagnosing diseases and studying tissue architecture for microorganism identification bright field microscopy helps identify and study microorganisms such as bacteria fungi and alkene blood cell analysis blood smears are absorbed using bright field microscopy for assessing different types of blood cells and their abnormalities microbiology for studying bacterial morphology it is used to observe and classify bacteria based on their shape Arrangement and staining characteristics yeast and fungal studies bright field microscopy assists in identifying and characterizing different types of yeast and fungi Education and Research sector classroom teaching bright field microscopy is commonly used for educational purposes to introduce students to the basics of microscopy and cellular structures in research it serves as a primary tool for primary observations and documentation of specimens in various research studies quality control and Industry food and beverage analysis it is used to examine food products for contamination spoilage and quality control pharmaceutical analysis brightfield microscopy assists in the inspection of pharmaceutical products for the quality detects and uniformity for Environmental Studies algal and protozoan Analysis brightfield microscopy helps identify and study microorganisms in water samples aiding in environmental monitoring in soil microbiology it is employed to observe soil microorganisms and their interactions Material Science for particle analysis brightfield microscopy is used to analyze particles contaminants and impurities in materials for quality control it assists in assessing the quality and properties of materials including fibers polymers and minerals medical diagnostics urine sediment analysis bright field microscope is used to examine urine sediments for the presence of cells crystals and other components hematology it aids in the valuation of blood cells and components for diagnosing blood Related Disorders Agriculture and plant Sciences plant tissue analysis bright fill microscopy helps study plant structures cells and tissues in various agriculture research applications now coming to the last type of bright field microscope that is Stereo microscopes another type of light microscope the stereo microscope is used for the observation of the surface of large specimens the microscope is used when 3D information is required a wide range of objectives and eyepieces are available for different applications the light source can be from above from below the specimen is circling the specimen using a ring light or from the side giving a dark Field Effect these different light angles serve to add contrast or Shadow to the images fixation and staining is not required in case of stereo microscopes binocular viewing stereo microscopes typically feature two eyepieces providing binocular vision this enhances depth perception and allows for comfortable extended use low magnification stereo microscopes provides relatively low magnification usually in the range of 5x 200x this allows for observing larger specimens while maintaining a wide field of view large working distance the distance between the objective lens and the specimen walking distance is greater in Stereo microscopes as you can see here in the diagram compared to the compound microscopes this is especially useful when manipulating or dissecting specimens or working under the microscope application of stereo microscopes dissection and manipulation stereo microscopes are idle for dissecting small organisms performing intricate procedures or manipulating delicate specimens their large working distances and 3D visualization capabilities make such tasks easier entomology entomologists use stereo microscopes to study insects and other small invertebrates enabling detailed examination of their external features botany stereo microscopes Aid in observing plant structures such as leaves flowers and Roots allowing researchers to study morphology and developmental stages paleontology paleontologists use stereo microscopes to examine fossils identifying and studying intricate details of ancient organisms quality control in Industries such as manufacturing and quality control stereo microscopes are used for inspecting products analyzing defects and Performing fine assembly work gemology gemologists use stereo microscopes to analyze gemstones for characteristics such as inclusions colors and cut in the next session we'll discuss dark field microscopy for any further query you can contact me through the given email ID thank you for watching [Music] thank you