Instrumentation 6

Microscopy is the study of objects or samples that are too small to be seen by the naked eye. There are several types of microscopy, each with its own advantages and limitations. Here are the main types of microscopy:

1. Optical microscopy: This is the most common type of microscopy, which uses visible light to illuminate a sample. Optical microscopy can be further divided into several subtypes, such as brightfield, darkfield, phase contrast, fluorescence, and confocal microscopy.

Optical microscopy is a technique that uses visible light to observe the sample under a microscope. It consists of several components, including an objective lens, an eyepiece lens, and a light source.

The working of optical microscopy involves the following steps.

• The sample to be viewed is prepared by fixing it onto a glass slide and adding a stain or dye to enhance its contrast.
• The light source, located beneath the sample, emits light that is directed through the condenser lens to focus the light onto the sample.
• The objective lens is placed above the sample and is used to magnify the image of the sample. The magnified image is formed by the refraction of light passing through the objective lens.
• The eyepiece lens, located above the objective lens, further magnifies the image formed by the objective lens and focuses the light onto the observer's eye. The image is formed by the interaction of the light waves passing through the sample with the lens.
• The observer can view the magnified image of the sample by looking through the eyepiece lens.

 

By adjusting the focus knob, the observer can bring the sample into sharp focus, while by adjusting the objective lens, they can change the level of magnification.

Optical microscopy is widely used in a variety of scientific and medical applications, including.

1. Biological research: Optical microscopy is an essential tool in studying cells, tissues, and organisms. It enables scientists to visualize cellular structures, study cell behavior and processes, and identify cellular abnormalities.
2. Medical diagnosis: Optical microscopy is commonly used in medical diagnosis for the detection of various diseases, including cancer, infections, and genetic disorders. It enables clinicians to examine tissue samples and identify abnormal cells.
3. Material science: Optical microscopy is used to investigate the properties of materials, including their structure, composition, and defects. It is used in fields such as metallurgy, nanotechnology, and semiconductors.
4. Forensic analysis: Optical microscopy is used in forensic analysis to identify and analyze trace evidence, including fibers, hairs, and other microscopic materials.
5. Quality control: Optical microscopy is used in the manufacturing industry to monitor and ensure product quality. It is used to examine the quality and consistency of materials, such as polymers, metals, and ceramics.

 

• Brightfield microscopy: This is the simplest form of optical microscopy, which uses a light source to illuminate a sample that is placed on a glass slide. The sample absorbs some of the light, causing it to appear darker against a bright background.

Brightfield microscopy is a common technique used in biological and medical research to observe stained or naturally pigmented specimens. It works by passing light through the sample and using a series of lenses to magnify the image.

The working of Brightfield microscopy can be explained in the following steps.

• A light source such as a halogen lamp or LED is used to illuminate the sample. The light passes through a condenser lens, which focuses and directs the light onto the specimen.
• The sample is prepared by staining it with dyes or by natural pigmentation. The sample is then placed on a glass slide and covered with a coverslip.
• The light passes through the sample and is then focused by the objective lens. The objective lens is located near the specimen and is responsible for the magnification of the image.
• The magnified image is then projected to the eyepiece lens, which further magnifies the image for the observer to see.
• The magnified image is formed as light passes through the sample and is diffracted or refracted, which creates the image that is viewed through the eyepiece.
• The observer can view the image through the eyepiece and adjust the focus and magnification as needed to observe different aspects of the sample.

 

Brightfield microscopy has a wide range of applications in biological and medical research, as well as in other fields. Some common applications include

1. Cell biology: Brightfield microscopy is often used to observe cell morphology and structure, as well as to track cell growth and behavior over time.
2. Histology: Brightfield microscopy is used to study tissue samples, including examining the morphology of cells and tissues, identifying pathological changes, and detecting abnormal cells.
3. Microbiology: Brightfield microscopy is used to study microorganisms, including bacteria, fungi, and protozoa. It is often used in clinical and diagnostic settings to identify pathogens and to study their structure and behavior.
4. Material science: Brightfield microscopy is used to examine the structure and properties of materials, including metals, ceramics, and polymers. It can be used to study surface defects, grain boundaries, and other features that affect material properties.
5. Quality control: Brightfield microscopy is often used in manufacturing and quality control settings to inspect products for defects or irregularities.
6. Education: Brightfield microscopy is commonly used in educational settings to teach students about cell biology, histology, and other topics.

 

• Darkfield microscopy: This type of microscopy is used to visualize samples that are transparent or lack contrast. A special condenser is used to direct light at an oblique angle, causing the sample to appear bright against a dark background.

Darkfield microscopy is a specialized type of optical microscopy that is used to visualize samples that are difficult to see using traditional brightfield microscopy. In darkfield microscopy, a special condenser is used to direct light at a steep angle onto the sample, causing the sample to appear bright against a dark background.

 

Here is the step-by-step working principle of darkfield microscopy 

• A darkfield condenser is placed under the sample stage of the microscope. This condenser blocks the direct light from the light source, allowing only oblique rays to pass through.
• The oblique light passes through the sample, reflecting off the edges and contours of the specimen.
• The scattered light is then captured by the objective lens of the microscope and directed to the eyepiece, creating an image that appears bright against a dark background.
• The contrast between the bright specimen and the dark background allows for improved visualization of samples that are difficult to see using brightfield microscopy.

 

Darkfield microscopy has a variety of application:

1. Microbial studies: Darkfield microscopy is commonly used to observe live microorganisms, such as bacteria and fungi, that are difficult to see using traditional brightfield microscopy.
2. Hematology: Darkfield microscopy can be used to observe red blood cells, white blood cells, and platelets in blood samples. 
3. Mineralogy: Darkfield microscopy is used to study the structure and properties of minerals. 
4. Material science: Darkfield microscopy is used to examine the structure and properties of materials, including metals, ceramics, and polymers.
5. Quality control: Darkfield microscopy is often used in manufacturing and quality control settings to inspect products for defects or irregularities.
6. Education: Darkfield microscopy is commonly used in educational settings to teach students about microbiology, hematology, and other topics.

 

• Phase contrast microscopy: This technique enhances the contrast of samples that have low refractive indices, such as living cells. It uses a phase plate to create a phase shift in the light that passes through the sample, resulting in a visible image.

Phase contrast microscopy is a type of optical microscopy that allows for the visualization of transparent or translucent specimens that would otherwise be difficult to see using traditional brightfield microscopy. In phase contrast microscopy, a special phase plate is used to transform the phase differences of light waves passing through a transparent specimen into differences in brightness or contrast, creating an image with high contrast and detail.

 

Here is the step-by-step working principle of phase contrast microscopy.

• The microscope is equipped with a specialized phase contrast condenser, which is designed to produce a hollow cone of light that illuminates the sample from the side.
• The phase plate is placed within the objective lens, in the path of the light that has passed through the sample. The phase plate converts the phase differences of light waves caused by the sample into amplitude differences.
• The amplitude differences are then transformed into contrast by the phase ring of the objective lens, which creates a sharp image with high contrast.
• The resulting image shows clear contrast between the transparent or translucent sample and its surroundings, allowing for detailed observation and analysis.

 

Phase contrast microscopy has a variety of applications, including. 

1. Cell biology: Phase contrast microscopy is commonly used in cell biology to study live cells, including their structure, function, and behavior.
2. Microbiology: Phase contrast microscopy is often used to observe microorganisms, including bacteria, fungi, and parasites, that are difficult to see using traditional brightfield microscopy.
3. Histology: Phase contrast microscopy is used to observe and analyze tissue samples, including blood and biopsy specimens.
4. Materials science: Phase contrast microscopy is used to study the structure and properties of materials, including polymers, fibers, and nanoparticles.
5. Quality control: Phase contrast microscopy is often used in manufacturing and quality control settings to inspect products for defects or irregularities.

 

• Fluorescence microscopy: This type of microscopy uses fluorescent dyes or proteins to label specific structures or molecules in a sample. When illuminated with a specific wavelength of light, the labeled structures emit fluorescence that can be detected and visualized using special filters and detectors.

Fluorescence microscopy is a type of optical microscopy that utilizes fluorescence to produce high-resolution images of cells, tissues, and other biological samples. Fluorescence microscopy works by exciting fluorescent molecules within the sample using a specific wavelength of light, causing them to emit light at a longer wavelength, which can be visualized and analyzed using specialized equipment.

 

Here is the step-by-step working principle of fluorescence microscopy.

• A fluorescent molecule or probe is introduced into the sample being studied. This can be done through a variety of methods, such as staining, genetic engineering, or chemical labeling.
• The microscope is equipped with a light source that emits a specific wavelength of light, known as the excitation wavelength, which is absorbed by the fluorescent molecule within the sample.
• The absorbed energy causes the fluorescent molecule to become excited and emit light at a longer wavelength, known as the emission wavelength.
• A specialized filter within the microscope is used to separate the excitation and emission wavelengths, allowing only the emitted light to be visualized and recorded.
• The resulting image shows the fluorescent molecules within the sample, allowing for high-resolution visualization and analysis.

 

Fluorescence microscopy has a wide range of applications, including.

1. Cell biology: Fluorescence microscopy is commonly used in cell biology to study cellular structures, functions, and interactions.
2. Immunology: Fluorescence microscopy is used to visualize the localization and distribution of immune cells and molecules within tissues and organs.
3. Neuroscience: Fluorescence microscopy is used to study the structure and function of neurons and other cells within the nervous system.
4. Cancer research: Fluorescence microscopy is used to visualize cancer cells and their behavior within tissues, allowing for better understanding and development of treatments.
5. Materials science: Fluorescence microscopy is used to study the structure and properties of materials, including polymers, nanomaterials, and semiconductors.

 

• Confocal microscopy: This is a specialized type of fluorescence microscopy that uses a laser to focus on a specific plane within a sample, allowing for high-resolution 3D imaging.

Confocal microscopy is a type of fluorescence microscopy that uses a laser to excite fluorescent molecules within a sample. Unlike conventional fluorescence microscopy, confocal microscopy uses a pinhole to exclude out-of-focus light, resulting in sharper images with improved contrast and resolution. Here is a detailed explanation of the working principle and applications of confocal microscopy:

 

Working Principle:

• A laser is used to excite fluorescent molecules within the sample, producing a signal that is emitted at a longer wavelength.
• A pinhole is used to exclude out-of-focus light, allowing only the in-focus light to be detected by a photodetector.
• The laser scans the sample in a series of 2D images, which are then reconstructed into a 3D image using specialized software.

 

Applications:

1. Biology: Confocal microscopy is widely used in biology to study the structure and function of cells, tissues, and organs. It is particularly useful for studying living cells and tissues in real-time, as well as for examining subcellular structures and organelles.
2. Neuroscience: Confocal microscopy is used in neuroscience to study the structure and function of neurons and other cells within the nervous system. It is particularly useful for studying neuronal morphology and connectivity.
3. Materials science: Confocal microscopy is used in materials science to study the structure and properties of materials, including polymers, semiconductors, and nanomaterials. It is particularly useful for studying surface topography and for characterizing the distribution of materials within a sample.
4. Medicine: Confocal microscopy is used in medicine to diagnose and monitor various diseases, including cancer, dermatological disorders, and cardiovascular disease. It is particularly useful for studying skin lesions and for detecting cancer cells within tissues.

 

2. Electron microscopy: This type of microscopy uses a beam of electrons instead of light to illuminate a sample. Electron microscopy can achieve much higher resolution than optical microscopy but requires specialized equipment and sample preparation.

Electron microscopy (EM) is a type of microscopy that uses a beam of electrons instead of light to visualize the sample. The high energy of the electrons enables them to achieve much higher resolution than traditional light microscopes. There are two main types of electron microscopy: transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

 

Working of Electron Microscopy.

• In a TEM, electrons are emitted from an electron source, focused by a series of lenses, and then passed through a very thin specimen. The electrons that pass through the specimen are then projected onto a fluorescent screen or a digital detector, producing a high-resolution image of the specimen's internal structure.
• In an SEM, a beam of electrons is focused onto the sample, which is coated with a thin layer of metal to improve conductivity. As the beam scans across the sample, it interacts with the sample's surface and generates various signals, including secondary electrons and backscattered electrons. These signals are then detected and used to generate a detailed 3D image of the surface structure of the sample.

 

Applications of Electron Microscopy.

1. Electron microscopy is widely used in the field of structural biology to visualize the internal structures of cells and tissues at high resolution. It is also used in materials science and nanotechnology to study the structure and properties of various materials, including metals, ceramics, and polymers. Additionally, it is used in the study of viruses, bacteria, and other microorganisms, and in the analysis of geological and environmental samples.

 

Some specific applications of electron microscopy include.

1. Visualizing the ultrastructure of cells and tissues, including organelles, membranes, and cytoskeletal elements.
2. Investigating the structure and properties of various materials, including metals, ceramics, and polymers, at the nanoscale.
3. Examining the surface structure and topography of various materials, including semiconductor chips, integrated circuits, and microelectronic devices.
4. Studying the structure and function of biological macromolecules, including proteins, DNA, and RNA.
5. Identifying and characterizing viruses, bacteria, and other microorganisms in clinical and research settings.

 

• Transmission electron microscopy (TEM): In TEM, a beam of electrons is transmitted through a thin section of a sample, producing an image with high resolution and contrast. (TEM) is a technique that uses a beam of electrons to produce images of thin sections of samples. Here is the working principle and applications of TEM:

Working principle:

• In TEM, a beam of electrons is generated and focused onto the sample using electromagnetic lenses. The electrons pass through the thin sample and are scattered, absorbed, or transmitted depending on the sample's density and thickness. The transmitted electrons are then collected by a detector on the other side of the sample, forming an image of the sample's internal structure. The resolution of TEM is extremely high, up to atomic scale, allowing for detailed examination of cellular and subcellular structures.

Applications:

1. TEM has a wide range of applications in biological and materials sciences. In biological sciences, TEM is used to study the ultrastructure of cells and tissues, including the arrangement of organelles, the morphology of viruses, and the structure of proteins. TEM is also used to study the fine structure of microorganisms, such as bacteria and fungi. In materials sciences, TEM is used to study the microstructure of materials, including metals, ceramics, and polymers. TEM is particularly useful for studying the atomic arrangements of crystals and interfaces, as well as defects and dislocations in materials. Additionally, TEM can be used to study nanoparticles and nanomaterials.

 

• Scanning electron microscopy (SEM): In SEM, a beam of electrons is scanned across the surface of a sample, producing a 3D image with high resolution and depth.

Scanning electron microscopy (SEM) is a type of microscopy that uses a focused beam of electrons to produce images of a sample's surface. Here is the working principle and applications of SEM:

Working principle:

• In SEM, a beam of electrons is generated and focused onto the sample using electromagnetic lenses. The electrons interact with the sample's surface, producing signals such as secondary electrons, backscattered electrons, and X-rays. These signals are then collected by detectors and used to form an image of the sample's surface. The resolution of SEM is typically in the range of a few nanometers, allowing for detailed examination of surface structures.

 

Applications:

1. SEM has a wide range of applications in many fields, including materials sciences, biology, and geology. In materials sciences, SEM is used to study the surface morphology, texture, and composition of materials, including metals, ceramics, and polymers. SEM can be used to observe the surface defects, fractures, and corrosion in materials. In biology, SEM is used to examine the surface structures of cells, tissues, and organisms, including the morphology of bacteria, viruses, and insects. SEM can also be used to study the surface features of fossils and minerals in geology.

 

3. Scanning probe microscopy: This type of microscopy uses a probe to scan the surface of a sample, producing high-resolution images of the sample's topography and properties.

Scanning probe microscopy (SPM) is a type of microscopy that uses a physical probe to scan the surface of a sample at the nanometer or atomic scale. The most commonly used SPM techniques are atomic force microscopy (AFM) and scanning tunneling microscopy (STM).

 

Working principle:

• In AFM, a sharp probe with a nanometer-sized tip is brought close to the surface of the sample, and a feedback mechanism is used to control the position of the probe as it scans across the surface. The probe interacts with the surface of the sample through interatomic forces, and the deflection of the probe is measured and used to create a topographic image of the surface.
• In STM, a metallic probe with a sharp tip is brought close to the surface of a conductive sample, and a voltage is applied between the tip and the sample. The current that flows through the tip and the sample is measured and used to create an image of the surface. STM is particularly useful for imaging conductive materials and for studying the electronic properties of surfaces.

 

Applications:

1. SPM has a wide range of applications in materials science, physics, chemistry, and biology. It can be used to study the topography, structure, and properties of surfaces and to manipulate and measure individual atoms and molecules. SPM is particularly useful for studying the properties of materials at the nanoscale, such as the roughness, elasticity, and adhesion of surfaces. In biology, SPM can be used to study the structure of proteins, DNA, and other biomolecules and to investigate cell surface interactions.

 

• Atomic force microscopy (AFM): In AFM, a small probe is scanned across the surface of a sample, measuring the forces between the probe and the sample's surface to produce a 3D image.

 

Atomic force microscopy (AFM) is a type of scanning probe microscopy that uses a tiny probe to scan the surface of a sample, generating a topographic image of the surface with atomic resolution. The AFM probe consists of a cantilever with a sharp tip at the end, which is scanned over the sample surface while measuring the interaction forces between the tip and the sample. The resulting topographic image is constructed by mapping the vertical displacement of the cantilever as it scans the surface.

The working principle of AFM involves the use of a piezoelectric material to move the cantilever in a precise manner. As the tip moves across the sample surface, the interaction forces between the tip and the sample cause a deflection of the cantilever, which is detected by a laser beam reflecting off the back of the cantilever. The amount of deflection is used to generate a topographic image of the sample surface.

AFM has a wide range of applications in materials science, surface chemistry, and biology. It can be used to image surfaces with atomic resolution, measure surface forces and mechanical properties of materials, and investigate surface reactions and interactions. AFM can also be used to study biological molecules such as proteins and DNA, and to image live cells and tissues.

 

Some of the specific applications of AFM include.

• Surface characterization: AFM can be used to measure surface roughness, topography, and mechanical properties of materials.
• Nanolithography: AFM can be used to fabricate patterns and structures on surfaces with nanometer-scale resolution.
• Biomolecular imaging: AFM can be used to image individual biomolecules, such as proteins and DNA, at high resolution.
• Cell imaging: AFM can be used to image live cells and tissues, providing information on their topography and mechanical properties.
• Material analysis: AFM can be used to study the composition and structure of materials, such as polymers, ceramics, and composites.

 

• Scanning tunneling microscopy (STM): In STM, a sharp probe is scanned across the surface of a sample, measuring the electrical current that flows between the probe and the sample's surface to produce a high-resolution image.

Scanning Tunneling Microscopy (STM) is a type of scanning probe microscopy that was invented in 1981 by Gerd Binnig and Heinrich Rohrer, who were awarded the Nobel Prize in Physics in 1986 for their work. STM is used to investigate the electronic and structural properties of surfaces at the atomic scale. It works by passing a fine metal probe (tip) over the surface of a sample while maintaining a constant current between the tip and the sample.

 

Working:

• STM works by scanning a sharp metallic tip across a conducting surface at a very small distance, typically in the range of 0.01-0.5 nm. The tip is usually made of tungsten or platinum-iridium, and it is held at a bias voltage relative to the sample. When the tip is brought close to the surface, the electric field between the tip and the surface causes the tunneling of electrons between the two. This tunneling current is measured and used to generate an image of the surface. 
• STM can be operated in two different modes: constant height mode and constant current mode. In constant height mode, the height of the tip is kept constant, and the current flowing through the tip is recorded as the sample is scanned. In constant current mode, the current is kept constant, and the height of the tip is adjusted to maintain this constant current as the sample is scanned.

Application:

1. STM has a wide range of applications in the field of materials science, nanotechnology, and surface science. It is used to investigate the atomic structure of surfaces, surface defects, and the properties of individual atoms and molecules. It can also be used to manipulate individual atoms and molecules on surfaces, which is important in the field of nanotechnology.

 

Some of the specific applications of STM are:

1. Surface topography imaging: STM can be used to obtain high-resolution images of the surface topography of materials at the atomic scale.
2. Surface electronic structure analysis: STM can be used to investigate the electronic properties of surfaces at the atomic scale. This is important in the study of surface chemistry, catalysis, and semiconductor materials.
3. Manipulation of atoms and molecules: STM can be used to manipulate individual atoms and molecules on surfaces. This has important applications in the field of nanotechnology.
4. Study of surface defects: STM can be used to investigate surface defects, such as vacancies, adatoms, and steps, which play an important role in many surface phenomena.
5. Study of biological systems: STM can be used to investigate biological systems, such as proteins and DNA, at the atomic scale. This has important applications in the field of biotechnology.

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