Last Updated: 21/08/2024
Overview of Microscopy
Microscopy is a cornerstone of scientific discovery, providing critical insights into the structure and function of biological specimens. This comprehensive overview delves into the intricacies of microscopes, including their history, objective lenses, maintenance, and various types, tailored for medical professionals, scientists, and experts.
The Basics of Microscopes
A microscope is an optical instrument that magnifies small objects, allowing detailed examination of specimens that are otherwise invisible to the naked eye. The principle of a microscope involves the use of lenses to bend light rays, creating an enlarged image of the specimen.
Robert Hooke - 1680
History of Microscopes
The history of microscopes dates back to the late 16th century. Hans and Zacharias Janssen, Dutch spectacle makers, are credited with creating the first compound microscope. In 1665, Robert Hooke improved upon this design and published "Micrographia," where he detailed his observations, including the cellular structure of cork, coining the term "cell."
Objective Lenses
Objective lenses are critical components of a microscope, determining the magnification and resolution of the image. Each microscope will only be comparable with specific specifications depending on the model and manufacture. As long as you know the specifics for your microscope you will be able to find compatible new or replacement lenses when needed. There are various types of objective lenses, each suited for different applications:
Some key specifications for lens compatibility:
The DIN number (focal tube length, 160mm common, more info Here).
The NA value (Numerical Aperture, high values usually require immersion oil, more info Here)
Lens Image distance (older microscopes have number values, current standard are infinity corrected represented by ∞ symbol. More info Here).
Coverslip distance (in mm for best performance).
Special design standards ( DIC etc. more info Here)
Magnification (number value provided such as 40x or 100x, and/or represented as a colour band such as blue, red or yellow).
Lens type (include a-chromatic, apochromatic, plan).
Plan vs. Infinity Corrected Objective Lenses
Plan Objective Lenses
Plan objective lenses are designed to correct for field curvature, an optical aberration where the focus of the microscope varies across the field of view. This means that Plan lenses provide a flat image across the entire viewing field, ensuring that all areas are in focus simultaneously. This correction is essential for applications requiring high-precision imaging, such as pathology, materials science, and any field where detailed analysis of the entire field of view is critical.
Key Features:
Flat Field Correction: Provides a uniformly focused image across the entire field of view.
High Resolution: Maintains sharpness and clarity from the center to the edges of the image.
Ideal for Detailed Work: Used extensively in fields requiring precise image analysis, such as histology and metallography.
Infinity Corrected Objective Lenses
Infinity corrected objective lenses are designed for modern microscopes that use an infinity optical system. In these systems, light exiting the objective lens travels parallel to the optical axis (i.e., at infinity) before being focused by an additional lens system, known as the tube lens, within the microscope. This design offers several advantages, including flexibility in adding optical components (like filters and prisms) between the objective and tube lens without affecting the focus.
Key Features:
Parallel Light Path: Allows for additional optical elements to be inserted without affecting image focus.
Flexibility: Ideal for advanced microscopy techniques such as fluorescence, phase contrast, and differential interference contrast (DIC).
High Numerical Aperture (NA): Supports high-resolution imaging with excellent light-gathering ability.
You can have both Plan and Infinity corrected lenses, they are not the same or incompatible.
Other Lens Types:
Achromatic Lenses: Correct chromatic aberrations for two colors and spherical aberration for one color.
Plan Lenses: Correct field curvature, providing a flat image across the field of view.
Apochromatic Lenses: Correct chromatic aberrations for three colors and spherical aberration for two colors, offering the highest resolution.
Maintenance of Objective Lenses
Proper maintenance of objective lenses is essential for optimal performance
Storage: Store microscopes in a clean, dry environment with controlled temperature and humidity to prevent mold growth.
Cleaning: Use an air blower to remove dust, followed by specialized lens cleaner and non-lint wipes. Avoid pure isopropyl alcohol as it can damage lens coatings.
Handling: Handle lenses with care, avoiding direct contact with glass surfaces to prevent scratches.
Environment: Use microscopes in clean, dust-free environments. Employ HEPA air purifiers to minimize airborne particles.
Types of Microscopes
Microscopes are essential tools in scientific research, enabling the observation of microscopic entities with exceptional detail. This overview will provide an in-depth look at various types of microscopes, explaining their working principles, underlying physics, and specific applications, offering a thorough understanding of their functionality and usage.
Optical Microscopes
Simple Light Microscope
A simple light microscope uses a single convex lens to magnify objects. The lens bends light rays to converge at a focal point, enlarging the image of the object being observed. This type of microscope is limited in its magnification and resolution due to its single lens design.
Physics: The convex lens in a simple microscope refracts light rays, causing them to converge and form an enlarged virtual image on the observer's retina. The magnification depends on the focal length of the lens, with shorter focal lengths providing higher magnification.
Applications: Often used in basic educational settings, simple light microscopes allow students to gain a fundamental understanding of microscopy. They are also utilized in situations requiring quick, preliminary inspections of specimens.
Compound Light Microscope
Compound light microscopes employ a system of lenses (objective and eyepiece) to achieve higher magnification and resolution. The objective lens creates a magnified real image, which is further enlarged by the eyepiece lens, producing a detailed virtual image.
Objective Lenses: These come in various magnifications (e.g., 4x, 10x, 40x, 100x) and are crucial for detailed imaging. The lenses are interchangeable, allowing for varying levels of magnification depending on the specimen's size and the required level of detail.
Illumination: Typically utilizes halogen or LED light sources. Light is directed through the specimen and focused by the condenser lens, then passes through the objective and eyepiece lenses to the observer’s eye, providing a clear image.
Physics: Compound microscopes use multiple lenses to refract and magnify light, enhancing both resolution and contrast. Techniques such as Kohler illumination ensure even lighting and improved image quality by aligning the light path optimally.
Applications: Widely used in biology and medical diagnostics, compound microscopes allow for the observation of cells, bacteria, and tissue samples. They are essential in fields like histology, microbiology, and cytology, where detailed analysis of small structures is required.
Phase Contrast Microscope
Phase contrast microscopes enhance the contrast of transparent specimens without the need for staining. This is achieved by exploiting differences in refractive index within the specimen, shifting the phase of light passing through it relative to light that does not pass through, creating high-contrast images.
Physics: Utilizes the principle of phase shift, where light waves passing through different parts of the specimen experience varying degrees of phase change. These differences are converted into changes in light intensity, enhancing the visibility of structures that would otherwise be invisible.
Applications: Ideal for live cell imaging, phase contrast microscopes are extensively used in fields like cytology and microbiology. They allow researchers to observe cellular processes in real-time without damaging or altering the specimens through staining.
Fluorescence Microscope
Fluorescence microscopes use high-intensity light sources (e.g., mercury or xenon lamps) to excite fluorescent molecules within the specimen. These molecules emit light at longer wavelengths, producing high-contrast images of specific structures within the sample.
Fluorophores: These are specialized stains or tags that emit light upon excitation. Fluorophores can be attached to specific cellular components, allowing researchers to visualize and study specific parts of the cell or tissue with high specificity.
Physics: Based on the principles of fluorescence and Stokes shift, where absorbed light is re-emitted at longer wavelengths. The emitted light is then filtered to eliminate excitation light, ensuring that only fluorescence from the specimen is observed.
Applications: Widely used in molecular biology and medical diagnostics, fluorescence microscopes are essential for techniques like immunofluorescence and GFP tagging. They allow for the study of cellular functions, protein localization, and molecular interactions.
Confocal Microscope
Confocal microscopes employ lasers and a pinhole aperture to eliminate out-of-focus light, producing sharp, high-resolution images. This technique allows for detailed optical sectioning and 3D reconstruction of specimens.
Physics: Utilizes point illumination and spatial pinholes to achieve optical sectioning. By scanning the laser across the specimen and collecting light through the pinhole, confocal microscopes significantly improve contrast and resolution, allowing for precise 3D imaging.
Applications: Essential in fields like developmental biology and neurobiology, confocal microscopes are used for detailed cellular imaging. They enable researchers to create detailed three-dimensional reconstructions of biological tissues, providing insights into complex cellular structures and functions.
Polarizing Microscope
Polarizing microscopes use polarized light to examine birefringent materials, such as crystals, minerals, and polymers. This technique reveals structural details that are not visible with standard light microscopy.
Physics: Birefringent materials split incident light into two rays with different refractive indices, which are analyzed using polarized light. This interaction creates interference patterns, enhancing the contrast of structures within the specimen.
Applications: Widely used in geology, material science, and chemistry, polarizing microscopes allow for the examination of mineral structures, crystalline phases, and the anisotropic properties of materials. They are essential for identifying and characterizing materials based on their optical properties.
Electron Microscopes Use electrons to take extremely precise images down to the atomic scale, utilizing multiple technologies. They are all expensive in the hundreds of thousands and even millions of dollars to build run and maintain. usually seen only in industrial labs and research settings such as university campuses.
Transmission Electron Microscope (TEM)
TEMs use a high-energy electron beam transmitted through an ultra-thin specimen, providing images at the molecular and atomic levels. Electrons interact with the specimen, creating detailed images based on electron density variations.
Physics: TEMs utilize electron scattering and diffraction. Electrons have much shorter wavelengths than visible light, allowing for much higher resolution imaging. The electron beam is transmitted through the specimen, and the resulting interactions create high-resolution images.
Applications: Critical in fields such as cell biology, virology, nanotechnology, and materials science, TEMs enable the visualization of cell ultrastructure, viruses, and nanomaterials at an atomic scale. They provide insights into the molecular organization and properties of specimens.
Scanning Electron Microscope (SEM)
SEMs scan a focused electron beam across the specimen's surface. Secondary or backscattered electrons emitted from the surface are detected to form high-resolution, three-dimensional images.
Physics: SEMs employ electron beam scanning and interaction with surface atoms. The emitted electrons provide topographical and compositional information, revealing detailed surface structures and material properties.
Applications: Widely used in material science, forensic science, and biology, SEMs allow for the examination of surface morphology, composition, and microstructure. They are essential for quality control, failure analysis, and research in various scientific fields.
Scanning Tunneling Microscope (STM)
STMs use a conductive tip that scans the specimen's surface at an atomic level. By measuring the tunneling current between the tip and the specimen, STMs provide atomic-scale images.
Physics: Based on quantum tunneling, where electrons tunnel through a barrier between the tip and the specimen. The tunneling current varies with the distance between the tip and the surface, allowing for atomic-scale resolution imaging.
Applications: Crucial in surface science, nanotechnology, and semiconductor research, STMs enable the study of surface atomic structure, electronic properties, and molecular interactions. They are instrumental in developing and characterizing nanoscale materials and devices.
Advanced Microscopes
Atomic Force Microscope (AFM)
AFMs use a cantilever with a sharp tip to scan the specimen's surface. Forces between the tip and the surface cause deflections in the cantilever, which are measured to create topographic maps.
Physics: AFMs rely on Van der Waals forces, electrostatic forces, and mechanical contact to measure surface properties at the nanoscale. The cantilever's deflections, caused by interactions with the specimen, are detected using laser beams and photodetectors.
Applications: Essential in nanotechnology, material science, and biology, AFMs provide high-resolution topographic maps of surfaces. They are used to study surface roughness, mechanical properties, and molecular interactions at the nanoscale.
Inverted Microscope
Inverted microscopes have the light source and condenser above the stage, with the objective lenses below. This design is ideal for observing live cells in culture dishes or larger specimens.
Physics: Inverted microscopes utilize similar principles to compound microscopes but are designed with the light path inverted for accessibility. This allows for easy manipulation and observation of specimens in larger containers, such as culture dishes.
Applications: Widely used in cell culture, developmental biology, and pharmacology, inverted microscopes are ideal for observing live cells, embryos, and larger specimens. They facilitate the study of cellular behavior, drug effects, and developmental processes.
Metallurgical Microscope
Metallurgical microscopes are designed to examine metal surfaces and structures using reflected light. They are essential for studying opaque specimens like metals, ceramics, and composites.
Physics: Utilizes reflected light microscopy, where light reflects off the specimen surface and is captured by the objective lens. This technique enhances the contrast of opaque materials, allowing for detailed examination of their microstructure.
Applications: Crucial in metallurgy, material science, and engineering, metallurgical microscopes are used to analyze the grain structure, phase distribution, and defects in metals and alloys. They are essential for quality control, failure analysis, and research in materials development.
Digital Microscope
Digital microscopes use a digital camera to capture images of the specimen, which are then displayed on a computer screen. They often come with software for image analysis and measurement.
Physics: Combines optical microscopy with digital imaging technology, allowing for real-time visualization and digital manipulation. The digital camera captures images through the optical system and converts them into digital signals for display and analysis.
Applications: Widely used in education, industrial inspection, and documentation, digital microscopes facilitate easy sharing and analysis of images. They are ideal for teaching, remote diagnostics, and detailed inspection of small components.
Stereo Microscope
Stereo microscopes, or dissecting microscopes, provide a three-dimensional view of the specimen using two separate optical paths. They are used for low-magnification observations and manipulation of samples.
Physics: Uses dual optical paths to create a stereoscopic effect, giving depth perception and a 3D view. This allows for a more natural observation of specimens, making it easier to manipulate and examine them in detail.
Applications: Essential in dissection, microsurgery, and circuit board inspection, stereo microscopes are used for tasks requiring low magnification and precise manipulation. They are widely used in biological research, electronics repair, and industrial inspection.
Specialized Imaging Techniques
Darkfield Microscopy
Darkfield microscopy enhances contrast in unstained specimens by illuminating the sample with light that does not directly enter the objective lens. This technique makes the specimen appear bright against a dark background.
Physics: Utilizes oblique light scattering, where light is scattered by the specimen and only scattered light reaches the objective lens. This enhances the visibility of structures that are otherwise difficult to see in brightfield microscopy.
Applications: Widely used in microbial observation and live cell imaging, darkfield microscopy is ideal for studying organisms and structures that are difficult to stain or that need to be observed in their natural state. It provides a clear view of motile cells and fine structures.
Differential Interference Contrast (DIC) Microscopy
DIC microscopy uses polarized light and prisms to produce high-contrast images of transparent specimens, providing a pseudo-3D effect.
Physics: Based on optical path length differences created by birefringence, DIC enhances contrast through interference. Light passing through different parts of the specimen experiences phase shifts, which are converted into intensity differences.
Applications: Essential in live cell imaging, cytology, and developmental biology, DIC microscopy allows for the observation of transparent and unstained specimens with high contrast and clarity. It is widely used to study cellular dynamics and morphology.
Super-Resolution Microscopy
Super-resolution microscopy techniques, such as STORM and PALM, break the diffraction limit of light to achieve higher resolution than conventional light microscopy.
Physics: Utilizes stochastic activation and precise localization of individual fluorescent molecules to surpass the diffraction limit. By repeatedly activating and imaging subsets of fluorophores, super-resolution techniques achieve nanoscale resolution.
Applications: Critical in molecular biology, neurobiology, and structural biology, super-resolution microscopy allows for the detailed study of molecular complexes, protein interactions, and cellular architecture at a resolution previously unattainable with standard light microscopy.
Microscope Recommendations
For beginners, a compound binocular microscope with a mechanical stage and at least 10x, 40x, 60x, and 100x plan achromatic infinity corrected objective lenses is recommended. Halogen lighting is ideal for higher magnifications, while LED lighting is suitable for general use.
Microscope Accessories
Essential microscope accessories include:
Cleaning Accessories: Kim Wipes, specialized lens cleaner (provided by microscope manufacturer or microscopy retailer), and air blowers or compressed air cans.
Immersion Oil: Necessary for 100x objective lenses.
Prepared Slides: Offer ready-to-view specimens, while blank slides require specimen preparation.
Camera Attachments: Integrated digital cameras or DSLR adapters for photo microscopy.
Using a Microscope
Using a microscope effectively involves several detailed steps and best practices to ensure clear, accurate imaging and to prevent damage to the instrument. Here’s a comprehensive guide:
Safety Precautions
Personal Protective Equipment (PPE): Always wear gloves, safety glasses, and a lab coat when handling biological specimens or potentially hazardous materials.
Handling Slides: Handle slides carefully to avoid cuts and contamination. Dispose of wet slides in the appropriate waste disposal containers.
Workspace Cleanliness: Ensure the workspace is clean and free of contaminants. Use disinfectant wipes to clean the workspace before and after any lab or microscopy sessions
Preparing the Workspace
NOTE: One important fact is that for all specialised instruments including microscopes, dust is the enemy and minimising the exposure of your equipment to dust will increase the time between professional servicing and cleaning, which can cost anywhere for a couple of hundred dollars up or possibly over $600. Avoiding contact with dust or touching the glass of a lens, avoiding breathing onto the glass (causing moisture) and using work practices that minimise chances of scratching the objective lens on coverslips/slides cannot be overstated.
Clean Environment: Ensure the workspace is clean and free of dust. Vacuum the floor with a HEPA filter-fitted vacuum cleaner. Wipe down surfaces with moist cloths to remove dust and disinfectant wipes to eliminate potential biological contaminants. Do not use outdoors, with open windows, or near the ocean.
Air Quality: Use a high-quality HEPA air purifier to clean the air. If on a budget, consider using a laminar flow hood or a small air purifier within an indoor tent to create a controlled environment. For necessary performance, purification systems for $1000 and over are often required.
Setting Up the Microscope
Initial Setup: Place the microscope on a stable, vibration-free surface. Ensure the area is well-lit but not directly exposed to sunlight, which can affect the microscope’s internal optics.
Turn On: Power on the microscope and set the objective turret to the lowest magnification, typically 4x or 10x.
Focusing the Microscope
Removing the Eyepiece: Remove an eyepiece and close the diaphragm until a small dot of light is visible through the eyepiece tube. Adjust the condenser screws to center the point of light, then replace the eyepiece and open the diaphragm.
Lower the Stage: Lower the mechanical stage as far down as it will go using the coarse focus knob to give ample space for slide placement.
Place the Slide: Carefully place the slide on the stage, securing it with the stage clips. Ensure the specimen is positioned directly over the light source.
Adjusting the Focus
Initial Focus: Slowly raise the stage with the coarse focus knob until the specimen is close to the objective lens. Switch to the fine focus knob for precise adjustments.
Avoiding Contact: Ensure there is a small gap between the objective lens and the slide to prevent scratching or breaking. Always move the stage away from the objective lens to find the correct focal point.
Fine-Tuning: Adjust the iris diaphragm and condenser to optimize light and contrast. Use the light intensity knob to fine-tune the illumination.
Changing Magnification
Lower the Stage: Always lower the stage before rotating the objective turret to avoid damaging the objective lenses or the slide.
Immersion Oil: When using a 100x objective lens, place a drop of immersion oil on the slide before raising the stage. This oil bridges the air gap between the lens and the slide, necessary for achieving a clear image at high magnification. NOTE: double check the objective when changing magnification for listed specifications to make sure it’s not a 40x lens which may be mistaken for a 100x lens made for immersion oil applications. Some 100x and lower magnification lenses are not sealed and immersion oil will enter the lens housing damaging it. “Oil” or “100x/oil” or something similar should be stamped on each lens for confirmation.
Repeat Focus Adjustments: For each new magnification, repeat the focusing steps, ensuring proper light and contrast adjustments.
Cleaning and Maintenance
After Use: Clean the body of the microscope with a disinfectant wipe. Use appropriate cleaning solutions and cloths for the lenses, especially after using immersion oil.
Storing the Microscope: Lower the stage, cover the microscope with a dust cover, and store it in a dust-free environment. If possible, store the microscope in a dedicated storage bag or case with elastic straps to secure it in place during transport.
By following these detailed steps and tips, you can ensure the longevity of your microscope and obtain high-quality, precise images for your research and analysis.
Tips for Using Microscopes to Study Different Tissue Types and Structures
When using microscopes to study various tissue types and structures, selecting the appropriate magnification and technique is crucial for obtaining clear, detailed images. Here are some specific tips to enhance your microscopy experience:
Low Magnification for Whole Structures:
Use low magnification (4x to 10x) to view entire structures or large specimens. This is particularly useful for initial scanning and locating areas of interest within a sample.
Examples: Whole organ sections, fibres, large tissue biopsies, and entire microorganism specimens.
Medium Magnification for Tissue Organization:
Medium magnification (10x to 40x) is ideal for examining the overall organization of tissues and identifying larger structural features.
Examples: Layer organization in skin, muscle fiber arrangement, and vascular patterns in tissues, structure of the mammary glands, lungs (Alveoli), kidneys (nephrons), liver (hepatic artery and lobules).
High Magnification for Cellular Details:
High magnification (40x to 100x) is necessary for observing detailed cellular structures, including cell morphology, organelles, and intracellular components.
Examples: Cell nuclei, red blood cells, some parasites, virus particles, individual spores, mitochondria, individual hyphae of mycelium and bacterial cell walls.
Phase Contrast for Transparent Cells:
Use phase contrast microscopy to enhance the contrast of transparent and unstained specimens. This technique is especially beneficial for observing live cells and thin tissue slices.
Examples: Live cell cultures, protozoa, and thin sections of transparent tissues like the cornea.
Fluorescence for Specific Molecules:
Employ fluorescence microscopy to study specific molecules within cells using fluorescent dyes or proteins. This method provides high specificity and contrast.
Examples: Immunofluorescence labelling of proteins, GFP-tagged molecules, and DNA staining with DAPI.
Confocal for 3D Imaging:
Utilize confocal microscopy for high-resolution, three-dimensional imaging of thick specimens. This technique allows for optical sectioning and detailed spatial analysis.
Examples: Thick tissue sections, biofilms, and complex cellular structures in 3D cultures.
Differential Interference Contrast for Living Cells:
Apply differential interference contrast (DIC) microscopy for live cell imaging. DIC provides high-contrast images with a pseudo-3D effect, ideal for observing dynamic processes.
Examples: Live cell movements, intracellular trafficking, and developmental processes in embryos.
Polarizing for Crystalline Structures:
Use polarizing microscopy to examine birefringent materials and crystalline structures. This technique enhances the visibility of structures that exhibit different refractive indices.
Examples: Crystals in biological tissues, muscle fibre orientation, and mineral deposits.
Electron Microscopy for Ultra-Structural Details:
Employ transmission electron microscopy (TEM) for ultra-structural details at the molecular and atomic levels. TEM provides the highest resolution for examining fine structures.
Examples: Viral particles, cell organelles, and nanomaterials.
Use scanning electron microscopy (SEM) for detailed surface imaging of specimens. SEM provides high-resolution images of surface morphology.
Examples: Surface topology of tissues, microbial structures, and material surfaces.
By choosing the appropriate magnification and microscopy technique, you can optimize the observation and analysis of different tissue types and structures, enhancing your research and diagnostic capabilities.
For further information related to the topics covered
Basic Types of Microscopes:
Websites of microscope manufacturers such as Nikon, Olympus, Leica, AMscope and Zeiss often have high-quality microscopes for sale along with compatible accessories, cleaning products and supplies.
Educational websites like Khan Academy or YouTube channels specializing in microscopy can provide visual guides and demonstrations. Microscopy Techniques:
Scientific journals and articles on platforms like PubMed or Google Scholar often contain detailed images and figures of microscopy techniques in action.
Websites like Microbehunter and MicroscopyU provide explanations of various microscopy methods.
Microscopy is an essential tool in scientific research, offering unparalleled insights into the microscopic world. Proper maintenance and understanding of microscopes and their components are crucial for obtaining accurate and high-quality images. By following this overview of Microscopy as well as the best practices and safety precautions, professionals can harness the full potential of microscopy in their work.
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