Microscopy and Specimen Preparation Chapter 3

Microscopy and Specimen Preparation

• Chapter 3 covers microscopy and specimen preparation techniques
• Light properties, types of microscopes, specimen preparation, and staining techniques are discussed
• Microscope development, from early examples by Galileo and Hooke to modern microscopes such as the British microscope (circa 1865), Leeuwenhoek's microscope (circa late 1600s), and Winkel-ZEISS Dissecting Microscope (circa 1927) are explored
• Leeuwenhoek's early microscopes could magnify over 200 times
• Hand-colored illustrations showcase the "animalcules" (microorganisms) Leeuwenhoek observed
• Modern microscopes discussed include compound light microscopes, scanning electron microscopes (SEM), and transmission electron microscopes (TEM)
• Microscopes use different resolution ranges; specimen size dictates the appropriate microscope

Units of Measurement

• Microscopes magnify small objects with different resolutions
• Size ranges for unaided human eyes and various microscopes (atomic force microscope, transmission electron microscope, light microscope, scanning electron microscope) are detailed
• Units of measurement include picometers, nanometers, micrometers, millimeters, centimeters, meters

Modern Microscopes

• Modern microscopes include compound light microscopes, scanning electron microscopes (SEM), and transmission electron microscopes (TEM)
• Diagrams of their components (light microscope, condenser lens, objective lens, projection lens, scanning electron microscope, electron detector, stage, etc.) are shown
• Light properties including reflection, transmission, absorption, and refraction are discussed
• Refractive index (RI) is a measure of the speed at which light passes through a material; it is important for immersion oil

Microscope Resolution

• Microscope resolution is the ability of a lens to separate or distinguish small objects that are close together
• Wavelength of light used is a major factor in resolution
• Shorter wavelengths lead to greater resolution. The shorter the wavelength, the higher the resolution

Effect of Wavelength on Resolution

• Resolution is the ability to see two items as separate, discrete units (not fuzzy or overlapped)
• Shorter wavelengths (high frequency) allow better resolution of details
• Diagrams illustrate the effect of wavelength on resolution, comparing images with high and low resolution/frequency

Analogy for the Effect of Wavelength on Resolution

• A clear explanation of the relationship between wavelength and resolution, using examples of different-sized objects (ball sizes) to demonstrate the concept that microscopes require short wavelengths for greater clarity.

Light Interactions with an Object

• Explains how light interacts with specimens (reflection, transmission, absorption, and refraction)

Light Properties (Refraction)

• Light bends (refracts) when passing between media with different densities
• Refractive index (RI) measures the speed of light through different materials. Values are given for glass, air, oil, and water.
• Immersion oil has a similar RI to glass (important for higher resolution)

Immersion Oil and Image Quality

• Immersion oil is used to minimize image distortion for microscopic viewing, by matching the refractive index of a specimen and the objective lens

Types of Microscopes

• Different types of light microscopes are discussed, including bright-field, dark-field, phase-contrast, differential interference contrast (DIC), and fluorescence microscopes, with examples
• Different types of electron microscopes are discussed, including scanning electron microscopes (SEM) and transmission electron microscopes (TEM), with examples
• Scanning tunneling microscopes (STM) are also mentioned, with images and descriptions

Light Microscopy

• Compound microscopes use two lenses to form the image
• Total magnification is determined by multiplying the magnifications of the ocular lens and objective lens
• Working distance is the distance between the lens and specimen (ideal for sharp focus)
• Microscopic field refers to the area visible through the microscope

Microscope Lenses

• Lenses focus light rays at focal points (F)
• Focal length (f) is the distance between the lens center and the focal point
• Lenses with shorter focal lengths allow for greater magnification

Bright-Field Microscope

• Creates a dark image against a brighter background
• Can be used for stained or unstained specimens (examples included)

Dark-Field Microscope

• Creates a bright image against a dark background
• Useful for observing live, unstained specimens, especially useful for prokaryotes (bacteria)

Phase-Contrast Microscope

• Enhances contrast between intracellular structures with minor refractive-index differences; shows interior structures of living cells
• Useful for observing living cells (e.g., bacterial endospores and inclusions)

Differential Interference Contrast Microscope (DIC)

• Creates an image by detecting differences in the refractive index and thickness of different specimen parts
• Displays live, unstained specimens in 3-dimensional (3D) appearance

Fluorescence Microscope

• Specimens are stained with fluorochromes
• Specimens are exposed to UV, violet or blue light
• A bright image is created from fluorescent light emitted by the specimen

Electron Microscopy

• Electron beams pass through the specimen
• Produces high magnification and high-resolution images
• Electron microscopy gives higher resolution than light microscopy due to the shorter wavelength of the electron beam.

Types of Electron Microscopes

• Scanning electron microscopes (SEM) use electrons reflected from the specimen surface to create a 3-dimensional image
• Transmission electron microscopes (TEM) use transmitted electrons to create an image of a specimen's internal structures, requires thin specimens
• Scanning Tunneling Microscopes (STM): create surface images (e.g., DNA, proteins) at the atomic level.

Preparation of Specimen (for Light Microscopy)

• Techniques used for preparing specimens for microscopy for light microscopy include wet mounts, and bacterial smears to increase the visibility of the specimen, enhance specific morphological features and preserve specimens.

Preparation of Specimen (for Electron Microscopy)

• Techniques used to prepare for electron microscopy include freeze-etching and shadow casting, to obtain properly prepared specimen for high-resolution images

Staining Techniques and Application

• Dyes are used to stain specimens to increase contrast and make internal and external structures more visible
• Ionizable dyes (basic dye, acidic dye) have charged groups
• Common staining types include simple stain, differential stain, Gram stain, acid-fast stain, and stains for specific structures like flagella, capsules, and endospores

Gram Staining

• Gram staining is a differential staining technique commonly used to distinguish between Gram-positive and Gram-negative bacteria based on cell wall structure
• Multi-step procedure with reagents and their application to organisms

Acid-Fast Staining

• Acid-fast staining is a differential staining procedure used to identify bacteria of the genus Mycobacterium (e.g., M. tuberculosis, M. leprae) which have waxy cell walls
• Multi-step procedure with reagents and their application to organisms.

Staining Specific Structures

• Bacterial structures such as flagella, capsules, and endospores can be stained using specialized techniques to improve visibility

Steps in Endospore Staining

• Procedure to follow when staining endospores using a multi-step procedure with reagents to be applied to the sample

End of Chapter 3

Explore the fascinating world of microscopy and specimen preparation techniques in Chapter 3. Dive into the properties of light, the evolution of microscopes from early examples to modern technology, and learn about various specimen preparation and staining methods. Illustrations of historical observations by Leeuwenhoek and modern microscopy techniques are also included.