Page 1: Introduction

• PowerPoint modified for BIO 275 (CVCC)

• Licensed by Rice University under Creative Commons.

• Access resources at OpenStax.

Page 2: Size Comparison

• Chart comparing size of molecules, prokaryotic and eukaryotic cells.

• Source: Arizona Biology site.

Page 3: Compound Light Microscope

• A compound light microscope uses multiple lenses for magnification.

• Microorganisms measured in micrometers (µm) or nanometers (nm).

• Antonie van Leeuwenhoek created the first effective light microscope.

• Uses glass lenses and visible light to magnify samples.

Page 4: Electromagnetic Spectrum

• Visible light is a part of the electromagnetic spectrum.

• Illustrated by NASA.

Page 5: Visible Light

• Visible light is between ultraviolet and infrared light in the spectrum.

• Relatively small range detectable by human eyes.

Page 6: ROYGBIV

• Visible light encompasses the colors: Red, Orange, Yellow, Green, Blue, Indigo, Violet (ROYGBIV).

• Violet has the shortest wavelengths, red the longest.

• Light travels in packets called photons.

Page 7: Wave Properties

• Wavelength: distance between wave peaks.

• Amplitude: height of a wave.

• Frequency: number of wavelengths in a timeframe.

Page 8: Human Eye Function

• Human eye uses a lens to focus light on the retina.

• Photoreceptors detect colors based on wavelength.

• Artificial lenses enhance visibility.

Page 9: Light Interactions

• Light can be reflected, absorbed, or transmitted.

• Reflection: wave bounce off a material.

• Absorption: energy captured; re-emission possible.

Page 10: Transmission

• Transparent materials allow light to pass; opaque materials do not.

• Example: Petri dish versus iron meteorite slice.

Page 11: Light Refraction

• White light disperses into spectrum when passing through a prism.

• Refraction occurs when light changes direction entering a new medium.

Page 12: Refraction Explained

• Refraction occurs at the interface of different media (e.g., air to water).

• Refractive index measures light-bending ability.

Page 13: Refraction and Reflection

• Light rays can either be refracted or reflected at medium boundaries.

Page 14: Lenses

• Compound light microscopes utilize curved lenses to focus light.

• Light waves converge at a focal point.

Page 15: Lens Refraction

• Convex lenses converge light for magnification.

• Concave lenses diverge light.

Page 16: Fluorescent Dyes

• Certain materials can convert non-visible radiation into visible light.

• Fluorescent dyes absorb UV/blue light and emit visible color.

Page 17: Phosphorescence

• Electrons emit energy as photons when returning to ground state.

• Phosphorescence describes this emission from excited states.

Page 18: Micrographs and Properties of Microscopes

• Micrographs showcase specimens under microscopes; key properties: magnification, resolution, contrast.

Page 19: Total Magnification

• Calculate total magnification by multiplying the objective lens and ocular lens.

• Example: 10x (ocular) x 100x (objective) = 1000x total magnification.

Page 20: Resolution

• Resolution measures clarity and distinguishes two points.

• Example of high versus low-resolution images.

Page 21: Resolving Power

• Light microscopes resolve structures ~0.2 µm; SEM ~10 nm; TEM ~0.2 nm.

• Poor resolution appears fuzzy.

Page 22: Wavelength Affects Resolution

• Shorter wavelengths lead to better resolution for smaller samples.

• Electron microscopes excel in resolution due to shorter wavelengths.

Page 23: Numerical Aperture

• Numerical aperture (NA) indicates lens's light-gathering ability; higher NA = better resolution.

Page 24: Contrast

• Transparent microbes are often hard to see; light and electron beams create contrast during imaging.

Page 25: Stains and Dyes

• Staining increases contrast; different dyes target various molecules.

Page 26: Microscope History

• Simple microscopes by eyeglass makers in late 1500s; Galileo’s contributions.

• Types: simple (one lens) and compound (multiple lenses).

Page 27: Zaccharias Janssen

• Potential inventor of the telescope and microscopes; historical evidence inconclusive.

Page 28: Leeuwenhoek and Galileo

• Anton van Leeuwenhoek first to observe microbes; simple microscopes had magnifications of 300x.

Page 29: Robert Hooke

• Hooke viewed cork cells and coined the term "cells" in 1665.

Page 30: Brightfield Microscopes

• Classroom microscopes produce dark images on bright backgrounds.

• Function involves illumination and light control via condensers and diaphragms.

Page 31: Brightfield Microscope Components

-Diagram detailing microscope parts including eyepiece, objective lenses, and stage.

Page 32: Chromophores

• Staining with methylene blue identifies cell structures; chromophores interact with light to show detail.

Page 33: Objective Lenses

• Different objectives based on magnification, numerical aperture, and dimensions.

Page 34: Oil Immersion Lens

• High magnification requires oil immersion to enhance resolution and minimize light bending.

Page 35: Objective Lens Problems

• Immersion oil matches refractive index of glass, improving optical quality.

Page 36: Oil Immersion and Slides

• Challenges faced when viewing specimens at high magnifications, solution: use of immersion oil.

Page 37: Limitations of Brightfield Microscopy

• Alternative microscopy types are used when traditional methods fail due to specimen visibility issues.

Page 38: Darkfield Microscopy

• Darkfield microscope uses an opaque disk to create bright images on dark backgrounds.

Page 39: Brightfield vs Darkfield Microscopy

• Darkfield increases contrast for viewing certain bacteria.

Page 40: Viewing with Darkfield

• Example of Treponema pallidum viewed brightly against a dark background.

Page 41: Phase-Contrast Microscopy

• Uses direct and diffracted light to produce contrast from transparent specimens.

Page 42: How Phase-Contrast Works

• In-phase and out-of-phase light waves combine for enhanced contrast in images.

Page 43: Brightfield vs Phase-Contrast

• Comparison of visibility and detail of unstained cells in different microscopy techniques.

Page 44: Differential Interference Contrast Microscopy

• Utilizes two light beams and prisms for high-resolution imaging and 3D appearance.

Page 45: DIC Microscopy Image

• Example of DIC microscopy showing a fungal structure.

Page 46: Fluorescence Microscopy

• Uses fluorochromes to absorb UV light and emit visible light for specimen visualization.

Page 47: Fluorescence Micrograph

• Bovine pulmonary artery endothelial cells under fluorescence microscopy.

Page 48: Immunofluorescence

• Technique identifies microbes by observing antibody-antigen complex formation.

Page 49: Fluorescent Antibody Technique

• Dye-tagged antibodies can detect pathogens like syphilis and Lyme disease.

Page 50: Direct vs Indirect Immunofluorescence

• Differentiates methods of staining and visualization using antibodies for microbial detection.

Page 51: Confocal Microscopy

• Scans multiple 2D images to create a 3D reconstruction; useful for thick specimens.

Page 52: Confocal Microscopy Example

• Micrograph showing cyanobacterium biofilm.

Page 53: Biofilm Description

• Biofilms formed by microbes aid survival and community support.

Page 54: Biofilm on Stainless Steel

• Example of biofilm growth on a surface.

Page 55: Electron Microscopy Overview

• Electron microscopes offer higher resolution using beams of electrons versus light.

Page 56: Transmission Electron Microscope

• TEM requires thin specimens for optimal visibility.

Page 57: TEM Micrographs

• Images typically display results of electron microscopy with possible artifacts.

Page 58: Normal TEM Photograph

• Visual of a standard transmission electron microscope.

Page 59: Electron Focusing

• Similar operation between electron (EM) and light microscopes but using electric fields instead.

Page 60: Preparing a Specimen

• Usage of an ultramicrotome for TEM preparation detailed.

Page 61: Scanning Electron Microscope Usage

• SEM produces 3D images by scanning specimens with a beam of electrons.

Page 62: TEM vs SEM Structures

• Comparison of electron microscope components for TEM and SEM.

Page 63: Micrographs Comparison

• Different imaging capabilities of TEM and SEM presented.

Page 64: Scanning Probe Microscopes

• STM and AFM allow viewing at atomic levels through direct specimen interaction.

Page 65: References

• Citations for microscopy images and techniques presented.

Page 66: Types of Electron Microscopes

• Overview of electron microscope functionality.

Page 67: Scanning Probe Microscopes Details

• Key features, uses, and magnifications of scanning tunneling and atomic force microscopy.

Page 68: Preparing a Wet Mount

• Wet mount techniques for viewing specimens under microscope detailed.

Page 69: Fixation Techniques

• Heat fixation and chemical fixation methods to prepare specimens.

Page 70: Smear Preparation

• Procedures for preparing smears from liquid or solid samples for microscopy.

Page 71: Slide Preparation Methods

• Overview of heat and chemical fixing in specimen preparation.

Page 72: Importance of Dyes

• Dyes enhance colors and contrast in microscopic samples.

Page 73: Basic Dyes

• Characteristics and common uses of basic dyes in microscopy.

Page 74: Methylene Blue Use

• Description of methylene blue dye properties and applications.

Page 75: Acidic Dyes

• Characteristics of acidic dyes and their role in providing negative stains.

Page 76: Other Stains

• Non-ionic stains and their solubility properties enhance specimen interaction.

Page 77: Positive and Negative Stains

• Differentiates between positive stains that color specimens and negative stains that color backgrounds.

Page 78: Staining Result Examples

• Micrographs show effects of different staining techniques on various bacteria.

Page 79: Simple vs Differential Staining

• Simple staining uses one dye; differential staining employs multiple dyes for complex views.

Page 80: Gram Staining

• Gram staining technique and its significance in differentiating bacteria types.

Page 81: Gram Positive vs Negative Bacteria

• Describes differences in cell wall composition affecting resistance to antibiotics.

Page 82: Gram Staining Procedure

• Step-by-step process highlighting key staining agents and their roles.

Page 83: Gram Staining Illustrated

• Visual representation of differential staining methods for classification.

Page 84: Micrograph Comparison with Gram Staining

• Example of stained gram-positive and negative bacteria for identification.

Page 85: Acid-Fast Staining

• Application of acid-fast staining in identifying bacteria with specific wall characteristics.

Page 86: Ziehl-Neelsen Staining

• Example of acid-fast stain visualizing Mycobacterium tuberculosis.

Page 87: Acid-Fast Stain Procedure

• Steps for staining acid-fast organisms using specific techniques and dyes.

Page 88: Endospore Staining

• Overview of endospore staining methods applicable to resistant bacteria.

Page 89: Capsule Identification Techniques

• Negative staining techniques used for visualizing bacterial capsules.

Page 90: Flagella Staining

• Staining procedures to visualize flagella on bacteria for identification purposes.

Page 91: Diverse Microscopy Techniques

• Overview of various microscopy approaches for viewing specific microorganisms.