Microscopy and Cell Structure and Function Laboratory Manual

Course Agenda, Assessment Schedule, and Administrative Deadlines

• Laboratory Focus and Primary Learning Objectives:

  • Mastery of basic microscopy techniques.

  • Identification of components and functional mechanisms of compound light microscopes and dissecting (stereo) microscopes.

  • Understanding fundamental characteristics, structural components, and cellular functions.

  • Classification and differentiation of cell types: Prokaryotes versus Eukaryotes, and Animal Cells versus Plant Cells.

• Administrative Deadlines and Assessment Schedule:

  • Pre-lab #2:

    • Submission: Due in Canvas by Sunday ($8/23$).

    • Availability Window: Accessible in Canvas from $12:00\text{ AM}$ on $8/17$ until $11:59\text{ PM}$ on $8/23$.

  • Quiz #1:

    • Submission: Due in Canvas by $8/27$.

    • Availability Window: Accessible in Canvas from $12:00\text{ AM}$ on $8/24$ until $11:59\text{ PM}$ on $8/27$.

    • Evaluated Course Content: Lab Safety and Syllabus/Assessment, Scientific Methods and Metric System, Microscopy, and The Cell (Structure and Function).

• Laboratory Agenda Structure:

  • Microscopy conceptual lecture.

  • Microscopy practical lab activities.

  • The Cell (Structure & Function) lecture.

  • Cell biology lab activities.

  • Laboratory bench cleanup protocol.

  • Review session and worksheet verification.

Overview and Taxonomy of Microscopes

• Definition of Microscope: An instrument designed to enlarge and visualize objects that are too small to be perceived by the unaided human eye.

• Major Categories of Microscopes:

  • Compound Light Microscope: Uses visible light and glass lenses to magnify thin, transparent specimens mounted on glass slides.

  • Dissecting Microscope (Stereomicroscope): Uses low-magnification light reflected off the surface of 3D objects.

  • Confocal Laser Scanning Microscope: Employs laser light to scan biological samples and create high-resolution optical sections.

  • Scanning Electron Microscope (SEM): Directs a beam of electrons across the surface of a specimen to produce high-resolution, three-dimensional surface imagery.

  • Transmission Electron Microscope (TEM): Passes electrons through ultra-thin specimen slices to reveal detailed internal ultrastructure.

• Historical Commemoration: Recognized in historical philately, such as the Stamps of Germany ($1980$).

Spatial Scale and Resolution Ranges for Biological Observation

• Comparative Dimensional Spectrum (Source: Molecular Biology of the Cell, 4th Edition, Figures 9-1 and 9-2):

  • Naked Eye Capabilities:

    • Dimensional Range: Approximately $20\text{ mm}$, $2\text{ mm}$, $0.2\text{ mm}$ ($1\text{ cm}$, $1\text{ mm}$, down to $100\text{ }\frac{1}{10}\text{ mm}$ / $100\text{ }\times 10^{-6}\text{ m}$).

    • Observable Structures: Large eukaryotic structures, macroscopic plant tissues, macroscopic animal tissues.

  • Light Microscope Capabilities:

    • Dimensional Range: $100\text{ }\frac{1}{10}\text{ mm}$ down to $0.2\text{ }\frac{1}{1000}\text{ mm}$ ($100\text{ }\times 10^{-6}\text{ m}$ down to $200\text{ nm}$ / $0.2\text{ }\times 10^{-6}\text{ m}$).

    • Observable Structures:

      • Plant cell: $\text{Size} \thicksim 100\text{ }\times 10^{-6}\text{ m}$

      • Animal cell: $\text{Size} \thicksim 10\text{ }\times 10^{-6}\text{ m}$ to $20\text{ }\times 10^{-6}\text{ m}$

      • Bacterium: $\text{Size} \thicksim 1\text{ }\times 10^{-6}\text{ m}$ to $2\text{ }\times 10^{-6}\text{ m}$

  • Electron Microscope Capabilities:

    • Dimensional Range: $0.2\text{ }\times 10^{-6}\text{ m}$ ($200\text{ nm}$) down to $0.2\text{ nm}$ ($0.1\text{ nm}$ / $1\text{ Å}$).

    • Observable Structures:

      • Bacterium: $\text{Size} \thicksim 1\text{ }\times 10^{-6}\text{ m}$

      • Virus: $\text{Size} \thicksim 100\text{ nm}$

      • Ribosome: $\text{Size} \thicksim 20\text{ nm}$

      • Globular protein: $\text{Size} \thicksim 10\text{ nm}$

      • Small molecule: $\text{Size} \thicksim 1\text{ nm}$

      • Atom: $\text{Size} \thicksim 0.2\text{ nm}$ ($0.1\text{ nm}$ / $1\text{ Å}$)

Essential Terminology of Microscopy

• Magnification: The degree or factor by which the apparent size of an object is enlarged. Expressed in numerical multiples such as $2\times$, $4\times$, and $10\times$, indicating that the object's visual image is scaled to twice, four times, or ten times its physical dimensions.

• Resolution: The limit of resolution or spatial resolving power that permits two separate, adjacent points to be distinguished as distinct entities. If two points are positioned closer together than the resolution threshold of the optical system, their images blur into a single feature. Resolution directly determines image clarity.

• Contrast: The difference in light intensity or visual threshold between the specimen and its immediate background. High contrast occurs when the surrounding field is illuminated bright while the object is dark. Adjusting contrast is critical in light microscopy to prevent bright background light from washing out translucent or lightly pigmented biological samples.

• Parfocal: A quality of a multi-objective lens system where the specimen remains approximately in focus when switching from a lower-power objective (e.g., $10\times$) to a higher-power objective (e.g., $40\times$ or $100\times$).

• Parcentral: A quality of an optical system where the center of the field of view remains centered when switching objectives from lower power (e.g., $10\times$) to higher power (e.g., $40\times$ or $100\times$).

Microscope Handling, Operational Protocols, and Troubleshooting

• Standard Startup and Focus Procedures:

  • Always initiate viewing with the lowest power objective lens, designated as the scanning objective ($4\times$).

  • When placing a glass slide onto or removing it from the mechanical stage, ensure the lowest power objective is selected and the stage is lowered to its absolute bottom position.

• Lens Maintenance Guidelines:

  • Clean optical glass components using specified lens paper exclusively.

  • Never use Kimwipes, paper towels, or tissues on optical lenses, as coarse fibers cause permanent scratching.

• Focus Control Hazards:

  • Never adjust the coarse focus adjustment knob while operating under high-power objective lenses ($40\times$ or $100\times$), as doing so risks driving the lens into the slide, smashing the specimen, and destroying the objective optics.

• Safety and Physical Handling:

  • Glass slides are highly fragile; handle with extreme caution.

  • Always transport a microscope using two hands: one securely gripping the arm, and the second fully supporting the underside of the base.

  • Troubleshooting Reference Media: Video guide on microscope basics and lab troubleshooting accessible at https://www.youtube.com/watch?v=FTtYmchpivw&t=95s.

Compound Light Microscope Anatomy and Optical Systems

• Mechanical and Optical Components:

  • Eyepieces (Oculars): The uppermost lenses through which the operator views the specimen; typically provide a base magnification of $10\times$

  • Revolving Nosepiece: The rotating housing that holds multiple objective lenses.

  • Objective Lenses: Magnifying lenses set into the nosepiece (e.g., $4\times$, $10\times$, $40\times$, $100\times$).

  • Mechanical Stage: The flat platform where the slide is clamped using stage clips.

  • Stage Adjustment Knobs: Coaxial controls that move the stage along the X-axis and Y-axis.

  • Arm: The vertical support connecting the head assembly to the base.

  • Base: The wide bottom support resting on the lab bench.

  • Coarse Focus Adjustment Knob: Large knob used to rapidly adjust the stage height for coarse focusing under low power.

  • Fine Focus Adjustment Knob: Small knob used to bring the image into sharp focus under medium and high power.

  • Condenser: An optical lens unit located beneath the stage that focuses light from the lamp onto the specimen.

  • Iris Diaphragm: An adjustable aperture built into the condenser to regulate the amount and angle of light passing through the specimen to control contrast.

  • Light Source: The built-in illuminator located in the base.

  • ON/OFF Switch: Controls power to the light source.

  • Voltage Control (Dimmer): Rotary dial regulating electrical current supplied to the illuminator to raise or lower light intensity.

• Oil Immersion Mechanics ($100\times$ Objective):

  • Requires immersion oil placed directly between the glass coverslip and the lens element.

  • Immersion oil matches the refractive index of glass, preventing light refraction into air, thereby maximizing light collection efficiency and enhancing image resolution.

  • Strict Warning: Never engage or attempt to view through the $100\times$ objective lens without applying immersion oil.

Mathematical Formulas for Microscope Magnification and Field of View

• Total Magnification Equation:     $\text{Mag}_{\text{total}} = \text{Mag}_{\text{obj}} \times \text{Mag}_{\text{ocu}}$

  • $\text{Mag}_{\text{total}}$: Total magnification power achieved.

  • $\text{Mag}_{\text{obj}}$: Magnification power of the active objective lens.

  • $\text{Mag}_{\text{ocu}}$: Magnification power of the ocular eyepiece lens (Standard value: $\text{Mag}_{\text{ocu}} = 10\times$).

• Field of View (FOV) Proportionality Equation:     $\text{LPD} \times \text{LPM} = \text{HPD} \times \text{HPM}$

  • $\text{LPD}$: Low-Power Field of View Diameter (measured physically with a millimeter scale).

  • $\text{LPM}$: Low-Power Objective Magnification (Note: use objective magnification value, not total magnification).

  • $\text{HPD}$: High-Power Field of View Diameter (calculated value).

  • $\text{HPM}$: High-Power Objective Magnification (Note: use objective magnification value, not total magnification).

• Step-by-Step FOV Calculation Procedure:

  1. Determine individual objective lens magnifications (low-power objective magnification and high-power objective magnification).

  2. Place a transparent millimeter ruler under the low-power objective and measure the low-power diameter ($\text{LPD}$) in millimeters.

  3. Rearrange the mathematical equation to solve for the unknown high-power diameter:        $\text{HPD} = \frac{\text{LPD} \times \text{LPM}}{\text{HPM}}$

Slide Terminology, Abbreviation Standards, and Preparation Methods

• Standardized Anatomical Abbreviations on Prepared Slides:

  • w.m.: Whole Mount (the complete organism or intact structure is mounted without sectioning).

  • x.s. / c.s.: Cross-Section (a transverse slice cut perpendicular to the long axis of the structure).

  • l.s.: Longitudinal Section (a slice cut parallel along the length of the long axis of the structure).

• Slide Preparation Categories:

  • Prepared Slides: Permanently preserved and stained specimens sealed under coverslips.

  • Wet Mount Slides: Temporary preparations made by mounting fresh tissue or living aqueous organisms in a drop of liquid (e.g., water or stain) under a coverslip.

Dissecting Microscope Anatomy and Features

• Structural and Optical Anatomy:

  • Diopter Adjustment Ring: Focus adjustment ring located on individual eyepieces to compensate for focal differences between the user's left and right eyes.

  • Eyepieces: Dual ocular lenses providing binocular viewing.

  • Stereo Head: Upper housing unit containing paired optical paths.

  • Rotating Objective Selector: Mechanism housing lower stereoscopic objective lenses.

  • Focus Knob: Adjusts vertical distance of the stereo head to bring specimen into sharp focus.

  • Lighting Controls: Dual switch system regulating top (reflected/incident) lighting and bottom (transmitted) lighting.

  • Top/Bottom Illumination Systems: Top light illuminates opaque specimens; bottom light illuminates translucent specimens.

  • Stage Plate: Reversible circular surface plate mounted in the base.

  • Stage Clips: Metal clips securing thick specimens or dishes to the stage plate.

• Aqueous Specimen Exploration Resource:

  • Virtual pond dip interactive link: http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/pond/ (Credit: Microscopy-UK).

Comparative Analysis: Compound vs. Dissecting Light Microscopes

• Illumination Geometry and Image Perspective:

  • Dissecting Microscope: Light source shines from above (reflected/incident light), producing a three-dimensional ($3\text{-D}$) surface perception.

  • Compound Microscope: Light source shines from below (transmitted light through specimen), producing a flat, two-dimensional ($2\text{-D}$) image.

• Magnification Capability Limits:

  • Dissecting Microscope: Low magnification range, typically between $\thicksim 40\times$ and $50\times$

  • Compound Microscope: High magnification range, up to $\thicksim 1000\times$

• Depth of Field Parameters:

  • Dissecting Microscope: High depth of field (allows large vertical cross-sections of 3D objects to remain simultaneously in focus).

  • Compound Microscope: Low depth of field (only very thin optical planes remain in focus at one time).

• Field of View (FOV) Dimensioning:

  • Dissecting Microscope: High (large physical surface area viewed).

  • Compound Microscope: Low (restricted, small physical area viewed).

Historical and Conceptual Visuals in Microscopy

• Historical Satirical Artwork:

  • Monster Soup commonly called Thames water: Etching created by William Heath ($1828$, referenced $1985$), depicting microscopic organisms found in public drinking water.

• Humorous Educational Concepts:

  • Illustrative visual: Life on a microscope slide, depicting microscopic organisms reacting in panic to an approaching cover glass: "Aaaa! Look out everyone! It's a coverslip!"

Microscopy Laboratory Procedures and Safety Protocols

• Personal Protective Equipment (PPE): Wear protective gloves at all times throughout the session.

• Calculations: Compute total magnification for each ocular and objective combination on the compound microscope.

• Optical Inversion Phenomenon: Observe a prepared slide of the letter "e" to witness spatial inversion (reversal along top-bottom and left-right axes).

• Field of View Assessment: Quantify FOV dimensions using a clear ruler and the proportional FOV equation.

• Depth of Focus Observation: Inspect a prepared slide containing multi-colored crossed threads under varying powers to determine vertical focus plane layering.

• Wet Mount Assembly Protocol:

  1. Place a drop of pond water containing live micro-organisms onto a clean glass slide.

  2. Lower a glass coverslip gently at an angle to eliminate trapped air bubbles.

  3. Post-Observation Cleanup: Clean and completely dry all reusable glass slides; carefully discard used glass coverslips into designated broken glass disposal bins.

• Stereomicroscope Manipulation: Examine macroscopic solid objects under stereoscopic illumination.

• Worksheet Review: Complete all review questions within the lab activity worksheet and present calculations directly to the Teaching Assistant (TA) for grading.

Core Principles of Cell Theory and Cytology

• Foundational Tenets of Cell Theory:

  1. All living organisms are composed of one or more cells.

  2. Cells are the fundamental, structural, and smallest functional working units of life.

  3. All cells arise exclusively from pre-existing cells through cellular division.

• Definition of Cytology: The specialized branch of biological sciences focused on the study of cellular structure, internal organization, and physiological function.

• Representative Cellular Diversity:

  • Amoeba proteus (Free-living single-celled eukaryotic protist)

  • Plant Stem Cells (Structural plant tissue cells)

  • Red Blood Cells (Specialized non-nucleated animal transport cells)

  • Nerve Cells (Specialized elongated signaling cells)

  • Bacteria (Prokaryotic single-celled organisms)

Classification and Structural Comparison of Prokaryotic and Eukaryotic Cells

• Prokaryotic Cell Profile:

  • Dimensions: Small physical size, approximately $1\text{ }\mu m - 10\text{ }\mu m$

  • Organellar Structure: Lacks membrane-bound organelles and lacks a true membrane-enclosed nucleus.

  • Genetic Material: Unbound nucleoid region containing circular DNA.

  • Internal Complexity: Extremely few internal structures (primarily cytosolic ribosomes).

  • Organizational State: Typically unicellular organisms.

  • Taxonomic Domains: Domain Bacteria and Domain Archaea.

  • Specific Examples: Salmonella, E. coli.

  • Anatomical Structures: Capsule, Cell Wall, Cytoplasmic Membrane, Ribosomes, Pili, Cytoplasm, Nucleoid, Flagella.

• Eukaryotic Cell Profile:

  • Dimensions: Larger physical size, approximately $10\text{ }\mu m - 100\text{ }\mu m$

  • Organellar Structure: Features linear membrane-enclosed organelles and a true nucleus.

  • Genetic Material: Compartmentalized within a double-membraned nuclear envelope.

  • Organizational State: Occur as unicellular, colonial, or complex multicellular organisms.

  • Taxonomic Domains/Kingdoms: Domain Eukarya (Kingdoms Protista, Fungi, Plantae, and Animalia).

  • Anatomical Structures: Microtubules, Chromatin, Mitochondrion, Nuclear Envelope, Nuclear Pore, Nucleus, Nucleolus, Centriole, Vesicle, Cytosol, Golgi Complex, Lysosome, Flagellum, Rough Endoplasmic Reticulum, Ribosomes, Plasma Membrane, Smooth Endoplasmic Reticulum.

• Direct Comparison Matrix: Prokaryotes vs. Eukaryotes:

  • Nucleus: Prokaryotes = Absent | Eukaryotes = Present

  • Location of DNA: Prokaryotes = Nucleoid region | Eukaryotes = Nucleus

  • Cell Wall: Prokaryotes = Always present | Eukaryotes = Absent in animal cells; present in plants, fungi, and some protists

  • Cell Membrane: Prokaryotes = Present | Eukaryotes = Present

  • Membrane-Bound Organelles: Prokaryotes = Absent | Eukaryotes = Present

  • Ribosomes: Prokaryotes = Present | Eukaryotes = Present

  • Cell Division Method: Prokaryotes = Binary fission | Eukaryotes = Mitosis and Meiosis

Comparative Organellar Composition: Animal vs. Plant Cells

• Structures Present in Animal Cells but Absent in Plant Cells:

  • Centrioles: Microtubule-organizing structures involved in spindle fiber formation during animal cell division.

  • Lysosomes: Membrane-bound vesicles containing hydrolytic enzymes for intracellular digestion.

  • Cilia or Flagella: Motile hair-like structures involved in cellular locomotion.

  • Small Vacuoles: Small, temporary storage vesicles.

• Structures Present in Plant Cells but Absent in Animal Cells:

  • Cell Wall: Rigid outer boundary composed primarily of cellulose that maintains structural integrity and protects against mechanical stress.

  • Chloroplasts: Plastid organelles containing chlorophyll that execute photosynthesis.

  • Large Central Vacuole: Massive water-filled organelle maintaining turgor pressure and storage.

  • Plasmodesmata: Cytoplasmic channels piercing cell walls to allow intercellular transport and communication between adjacent plant cells.

• Interactive Learning Link: Interactive cellular architecture animations accessible at https://www.cellsalive.fun/cellsalive_files/cells/3dcell.htm (Credit: Cells Alive).

Subcellular Structures and Organelle Functionality

• Outer Boundary Structures:

  • Cell Membrane (Plasma Membrane):

    • Double-layer (phospholipid bilayer) fluid outer boundary of the cell.

    • Controls movement of substances into and out of the cell.

    • Structure Review Media: https://www.youtube.com/watch?v=vwAJ8ByQH2U.

  • Cell Wall:

    • Rigid outer layer found surrounding plant cells and bacteria.

    • Provides structural support, rigidity, and mechanical protection.

• Internal Organelles and Structural Compartments:

  • Nucleus: Known as the "control center of the cell"; houses hereditary genetic material (chromatin/DNA).

  • Nucleolus: Specialized region located inside the nucleus containing ribosomal RNA (rRNA) responsible for building protein-synthesizing machinery.

  • Cytoplasm: Gel-like matrix (cytosol) enclosed by the cell membrane holding organelles.

  • Mitochondria: Referred to as the "powerhouse of the cell"; double-membrane organelles generating cellular energy (ATP).

  • Endoplasmic Reticulum (ER):

    • Network of folded membranes transporting materials throughout the cell.

    • Smooth Endoplasmic Reticulum: Lacks attached ribosomes; involved in lipid synthesis and detoxification.

    • Rough Endoplasmic Reticulum: Studded with outer surface ribosomes; involved in processing proteins.

  • Ribosomes: Non-membrane-bound molecular complexes of proteins and RNA that conduct protein synthesis. Thousands exist freely suspended or bound within every living cell.

• Plastids in Plant Cells:

  • Specialized double-membraned storage and metabolic organelles found in plant cells and photosynthetic algae.

  • Contain internal circular double-stranded DNA ($\text{dsDNA}$).

  • Responsible for food synthesis and storage; frequently store biological pigments.

  • Chloroplast: Photosynthetic organelle containing green pigments; observed in aquatic plants such as Elodea.

  • Amyloplast: Non-pigmented leucoplast organelle specialized for starch storage; observed in potato tissue (Solanum tuberosum).