Comprehensive Study Guide to Light and Electron Microscopy and Histological Techniques

Fundamentals of Light Microscopy and Optical Parameters

Image Formation Characteristics:
- The image generated by a classical transmitted-light microscope is:
  - Virtual (obraz pozorny / urojony): Formed by the apparent backward divergence of rays exiting the ocular lens; cannot be projected directly onto a screen without auxiliary lenses.
  - Magnified (powiększony): Enlarged relative to the specimen's actual physical dimensions.
  - Inverted (odwrócony): Reversed both upside-down and left-to-right.
Total Magnification Calculation:
- Total magnification is determined by multiplying the linear magnification of the objective lens, the magnification of the eyepiece (ocular), and any intermediate optical magnification factor present: $\text{Total Magnification} = \text{Magnification}_{\text{objective}} \times \text{Magnification}_{\text{eyepiece}} \times \text{Magnification}_{\text{intermediate}}$
- Standard optical light microscopes provide total magnifications within the range of $10\times$ to $1500\times$ .
Numerical Aperture ( $An$ / $NA$ ):
- A critical dimensionless index characterizing the light-gathering capacity and resolve limit of an objective lens: $An = n \times \sin(\alpha)$
  - $n$ : The refractive index of the optical medium filling the space between the front lens and the cover glass (e.g., air, water, or immersion oil).
  - $\alpha$ : The angular semi-aperture, defined as the angle enclosed between the central optical axis and the marginal ray captured by the objective lens.

Ray path and acceptance angle alpha in an objective lens

Limit of Resolution ( $E$ ):
- Defined as the minimum separation distance between two specimen points at which they can still be distinguished as separate entities.
- Resolution power is inversely proportional to this distance; smaller numerical values of $E$ denote superior resolving power.
- Calculated using Abbe's formula: $E = \frac{0.5 \times L}{An}$
  - $L$ : The wavelength of the illuminating light (average wavelength of visible light is taken as $0.55\,\mu\text{m}$ or $550\,\text{nm}$ ).
- Standard parameters across objective types:
  - Dry objectives: $An = 0.95$ , yielding a resolution limit of $E = 0.3\,\mu\text{m}$ .
  - Water immersion objectives: $An = 1.2$ , yielding a resolution limit of $E = 0.24\,\mu\text{m}$ .
  - Oil immersion objectives: $An = 1.42$ , yielding the optimal optical resolution limit of $E = 0.19\,\mu\text{m}$ .
Scale of Biological Structures and Resolving Limits:
- Limit of resolution of the unaided human eye: $0.2\,\text{mm}$ ( $200\,\mu\text{m}$ ).
- Typical eukaryotic cell size: approximately $20\,\mu\text{m}$ .
- Typical organelle and bacterial dimensions: approximately $2\,\mu\text{m}$ .
- Limit of resolution of the light microscope: $200\,\text{nm}$ ( $0.2\,\mu\text{m}$ ).
- Typical dimensions of viruses and macromolecular complexes: approximately $20\,\text{nm}$ .
- DNA double helix diameter and small molecules: approximately $2\,\text{nm}$ .
- Limit of resolution of the transmission electron microscope (TEM): $0.2\,\text{nm}$ .
- Individual atoms: below $0.2\,\text{nm}$ , resolvable with atomic force microscopy (AFM).
- Metric length conversion scale:
  - $1\,\text{meter}\,(1\,\text{m}) = 10^0\,\text{m}$
  - $1\,\text{decimeter}\,(1\,\text{dm}) = 0.1\,\text{m} = 10^{-1}\,\text{m}$
  - $1\,\text{centimeter}\,(1\,\text{cm}) = 0.01\,\text{m} = 10^{-2}\,\text{m}$
  - $1\,\text{millimeter}\,(1\,\text{mm}) = 0.001\,\text{m} = 10^{-3}\,\text{m}$
  - $1\,\text{micrometer}\,(1\,\mu\text{m}) = 0.000001\,\text{m} = 10^{-6}\,\text{m}$
  - $1\,\text{nanometer}\,(1\,\text{nm}) = 0.000000001\,\text{m} = 10^{-9}\,\text{m}$

Biological scale and resolution limits of microscopic systems

Optical Components and Microscope Classifications

Components of the Transmitted Light Optical Train:
- Illumination Device / Light Source:
  - Construction: Halogen bulbs, light-emitting diodes (LED), standard incandescent bulbs, or adjustable planar/concave mirrors in older instruments.
  - Function: Generates and projects the primary beam of visible light.
- Condenser (Abbe Apparatus):
  - Construction: Multi-lens gathering system mounted beneath the specimen stage.
  - Function: Focuses diffuse light from the source into a concentrated, convergent cone that evenly illuminates the field of view.
- Objective Lens:
  - Construction: Highly complex, compound multi-element glass lens system.
  - Function: Primary image-forming component; magnifies the specimen and projects an intermediate real image into the optical tube.
- Intermediate Optical System:
  - Construction: Beam-splitting prisms, mirror sets, and transfer lenses.
  - Function: Bends the optical axis to match the ergonomic inclination of the binocular head; splits light paths between oculars and documentation cameras; provides auxiliary intermediate magnification in certain designs.
- Eyepiece (Ocular):
  - Construction: Pair of plano-convex glass lenses.
  - Function: Receives and further magnifies the real intermediate image from the objective, producing the final enlarged virtual image observed by the user.
Taxonomy of Light Microscopy Systems:
- Classical Transmitted Light Microscopes:
  - Standard Upright Microscope: Standard configuration with an overhead objective turret and sub-stage illumination.
  - Inverted Microscope: Objectives are positioned beneath the specimen stage while the condenser and illuminator sit above; used for living cell cultures in flasks, Petri dishes, and microtiter plates.
- Modifications of Classical Light Microscopy:
  - Darkfield Microscope: Employs a specialized darkfield condenser that blocks central unscattered rays; only light scattered by sample boundaries enters the objective, rendering specimens luminous against a pitch-black background.
  - Phase-Contrast Microscope: Translates minute phase shifts caused by cellular refractive index differences into visible amplitude (brightness) shifts; enables visualization of unstained living cells.
  - Interference Microscope (DIC / Nomarski): Uses polarized light and birefringent prisms to split and recombine light beams, generating pseudo-three-dimensional relief contrast of living, unstained cells.
  - Polarizing Microscope: Utilizes a polarizer and an analyzer to detect optically anisotropic (birefringent) structures such as crystalline inclusions, collagen fibrils, and cytoskeletal filaments.
- Fluorescence Microscope:
  - Utilizes short-wavelength excitation light; fluorophores in the specimen absorb this energy and emit light at longer wavelengths, separated by dichroic beam-splitters.
- Confocal Laser Scanning Microscope (CLSM):
  - Employs focused laser beams and pinhole apertures to eliminate out-of-focus light flare, generating high-resolution optical sections for 3D reconstructions.

Objective Lenses: Mechanics, Classifications, and Markings

Operational Principles of Dry vs. Immersion Objectives:
- Dry Objectives:
  - Designed for low and intermediate magnifications ( $4\times$ - $5\times$ , $10\times$ , $20\times$ , $40\times$ ).
  - Air ( $n = 1.00$ ) separates the cover glass ( $n \approx 1.515$ ) from the lens. Rays exiting glass into air at high angles undergo severe refraction away from the normal, and rays exceeding the critical angle undergo total internal reflection, escaping collection.
- Immersion Objectives:
  - Designed for high magnifications ( $60\times$ , $100\times$ ).
  - The air gap is replaced with an immersion fluid (immersion oil, $n \approx 1.515$ ) matching the refractive index of glass.
  - Eliminates refraction and reflection at the glass-air boundary, preventing light loss, preserving wide ray angles ( $\alpha$ ), and maximizing numerical aperture and resolution.

$Refraction in dry objective versus light conservation in oil immersion objective$

Objective Barrel Markings and Identification:
- Correction Class Inscriptions:
  - Mono. (Monochromatic): Corrected for a single specific wavelength of light.
  - Unmarked or A (Achromat): Standard correction; corrected chromatically for two visible wavelengths (red and blue) and spherically for green.
  - Apo. (Apochromat): High-tier correction; corrected chromatically for three to four wavelengths and spherically for two or three, delivering high optical fidelity across the spectrum.
  - Plan. (Planachromat): Incorporates flat-field correction to eliminate field curvature aberrations, ensuring the image remains sharp from the center to the outermost edges.
  - Plan Apo. (Planapochromat): Combines comprehensive apochromatic chromatic correction with complete planar field-flattening.
- Magnification Inscription: Nominal magnification power indicated as a numeral followed by an "x" (e.g., 4x, 10x, 20x, 40x, 60x, 100x).
- Numerical Aperture ( $An$ ): Engraved alongside the magnification separated by a slash (e.g., 60x/1.40, 40/0.65, 100/1.25).
- Immersion Medium: Engraved explicitly when required (e.g., Oil, W or Water); dry objectives feature no immersion designation.
- Optical Contrasting Compatibility: Specialized abbreviations designate optical design compatibility (e.g., DIC H indicates differential interference contrast compatibility).
- Mechanical Tube Length: Denotes the required optical distance from objective shoulder to ocular seat in millimeters (traditionally 160). In infinity-corrected systems, it is marked with the infinity symbol (\infty), indicating compatibility with modern infinity optics regardless of tube length.
- Coverslip Thickness Requirement: Designated in millimeters, standardly 0.17 ( $0.17\,\text{mm}$ , equivalent to standard No. 1.5 coverslips). Marked with a hyphen (-) if the objective is insensitive to coverslip variations or intended for uncovered preparations.
- Working Distance (WD): The physical clearance separating the front lens surface from the upper face of the coverslip when the specimen is in focus, indicated in millimeters or centimeters (e.g., WD 0.21 denotes $0.21\,\text{mm}$ ; or $0.15\,\text{cm}$ on certain $60\times$ lenses).

Objective barrel markings and technical specifications

Standard Objective Color Ring Codes:
- Red ring: Low magnification ( $4\times$ - $5\times$ ).
- Yellow ring: Low-medium magnification ( $10\times$ ).
- Green ring: Intermediate magnification ( $20\times$ ).
- Blue ring: High dry and high immersion magnification ( $40\times$ and $60\times$ ).
- White ring: Highest magnification immersion ( $100\times$ ).
Functional Trends with Increasing Objective Magnification:
- Working distance decreases.
- Numerical aperture ( $An$ ) increases.
- Resolving capacity increases (the resolution limit $E$ decreases to smaller distances).
- Front lens diameter decreases markedly.
- Light collection diminishes due to smaller front apertures, requiring increased light source intensity.
Specialty Objectives:
- Fluorescence Objectives: Feature optical glass formulations providing high transmission of short-wavelength ultraviolet light down to $340\,\text{nm}$ .
- Long Working Distance (LWD) Objectives: Feature elongated focal lengths to allow imaging through thick culture vessels in inverted microscopes.

Principles of Electron Microscopy: TEM and SEM

Transmission Electron Microscopy (TEM):
- Operational Mechanism:
  - Operates analogously to transmitted light microscopy: an accelerated beam of electrons passes through an ultrathin tissue specimen.
  - Contrast is generated by electron scattering; atomic nuclei within the specimen deflect electrons via elastic and inelastic scattering.
  - Scattered electrons are blocked by objective apertures and fail to hit the screen, forming dark regions (electron-dense). Unscattered electrons pass freely, striking the fluorescent screen or digital detector to form bright regions (electron-lucent).
- Instrument Construction:
  - Hermetically sealed vertical metal column maintained under high vacuum (reaching up to $-100\,\text{atmospheres}$ ) by linked mechanical and turbomolecular vacuum pumps.
  - High-voltage transformers converting standard electrical power into high-potential, low-amperage current to accelerate electrons.
  - Electron gun situated at the top of the column emitting high-energy primary electrons.
  - Electromagnetic coils functioning sequentially as condenser, objective, and projector lenses.
  - Fluorescent observation screen, viewing binocular loupe, and photographic plate/digital camera detector located at the column base.
- Contrast Enhancement in Biological Tissues:
  - Native living cells consist predominantly of light chemical elements ( $\text{C}$ , $\text{H}$ , $\text{O}$ , $\text{N}$ ) that exhibit negligible electron-scattering capacity, resulting in pale, low-contrast images.
  - Contrasting requires introducing heavy metal salts with high atomic weights (uranium, lead, osmium) that bind selectively to specific macromolecules, yielding sharp, high-contrast, black-and-white micrographs.
Scanning Electron Microscopy (SEM):
- Operational Mechanism:
  - A finely focused electron probe does not traverse the specimen; instead, it rasters across the specimen surface point-by-point.
  - The primary beam knocks secondary electrons out of the surface atoms.
  - Secondary electrons are collected by an electron collector/detector, amplified electronically, and reconstructed synchronously on a digital display monitor.
- Applications and Preparative Differences:
  - Used to investigate three-dimensional surface morphology, spatial contours, and structural topology of cells and tissues.
  - Generates plastic, pseudo-three-dimensional relief imagery.
  - Specimens are dehydrated and sputter-coated with a thin conductive film of carbon and heavy metals (e.g., gold) to prevent electrical charge accumulation.

Structural ray path comparison of Transmission Electron Microscope versus Scanning Electron Microscope

Comparative Analysis: Light Microscope (LM) vs. Transmission Electron Microscope (TEM):
- Magnification: LM reaches up to $1500\times$ ; TEM achieves $1\,000\,000\times$ or higher.
- Maximum Resolution Limit: LM resolves to $200\,\text{nm}$ ( $0.2\,\mu\text{m}$ ); TEM resolves to $0.2\,\text{nm}$ .
- Image Radiation Source: LM uses visible light rays; TEM uses an accelerated electron beam.
- Focusing Elements: LM utilizes ground glass lenses; TEM uses electromagnetic coil fields.
- Observation Mechanism: LM viewed directly through glass oculars; TEM visualized on a fluorescent screen and digital display monitor.
- Specimen Support: LM uses transparent glass slides; TEM uses fine $3\,\text{mm}$ copper or nickel grids.
- Specimen Viability: LM can observe living cells; TEM strictly cannot (samples are destroyed by the high vacuum and ionizing radiation).
- Processing Requirements: LM does not always require extensive processing (can use wet mounts); TEM always requires intricate chemical processing.
- Image Coloration: LM yields full natural or histological color; TEM produces strictly monochromatic black-and-white imagery.

Biological Specimen Preparation for Light Microscopy: The Paraffin Technique

Essential Criteria for Diagnostic Light Microscope Preparations:
- Transparency: The sample must be thin enough to permit unimpeded light transmission. Thinner sections yield higher optical resolution.
- Contrast / Staining: Native cells exhibit near-identical optical density; differential staining establishes visual contrast between organelles and tissues.
- Structural Preservation: Morphology must reflect the living tissue state with high fidelity, preventing post-mortem degradation artifacts.
Primary Types of Microscopic Preparations:
- Sections (Skrawki): Ultra-thin tissue slices cut from solid organs.
- Smears (Rozmazy): Monolayers spread from body fluids containing suspended free cells (peripheral blood, red bone marrow aspirates, cerebrospinal fluid, exudates, and mucosal secretions).
Sequential Stages of the Paraffin Technique:
- 1. Material Sampling:
  - Vital sampling: Harvested via diagnostic biopsy or surgical excision.
  - Post-mortem sampling: Harvested as rapidly as possible following cardiac arrest to halt autolytic degradation.
  - Specimen sizing: Cut using sharp razor blades into blocks no larger than $1\,\text{cm} \times 1\,\text{cm} \times 1\,\text{cm}$ or $2\,\text{cm} \times 0.5\,\text{cm} \times 0.5\,\text{cm}$ to guarantee thorough chemical penetration.
- 2. Fixation:
  - Objectives: Denature metabolic and lysosomal enzymes to halt autolysis (self-digestion) and secondary bacterial putrefaction; stabilize tissues against harsh downstream processing reagents.
  - Mechanism: Forms covalent cross-links between polypeptide chains, generates methylene bridges ( $-\text{CH}_2-$ ) via formaldehyde, or binds protein sulfhydryl ( $-\text{SH}$ ) groups. This denatures protein secondary, tertiary, and quaternary structures while preserving overall tissue architecture.
  - Fixatives: Simple fixatives include formalin, ethyl alcohol, and acetone. Compound mixtures include Bouin's fluid (formalin, picric acid, glacial acetic acid) and AFA (alcohol, formalin, acetic acid).
  - Immersion Fixation: Tissue block is submerged in a fixative volume $50\times$ to $100\times$ greater than the tissue volume. Fixative diffuses from the periphery inward; slow core penetration can lead to autolytic artifacts in thick specimens. Processing time ranges from 1 hour to several days.

Diffusion gradient of fixative in tissue block over time

    *   *Perfusion Fixation:* Performed via catheterization of an organ's arterial supply (or transcardially in small animals). Physiological saline is infused to clear blood, followed immediately by fixative, which drains through opened venous returns. Fixative reaches tissues instantly through the capillary bed, completing fixation in  $5$  to  $15\,\text{minutes}$ .

*   **3. Washing (Płukanie):**
    *   Eliminates excess unbound fixative.
    *   Conducted in running tap water for aqueous fixatives, or in ethyl alcohol for compound fixatives containing  $\ge 70\%$  ethanol.

*   **4. Dehydration (Odwadnianie):**
    *   Removes all free tissue water and fixative moisture, replacing it with alcohol.
    *   Carried out in an ascending ethanol series:  $30\%$ ,  $50\%$ ,  $60\%$ ,  $70\%$ ,  $80\%$ ,  $90\%$ ,  $96\%$ , and  $100\%$  absolute ethanol to avoid osmotic shock.

*   **5. Clearing / Intermediate Fluids (Prześwietlanie):**
    *   Removes absolute alcohol, which is immiscible with paraffin, replacing it with an organic solvent that dissolves in molten paraffin.
    *   Reagent: Xylene.
    *   The specimen becomes translucent ("cleared") as xylene's refractive index approaches that of dehydrated tissue proteins.

*   **6. Paraffin Infiltration and Embedding (Zatapianie w parafinie):**
    *   *Infiltration:* Specimen is incubated in molten paraffin inside an oven maintained a few degrees above the paraffin melting point.
    *   Paraffin grades vary by hardness and melting point (e.g.,  $45^\circ\text{C}$  for soft paraffin,  $60^\circ\text{C}$  for hard paraffin).
    *   *Embedding:* Infiltrated tissue is oriented inside a molding cassette, covered with molten paraffin, and chilled to solidify into a hard paraffin block.

Paraffin embedding cassette and solidified tissue block

*   **7. Sectioning (Krojenie):**
    *   Performed on microtomes equipped with high-grade steel knives.
    *   *Rotary microtome:* Specimen block moves vertically against a stationary knife.
    *   *Sledge (sliding) microtome:* Knife moves horizontally across a stationary specimen block.
    *   *Thickness:* Sections are cut at a thickness of  $4$  to  $7\,\mu\text{m}$ .
    *   *Ribbon processing:* Slicing generates continuous ribbons of sections. These ribbons are separated and floated onto a warm water bath ( $40^\circ\text{C}$ ) to soften the paraffin and stretch out compression folds, then collected onto glass slides and dried.

*   **8. Staining (Barwienie):**
    *   *Classification of Histological Dyes:*
        *   **Basic (Nuclear) Dyes:** Positively charged dyes that bind negatively charged acidic cellular components (DNA and RNA in nuclei, nucleoli, rough endoplasmic reticulum, and ribosomes). Example: Hematoxylin, which stains acidic structures deep purple/violet.
        *   **Acidic (Cytoplasmic) Dyes:** Negatively charged dyes that bind basic cytoplasmic proteins and organelles. Example: Eosin, which stains basic structures pink/red.
        *   **Lipid-Soluble (Neutral) Dyes:** Non-ionic dyes (e.g., Sudan stains) that partition into hydrophobic lipids. Can only be applied to frozen sections, as routine paraffin clearing solvents dissolve all neutral lipids.
    *   *Special and Routine Staining Regimens:*
        *   *Hematoxylin and Eosin (HE):* Universal routine histological stain.
        *   *Giemsa Stain:* Used for hematological smears of blood and bone marrow.
        *   *Azan-Mallory Stain:* Stains connective tissue fibers and collagen blue.
        *   *Masson's Trichrome Stain:* Stains collagen fibers green.
        *   *Silver Impregnation:* Silver nitrate ( $\text{AgNO}_3$ ) reduces to metallic black silver precipitate on argyrophilic structures (type III collagen / reticular fibers).

*   **Detailed Hematoxylin and Eosin (HE) Protocol:**
    1.  Deparaffinization in xylene ( $2\times$ ) to remove paraffin.
    2.  Rehydration through a descending ethanol series ( $100\% \rightarrow 50\%$ ).
    3.  Rinsing in distilled water.
    4.  Nuclear staining with hematoxylin.
    5.  Rinsing in water.
    6.  Differentiation in dilute acid-alcohol solution to remove excess, non-specific dye.
    7.  Rinsing in water.
    8.  Cytoplasmic counterstaining with eosin.
    9.  Rinsing in water.
    10. Dehydration through an ascending ethanol series ( $80\% \rightarrow 100\%$ ).
    11. Clearing in xylene ( $2\times$ ).

*   **9. Mounting (Zamykanie):**
    *   The stained section is covered with a coverslip using mounting resins such as Canada balsam or DPX.
    *   These resins match the refractive index of glass ( $n \approx 1.5$ ), preventing light scattering and preserving specimens for decades.

Specimen Preparation for Transmission Electron Microscopy (TEM)

1. Material Sampling:
- Harvested via in vivo surgical biopsy or strictly within $3\,\text{minutes}$ post-mortem.
- Tissue pieces are cut with sharp razor blades to dimensions not exceeding $1\,\text{mm} \times 1\,\text{mm} \times 1\,\text{mm}$ .
2. Primary Fixation:
- Buffer Vehicle: Uses $0.1\,\text{M}$ phosphate or cacodylate buffer to maintain physiological conditions ( $\text{pH} = 7.2$ - $7.4$ , osmolarity, and ionic strength).
- Fixative Components:
  - Paraformaldehyde: Rapidly penetrates tissue; forms loose cross-links that preserve protein structure and glycogen without fixing lipids.
  - Glutaraldehyde: Penetrates slowly; forms strong, permanent cross-links with proteins and binds some lipids.
- Standard Mixture: $2.5\%$ glutaraldehyde and $1\%$ paraformaldehyde in $0.1\,\text{M}$ phosphate buffer at $\text{pH} = 7.4$ .
- Application: Immersion or vascular perfusion for $2\,\text{hours}$ at $4^\circ\text{C}$ or room temperature.
3. Washing:
- Conducted using the vehicle buffer (e.g., $0.1\,\text{M}$ phosphate buffer) to clear unbound fixative.
- Duration ranges from a standard $24\,\text{hours}$ up to $7$ - $14\,\text{days}$ during specimen transport.
4. Secondary Fixation and Pre-contrasting:
- Reagent: $1\%$ - $2\%$ Osmium tetroxide ( $\text{OsO}_4$ ) in distilled water or phosphate buffer.
- Function: Cross-links and stabilizes unsaturated lipids and cell membranes, preventing their extraction by organic solvents during dehydration, while imparting heavy osmium atoms for initial electron density.
- Application: Immersion for $2\,\text{hours}$ at room temperature.
5. Dehydration:
- Carried out in an ascending ethanol series: $30\%$ , $50\%$ , $60\%$ , $70\%$ , $80\%$ , $90\%$ , $96\%$ , and $100\%$ absolute ethanol.
6. Intermediate Solvents:
- Specimens are passed through organic solvents miscible with both ethanol and embedding resins, such as acetone or propylene oxide.
7. Resin Infiltration and Embedding:
- Media: Synthetic embedding resins, including epoxy resins (EPON 812), polyester resins, or methacrylates.
- Infiltration: Specimen is soaked in a $1:1$ mixture of intermediate solvent and liquid resin for $24\,\text{hours}$ .
- Embedding and Polymerization: Tissue is transferred to capsules filled with pure liquid resin and heat-cured in an oven at $60^\circ\text{C}$ to $80^\circ\text{C}$ for $24$ to $48\,\text{hours}$ , yielding hard plastic blocks.
8. Trimming, Sectioning, and Grid Mounting:
- Semi-thin Sectioning:
  - Cut at a thickness of $0.5$ to $2\,\mu\text{m}$ on an ultramicrotome.
  - Dried onto glass slides, stained with Toluidine blue, and examined under a light microscope to locate target regions of interest.
- Block Trimming:
  - Under a stereomicroscope, the resin block face is trimmed down into a small trapezoidal pyramid whose apex matches the targeted region, with edge lengths not exceeding $0.3\,\text{mm}$ .
- Ultrathin Sectioning:
  - Cut at a thickness of $20$ to $60\,\text{nm}$ on an ultramicrotome using glass or diamond knives (steel knives are insufficiently sharp and hard).
  - Sections float onto the surface of a water-filled boat attached to the knife edge, and are transferred with fine tweezers onto a $3\,\text{mm}$ circular copper or nickel mesh grid.
9. Post-Staining (Heavy Metal Contrasting):
- To enhance contrast beyond the initial osmium treatment, grids are stained with heavy metal salt solutions:
  - Uranyl acetate: Binds nucleic acids (DNA and RNA) and proteins.
  - Lead citrate: Binds membranes and general cytoplasmic proteins.