Imaging cells using microscopy

Magnification

Ratio of image size to actual size

Resolution

Minimum distance between two distinguishable points (Light microscope limit ≈ 0.2 µm)

Contrast

Differences in brightness or color between parts of the sample

Light microscopes

• Invented: 17th century

• Major Advantage: Can view living cells

• Major Limitation: Limited resolution

  • Dissecting microscope

  • Compound brightfield microscope

• Total Magnification: Ocular lens × Objective lens

Dissecting light microscope

up to ~70× (surface features)

Compound brightfield light microscope

• 400–1000× (internal features)

• light passes through transparent specimen

• most common LM

Phase Contrast LM

• Refracted/unrefracted light

• Live transparent cells

Differential Interference Contrast (DIC) LM

• 2 beams of light

• High-contrast images of living cells

Fluorescent LM

• Fluorescent-tagged molecules

• Detect specific proteins (uses antibodies)

Confocal LM

• Laser-based optical sectioning

• 3D imaging, thick specimens (e.g. embryos

Deconvolution LM

• Computational

• Removes out-of-focus light

Super-resolution LM

• Tracks individual molecules

• Breaks diffraction limit

LM sample preparation

• Whole mounts – Small transparent specimens

• Tissue Sections – Most tissues require sectioning

• Fixation – Prevents decay (chemical fixatives)

• Dehydration/Clearing – Removes water before wax

• Embedding – In wax

• Sectioning – Thin slices (~5 µm) via microtome

• Staining:

  • Eosin: Cytoplasm

  • Haematoxylin: Nuclei

EM

• Invented: 1930s (Germany, Ruska & Knoll)

• Advantages: Much greater resolution due to short electron wavelength (~0.08 nm)

• Limitations:

  • Needs vacuum

  • Samples must be dead

  • Requires complex prep

TEM

• Function: Electrons pass through thin specimen sections

• Resolution: Up to ~0.08 nm

• Structure: Similar to LM but uses magnetic lenses

• Image: Viewed via fluorescent screen or digital camera

TEM Sample preparation

• Whole Mounts – For bacteria/viruses

• Fixation – Glutaraldehyde (proteins), Osmium tetroxide (lipids)

• Dehydration – Ethanol series

• Embedding – In plastic (e.g., epoxy resins)

• Sectioning – ~50 nm slices (ultramicrotome)

• Staining – Heavy metals (e.g., lead) for contrast

SEM

• Function: Electron beam scans surface

• Image: 3D, shows topology (depth of field is high)

• Signal: Reflected electrons collected and displayed

SEM sample preparation

• Fixation – Same as TEM

• Dehydration – Replace water with ethanol

• Critical Point Drying – Prevents shrinkage

• Coating – Thin gold layer to protect from beam

Cell fractionation

• Purpose: Isolate and study organelles/proteins

• Steps:

  1. Homogenization – Break cells open

  2. Differential Centrifugation – Separate organelles by size/density

Cell fractionation uses

• Protein Enrichment – Increase concentration of target protein

• Protein Characterization – Locate protein inside the cell

• Protein Translocation – Study movement of proteins (e.g., signaling to nucleus)