M3L2 - Visualizing the Cell

Limitations of Human Eye

• Our limits are a fifth of a mm

  • Cannot differentiate between objects closer than that

• We can see with the unaided eye:

  • nerve cells

  • single cell of some eggs

Types of Microscopes (2)

• Light Microscope

• Electron Microscope

Light Micrscopy

• Uses light to illuminate the object 

• Allows visualization of most prokaryotic cells and organelles inside eukaryotic cells 

• Limitation: Objects less than 200nm

Electron Microscopy

• Uses a beam of electrons to illuminate objects

• Visualizes small bacteria, viruses, or large proteins, protein complexes, ribosomes, lipids, and small molecules

Limit of Resolution (D)

• Wavelength of source of illumination limits our ability to see small objects

  • Small wavelength = smaller D = better resolution

• Resolution: The ability to see an object/amt of detail we can observe with clarity

• Microscopy resolution (D): The smallest distance between 2 objects where they still appear distinct 

  • Small D = better resolution 

• Better to minimize refraction of illuminating light

Resolution (D) Formula 

• Formula: D = 0.61(λ) / NA 

  • λ = wavelength of light

    • Shorter wavelength = higher resolution

  • NA = Numerical Aperture 

    • How much light bends as it passes from the objective lens through air to the specimen.

    • closer to 1 = better

• Wavelength of source of illumination limits our ability to see small objects

  • Small wavelength = smaller D = better resolution

• Better to minimize refraction of illuminating light

Improving Resolution (Decreasing D) 

• Light microscopy

  • Wavelength: 450nm

  • Microscope objective NA = 0.94

    • (closer to 1 = good) 

  • D = 292nm 

    • Objects closer than this distance can’t be discerned 

• Bright field microscopy

  • Resolving power of 0.2 um 

  • Can magnify 1000x

• UV light source

  • λ = 200 - 300nm

  • D will be smaller (better resolution)

• Electron Microscope

  • D = 0.1 - 10nm

Brightfield Microscopy: Blood Smear

• Samples can be live or fixed, and stained or unstained 

• Blood smear can be stained with whole cell dye and seen

  • Distinguishable between biconcave shape of typical RBC (lighter stain in middle) 

  • You can see sickled RBC (sickle cell anemia sample) 

• Scale bar:

  • Magnification is 535x

  • Length of scale bar is 25μm

  • Diameter of RBC = about 8μm

Brightfield Microscopy: Tissues

• Samples embedded in paraffin to slice thin sections

• This is a stained (with PAS)  small intestine section of a mouse 

• Observations:

  • Surface columnar epithelial cells form a single, even row.

  • Nuclei or intestinal cells are stained blue 

  • Magenta cells are specialized goblet cells (GC) 

  • They stain strong as they’re right in polysaccharides and glycogen

  • Microvilli cover the main columnar epithelial cells

Phase Contrast AND Nomarski (DIC) microscopy 

• Complementary techniques C

• Produces high contrast images of unstained and unfixed transparent biological specimens 

• Both rely on enhancing difference in density of different region of specimen 

• They both allow live specimens to be visualized

  • Allows view of the dynamics of a living system

Phase contrast microscopy VS Nomarski (DIC) microscopy

• Phase: Favours clear visualization of internal cellular structures

  • Creates ‘halo’ around external surface

  • Obscures some visualization 

• Nomarski: Clear, sharper edges and surfaces and cell structures 

  • Clearer view of periphery of cell 

  • Can see better proximity between neighbouring cells (to study intracellular events) 

Immunofluorescence Microscopy

• Reveals the locations of specific molecules in the cell 

• Uses fluorescent dyes or antibodies to tag target molecules.

• A primary antibody binds specifically to the molecule/antigen of interest (e.g., antigen B).

• A secondary antibody, covalently attached to a fluorophore, binds the primary antibody.

• The fluorophore is excited by UV light, emitting fluorescence that marks the location of the target protein.

• Example: CD44 (a cell membrane protein) labeled with a green fluorophore, and DAPI (DNA-specific dye) labels nuclei blue, clearly showing cell membranes and nuclei.

How can we visualize unique molecules in a cell beyond general stains?

• Some stains are specific for biomolecule classes (e.g., DNA, polysaccharides, proteins).

• To detect a single specific protein, more advanced techniques are needed.

• Analogy: Like city lights in a satellite image reveal where people are and what they’re doing, fluorescent tagging marks the presence and distribution of specific molecules.

How does fluorescence microscopy compare to Nomarski in visualizing mitosis?

• Nomarski microscopy shows compact chromosomes in metaphase but lacks detailed visualization of internal structures.

• Fluorescence microscopy with DAPI (blue) and β-tubulin antibodies (green) reveals microtubules and the mitotic spindle, details invisible in Nomarski images.

• Because antibodies must access internal structures, cells must be fixed and processed, so immunofluorescence is used on non-living cells.

Fluorescence Microscopy: GFP Protein

• Looks at the dynamics of living cells instead of just fixed

• Green Fluorescent Protein (GFP) is a natural fluorescent protein from jellyfish.

• Recombinant DNA technology fuses the gene for GFP with a protein’s gene, creating a protein-GFP fusion.

• Once expressed in cells, the fusion protein is now fluorescent

  • reveals the location and behavior of the protein

• Ex. Tubulin protein fused to GFP within a cell

  • It shows its location and how microtubules are organizes into a mitotic spinde 

Confocal Laser Scanning Microscopy

• Obtains high resolution images from fluorescently labelled samples

• Creates optical sections while keeping tissues/cells intact

• It excited only the fluorophores in a thin section with a specialized laser 

  • Eliminate background fluorescence above and below region of interest 

  • A clear and detailed image is made 

• Ex. 2 images of BPAE cells 

  • DNA in nucleus stained with DAPI 

  • Actin tagged in green 

  • Antibody to visualize protein specific for GA is red 

  • Both images have actin easily seen but confocal image shows inc quality and clarity of the actin fillaments

Doconvolution Microscopy

• Creates a clear image similar to confocal microscopy by using traitional fluorescence microscopy 

• An image from a conventional fluoresence microscope is processed using computer algorithms

  • Subtracts the fluoresence that is out of focus above/below 

• Isolated digital sections of fluorescent images 

Transmission Electron Microscopy (TEM)

• Regular Electron Microscope:

  • λ = 0.005nm

  • D = 0.1nm

• TEM

  • beam is directed through thinly slices specimen to form an image 

  • Stained/dense areas appear dark

  • Unstained/sparse areas appear light 

• 52,000x magnification compared to brightfield image 

Scanning Electron Microscope (SEM)

• Uses beam of electrons to illuminate sample

• Beam is focused over surface of specimen coated in a thin layer of metal

• Produces 3D surface morphology image