Physics: Wavelength and Magnification Concepts

Wavelength and Energy

• Violet light has a wavelength of 390 nm.
• Green light has a wavelength of 550 nm.
• Red light has a wavelength of 750 nm.
• As the wavelength of light decreases, the energy of the light increases.

Magnification & Resolution

• Magnification is the ability to enlarge objects.
• Resolving power is the ability to show detail.

Enhancing Resolving Power

• Resolving power can be enhanced by increasing the numerical aperture.
• Numerical aperture can be increased by:
  • Altering the sub-stage condenser to increase the angle of light incidence.
  • Widening the cone of light to allow more light to pass through the lens.
  • Increasing the refractive index through specialised lenses or immersion oil.
• The refractive index is the light-bending ability of a medium. Water has a refractive index of 1.0 and oil has a refractive index of 1.5.

Refractive Index: Path of Light Through Different Mediums

• Through air:
  • Smaller angle of incidence.
  • Angle of refraction bends away from the normal line (larger angle of refraction).
  • Smaller refractive index (RI) value.
  • Smaller numerical aperture (NA).
• Through Oil:
  • Large angle of incidence.
  • Angle of refraction approaches the normal line (smaller angle of refraction).
  • Larger refractive index (RI) value.
  • Larger numerical aperture (NA).

Effect of Magnification

• High magnification can produce an image that is too large to fit within the field of view.
• High magnification can decrease the resolving power.

Resolution: Light Microscopy

• With light microscopy, resolution levels are between 100-200 nm (0.1-0.2 µm).
• The magnification range for light microscopy is 1000-1500X.

Resolution: Electron Microscopy

• Electron microscopy uses a beam of electrons to form an image.
• Electron waves are 100,000 times shorter than the waves of visible light.
• Electron microscopy has significantly higher resolution.

Electron Microscopy: Resolution and Magnification

• Resolution for electron microscopy is between 0.1-2 nm.
• Magnification for electron microscopy ranges from 1000X to 400-500,000X.

Techniques of Microscopy

Differential Interference Contrast (Nomarski)

• A technique used to enhance contrast in unstained, transparent samples.
• Small phase changes in light rays are transformed into brightness or light intensity differences.

Fluorescence Microscopy

• Combines magnification with visualizing fluorescence.
• Requires a powerful light source, specialized filters, and fluorescently labelled samples.

Scanning Electron Microscopy (SEM)

• Produces a 3D image.
• Uses a focused beam of electrons to scan the surface of a sample.

Transmission Electron Microscopy (TEM)

• Produces a 2D image.
• Uses a beam of electrons that are transmitted through the sample.

Cell Staining

• Cell staining is a technique used to enhance visualization of cells and their components under a microscope.
• Different stains can target specific cell components.
• Some stains are used on fixed (non-living) cells, some are used on living cells, and some are used on both.

Why Stain Cells?

• To enhance visualization.
• To highlight metabolic processes.
• To differentiate between live and dead cells in a sample.
• To enumerate cells to determine biomass in an environment.

Example Stains

• Sudan Black: accumulates in intracellular lipid globules, staining them red – used on living cells.
• Ethidium bromide: stains unhealthy cells red-orange – used on living cells.
• Osmium tetroxide: stains lipids black – used in optical microscopy.
• Fuchsin: stains collagen, smooth muscle, or mitochondria.
• Hematoxylin: stains nuclei blue-violet or brown.
• Hoechst stains: both 33258 and 33342 stain DNA in living cells.
• Rhodamine: a fluorescent stain used on proteins in fluorescence microscopy.
• Safranin: stains nuclei and colours collagen yellow.

Preparation of Samples

Wet Mount

• Allows examination of characteristics of live cells: motility, shape, and arrangement.
• Allows for immediate observation.
• Can be stained or unstained.
• Example: blood smear.

Dry Mount

• Made by drying and heating a film of a specimen.
• Allows for repeated observations.
• Only used with dead specimens.
• Can be stained.
• Example: prepared slide for pathology and biological research.

Microscope Field of View

• The field of view is the circular area that you can see when looking through a microscope.
• The size of the field of view changes depending on the magnification of the objective lens.

Measuring Field of View

• It is necessary to measure the field of view to determine the size of objects in the microscopy field of view.
• The field of view can be measured by observing a ruler under the microscope.

Size of Cells

• It is necessary to measure the field of view because cells come in a variety of sizes.
• Cell size depends on the function the cell performs.
• Cell shapes can also vary significantly:
  • Neurons are long.
  • Parenchyma cells are equidimensional.
  • Cell membranes are flexible.
  • Cell walls are rigid.

The Cytoskeleton

• The cytoskeleton is a network of protein-based fibers.
• The cytoskeleton is responsible for:
  • Cell shape and support.
  • Cell movement.
  • Organelle movement.
• The cytoskeleton is composed of three types of protein filaments:
  • Microtubules: 25 nm in diameter, made of tubulin dimers.
  • Microfilaments: 7 nm in diameter, made of actin subunits.
  • Intermediate Filaments: 8–12 nm in diameter, made of fibrous subunits (keratins coiled together).

Significance of Cell Size

• The maximum size a cell can grow to is limited by its surface area to volume ratio.
• As a cell grows, its volume increases faster than its surface area.
• This means that the cell's surface area eventually becomes too small to support the metabolic needs of the growing cell.

Origin of Life

• Scientists believe that life originated from a "primordial soup" of organic molecules on early Earth.
• The conditions on early Earth—such as the presence of volcanic activity, lightning, and the absence of a protective ozone layer—would have provided the energy needed for these molecules to form.
• Protobionts are thought to be the evolutionary precursors of prokaryotic cells.
• Protobionts may have originated as microspheres enclosed by lipidic membranes.

Endosymbiosis Theory

• The endosymbiosis theory proposed by Lynn Margulis explains the origin of eukaryotic cells.
• This theory states that mitochondria and plastids (chloroplasts and related organelles) were once free-living prokaryotic cells that were engulfed by larger cells.
• These engulfed cells were not digested, but instead began to live within the host cell.
• Over time, the engulfed cells and the host cells became dependent on each other, eventually evolving into a single organism.

Evidence Supporting Endosymbiosis

• Mitochondria and plastids have their own DNA and ribosomes.
• The DNA and ribosomes of mitochondria and plastids are more similar to prokaryotic DNA and ribosomes than to eukaryotic DNA and ribosomes.
• Mitochondria and plastids divide independently of the host cell.

Basic Features of All Cells

• Plasma membrane: A selective barrier that encloses the cell (in plant and bacteria cells, it’s called a cell wall).
• Cytosol: A jelly-like fluid that supports organelles and other cellular components.
• Cytoplasm: The cytosol and all of the organelles, excluding the nucleus.
• Ribosomes: Organelles where protein synthesis takes place.
• DNA: The genetic material contained in one or more chromosomes.

Differential Centrifugation

• Differential centrifugation is a technique used to separate cell organelles based on their size and density.
• First, the cell membrane is ruptured to release the cell's components using a homogenizer.
• The resulting mixture is called a homogenate.
• The homogenate is centrifuged at increasing speeds and time intervals.
• This separates the components of the cell by density and size. The most dense organelles sediment first.

This quiz covers key topics related to the wavelength and energy of light, magnification, and the resolving power of optical instruments. You will explore how modifying settings like numerical aperture and refractive index can enhance visibility and detail in microscopy. Test your understanding of these fundamental concepts in physics!