Cell Biology Unit 2: Microscopes

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Questions and Answers

What is the primary advantage of a transmission electron microscope (TEM) over a light microscope?

It uses fluorescent molecules.

It can visualize bacteria directly.

It has significantly higher resolution. (correct)

It requires living samples.

Which organelle size range falls within 0.1 µm to 2 µm?

Photons

Molecules

Organelles (correct)

Cells

What happens to fluorescent molecules upon excitation?

They lose energy and turn dark.

They absorb all wavelengths equally.

They become chemically inert.

They emit light of a longer wavelength. (correct)

Which equation correctly represents the energy of a photon?

E = hc / λ Signup and view all the answers

During fluorescence microscopy, the emitted light is observed through a filter that allows which type of light to pass?

Light of the emitted wavelength only Signup and view all the answers

What occurs after the energy of the excited state S1' is partially dissipated?

A photon of energy is emitted. Signup and view all the answers

What is the function of the first barrier filter in a fluorescence microscope?

To pass through blue light with a wavelength between 450 and 490 nm. Signup and view all the answers

Which fluorescent molecule emits red light when activated by light of the appropriate wavelength?

Tetramethylrhodamine Signup and view all the answers

What is the purpose of the beam-splitting mirror in a fluorescence microscope?

To reflect light above 510 nm while absorbing light below that wavelength. Signup and view all the answers

When fluorescein is excited, what type of light is emitted?

Green light. Signup and view all the answers

What is the primary function of the second barrier filter in confocal microscopy?

To cut out unwanted fluorescent signals Signup and view all the answers

How does confocal microscopy improve image quality compared to conventional fluorescence microscopy?

It eliminates depth-related blur by focusing on a single point of light Signup and view all the answers

What role does the dichroic mirror play in confocal microscopy?

It reflects only specific wavelengths of light Signup and view all the answers

Which of the following enhances the sensitivity of immunofluorescence microscopy?

Using secondary antibodies that recognize primary antibodies Signup and view all the answers

What does fixation do during the preparation of samples for microscopy?

Locks proteins in place while preserving cell structure Signup and view all the answers

What type of light does the laser provide in confocal microscopy?

Coherent monochromatic light Signup and view all the answers

What is the consequence of emitted light from areas outside the plane of focus in conventional fluorescence microscopy?

It causes a blurry image Signup and view all the answers

Which wavelength range does the second barrier filter allow through in confocal microscopy?

520 to 560 nm Signup and view all the answers

What is one main concern when using glutaraldehyde as a cross-linking reagent in fluorescence microscopy?

It autofluoresces and may interfere with fluorescence signal. Signup and view all the answers

Which organic solution is used for dehydration in the sample preparation process?

Acetone Signup and view all the answers

What is the limit of resolution of a light microscope using blue light with a wavelength of 450 nm and immersion oil objective?

194 nm Signup and view all the answers

What is necessary to ensure when using multiple primary antibodies in immunofluorescence microscopy?

The primary antibodies should be from different host species. Signup and view all the answers

What is the function of Triton X-100 in sample preparation for immunofluorescence?

It serves as a permeabilizing agent. Signup and view all the answers

Which light microscopy technique requires a phase plate to enhance image contrast?

Phase-contrast microscopy Signup and view all the answers

Which component is NOT used in a transmission electron microscope (TEM)?

Glass lenses Signup and view all the answers

What type of fluorescent probe is used to visualize DNA during immunofluorescence microscopy?

DAPI Signup and view all the answers

What is a key difference between light microscopes and transmission electron microscopes (TEMs)?

TEMs use electrons to form images. Signup and view all the answers

To create a composite image of multiple cellular components, what is essential during imaging?

Overlaying images acquired with different filter sets. Signup and view all the answers

What should be ensured about the fluorescent antibodies during an immunofluorescence experiment?

They should emit light at different wavelengths. Signup and view all the answers

What is the approximate size limit for objects to be resolved by a light microscope?

0.2 µm Signup and view all the answers

What consequence can occur if membranes are not permeabilized properly during antibody staining?

Antibodies will not penetrate the cells. Signup and view all the answers

Which microscopy technique is best suited for observing thicker samples?

Differential interference contrast (DIC) Signup and view all the answers

Which statement is true regarding the resolution capabilities of light microscopes?

Resolution is ultimately limited by the wavelength of light. Signup and view all the answers

What is the purpose of the fluorescent dye in fluorescence-activated cell sorting (FACS)?

To detect the presence of specific proteins on cell surfaces Signup and view all the answers

What happens to the electron beam in a transmission electron microscope?

It is projected downwards through a vacuum. Signup and view all the answers

What must happen to the cells when they reach 100% confluency?

They must be passaged to a new dish Signup and view all the answers

How many times can primary cells typically be passaged before they stop dividing?

What is a common characteristic of transformed cell lines?

They can grow indefinitely Signup and view all the answers

Which of the following is an essential component of the growth media for cultured cells?

Glucose Signup and view all the answers

What addition is necessary for the immortalization of primary cells?

DNA that expresses telomerase Signup and view all the answers

Which of the following cell lines is derived from human epithelial cells?

HeLa Signup and view all the answers

What role does transferrin play in the growth media for cultured cells?

It transports iron Signup and view all the answers

Study Notes

Cell Biology - Unit 2: How Cells Are Studied - I

Compound Microscope:
- Most common microscope in use today.
- Contains multiple lenses to magnify specimen images.

Optical Pathway in a Modern Compound Optical Microscope

Ocular Lens (Eyepiece): Collects and magnifies light from the objective lens.
Reflecting Prism: Directs light from the objective lens to the ocular lens.
Objective Lens: Focuses light from the specimen onto a focal plane, creating magnification.
Specimen: The object being viewed.
Condenser Lens: Focuses light onto the specimen; doesn't magnify.
Iris Diaphragm: Adjusts the amount of light entering the condenser.
Light Source: Provides illumination for the specimen.

Magnification vs. Resolution

Magnification: The product of the objective and ocular lens magnifications.
- 100X objective lens * 10X ocular lens = 1000X total magnification
Resolution: The ability to distinguish two nearby points as separate entities.
- High resolution is more important than high magnification.

Resolution (D)

Resolution is determined by the objective lens and its ability to gather light from the specimen.
The light enters as a cone due to diffraction.
Higher resolution corresponds to a smaller value of D.
Equation for resolution limit:
- D = 0.61λ / n sin α
  - λ = wavelength of light
  - n = refractive index of the medium between the specimen and objective lens
  - α = half the angle of the cone of light

Improving Resolution

Increasing α (numerical aperture) improves resolution because:
- Increasing a increases the denominator in the resolution formula, decreasing D.
Increasing the refractive index (n) of the medium between the specimen and the objective lens improves resolution (e.g., using oil immersion increases resolution by about 50%).
Shorter wavelengths of light improve resolution: Visible light has a shorter wavelength in the blue region than in red, leading to better resolution with blue light.

Types of Light Microscopy

Brightfield: No additional contrast other than the specimen's natural contrast.
Phase-contrast: Allows visualization of transparent materials, such as living cells. Refracted light is combined with transmitted light, producing contrast variations. Requires a phase plate.
Differential Interference Contrast (DIC): Produces 3-D appearance and high contrast; good for thicker specimens; relies on interference between polarized light.

Transmission Electron Microscopy (TEM)

Principle: Uses a beam of electrons instead of light.
Magnification: Much higher than light microscopy (e.g., 100,000x).
Resolution: Much higher than light microscopy (much smaller value for D).
Specimen Preparation: Specimen must be extremely thin (50-100 nm). Cut with an ultramicrotome.
Electron Optics: Uses electromagnetic coils to focus the electron beam, not glass lenses.
- Cathode emits electrons, accelerated by a voltage difference toward an anode.
Optical Path in TEM:
- Electromagnetic condenser lens focuses electrons onto specimen.
- Electromagnetic objective lens magnifies image in focal plane.
- Electromagnetic projector lens magnifies image again, projecting onto detector.
Resolution Limit: D= 0.2nm for TEM, compared to ~200nm for light microscopy.

Fluorescence Microscopy

Principle: Fluorescent molecules absorb light at one wavelength (excitation) and emit light at a longer wavelength (emission).
Filtering: Observing specimen by illuminating it with excitation wavelength and viewing with filter that only allows emitted wavelength (i.e., the fluorescence).
Optical path in fluorescence microscopy:
- light source --> first barrier filter -->specimen --> objective lens --> beam-splitting mirror --> second barrier filter --> eye/camera
Three-stage process:
- excitation: molecule absorbs energy from light source at excitation wavelength.
- energy dissipation: extra energy from absorption is released by part of molecule undergoing vibrations, and is relased as heat.
- fluorescence emission: molecule returns to ground state, emitting light at the emission wavelength.
Confocal Microscopy:
- Improves fluorescence microscope images
- Collects light only from a single point of focus within the specimen, eliminating light from out-of-focus areas thus producing sharper, 3-D images.

Immunofluorescence Microscopy & Immunogold Electron Microscopy

Immunofluorescence: Uses antibodies to target specific proteins of interest. Secondary antibodies are conjugated to fluorescent molecules for detection.
Immunogold electron microscopy: Uses electron-dense gold particles conjugated to antibodies to target specific proteins. Gold particles can be determined using electron microscopy.

Tissue Culture

Cell Isolation: Disrupting cell-cell contacts using proteases (e.g., trypsin), EDTA, or collagenase.
Culturing: Plating cells in a culture dish and allowing them to adhere and multiply. Differentiating adherent cells and non-adherent cells.
Cell sorting: Fluorescence-activated cell sorting (FACS) used to separate different cell types.

Other relevant concepts/ideas

Cells, organelles, molecules, units of measurement (e.g., microns, nanometers) are scaled appropriately to emphasize the different resolution levels of microscopy.
Common cell lines (e.g., HeLa, 293, CHO, MDCK) are mentioned.
Various fluorescent proteins (e.g., GFP, RFP) are discussed for visualizing different cells.
Fixation of cells/tissues for microscopy and permeabilization are highlighted.

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Description

Explore the intricacies of modern compound microscopes in this quiz on Cell Biology Unit 2. From understanding the optical pathways to differentiating magnification from resolution, this quiz will test your knowledge of how cells are studied under various lenses and magnification techniques.