Cell Biology Unit 2: Microscopes
42 Questions
0 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to Lesson

Podcast

Play an AI-generated podcast conversation about this lesson

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?

<p>E = hc / λ (B)</p> 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?

<p>Light of the emitted wavelength only (C)</p> Signup and view all the answers

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

<p>A photon of energy is emitted. (D)</p> Signup and view all the answers

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

<p>To pass through blue light with a wavelength between 450 and 490 nm. (D)</p> Signup and view all the answers

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

<p>Tetramethylrhodamine (D)</p> Signup and view all the answers

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

<p>To reflect light above 510 nm while absorbing light below that wavelength. (B)</p> Signup and view all the answers

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

<p>Green light. (A)</p> Signup and view all the answers

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

<p>To cut out unwanted fluorescent signals (D)</p> Signup and view all the answers

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

<p>It eliminates depth-related blur by focusing on a single point of light (D)</p> Signup and view all the answers

What role does the dichroic mirror play in confocal microscopy?

<p>It reflects only specific wavelengths of light (C)</p> Signup and view all the answers

Which of the following enhances the sensitivity of immunofluorescence microscopy?

<p>Using secondary antibodies that recognize primary antibodies (A)</p> Signup and view all the answers

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

<p>Locks proteins in place while preserving cell structure (B)</p> Signup and view all the answers

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

<p>Coherent monochromatic light (D)</p> Signup and view all the answers

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

<p>It causes a blurry image (B)</p> Signup and view all the answers

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

<p>520 to 560 nm (B)</p> Signup and view all the answers

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

<p>It autofluoresces and may interfere with fluorescence signal. (B)</p> Signup and view all the answers

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

<p>Acetone (D)</p> 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?

<p>194 nm (B)</p> Signup and view all the answers

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

<p>The primary antibodies should be from different host species. (B)</p> Signup and view all the answers

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

<p>It serves as a permeabilizing agent. (C)</p> Signup and view all the answers

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

<p>Phase-contrast microscopy (B)</p> Signup and view all the answers

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

<p>Glass lenses (B)</p> Signup and view all the answers

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

<p>DAPI (B)</p> Signup and view all the answers

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

<p>TEMs use electrons to form images. (A)</p> Signup and view all the answers

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

<p>Overlaying images acquired with different filter sets. (D)</p> Signup and view all the answers

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

<p>They should emit light at different wavelengths. (C)</p> Signup and view all the answers

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

<p>0.2 µm (C)</p> Signup and view all the answers

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

<p>Antibodies will not penetrate the cells. (C)</p> Signup and view all the answers

Which microscopy technique is best suited for observing thicker samples?

<p>Differential interference contrast (DIC) (C)</p> Signup and view all the answers

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

<p>Resolution is ultimately limited by the wavelength of light. (D)</p> Signup and view all the answers

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

<p>To detect the presence of specific proteins on cell surfaces (A)</p> Signup and view all the answers

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

<p>It is projected downwards through a vacuum. (C)</p> Signup and view all the answers

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

<p>They must be passaged to a new dish (D)</p> Signup and view all the answers

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

<p>25-40 times (D)</p> Signup and view all the answers

What is a common characteristic of transformed cell lines?

<p>They can grow indefinitely (C)</p> Signup and view all the answers

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

<p>Glucose (B)</p> Signup and view all the answers

What addition is necessary for the immortalization of primary cells?

<p>DNA that expresses telomerase (C)</p> Signup and view all the answers

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

<p>HeLa (D)</p> Signup and view all the answers

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

<p>It transports iron (C)</p> Signup and view all the answers

Flashcards

Resolution

The smallest distance two points can be apart and still be distinguished as separate entities.

Electron microscope (TEM)

A microscope that uses a beam of electrons to illuminate and magnify a sample.

Fluorescence microscopy

A type of microscopy where a fluorescent molecule absorbs light at one wavelength (excitation) and emits light at a longer wavelength (emission).

Photon energy and wavelength

The energy of a photon of light is inversely proportional to its wavelength.

Signup and view all the flashcards

Fluorescence process

A three-stage process involving excitation, emission, and fluorescence.

Signup and view all the flashcards

Fluorescence Emission

When a fluorescent molecule absorbs light energy, it enters an excited state (S1'). It then partially dissipates this energy, reaching a relaxed state (S1). Finally, the molecule emits a photon of light, returning to its ground state (S0).

Signup and view all the flashcards

Excitation Wavelength

A specific wavelength of light that causes a fluorescent molecule to emit light. For example, blue light can excite fluorescein to emit green light.

Signup and view all the flashcards

Emission Wavelength

A specific wavelength of light emitted by a fluorescent molecule after absorbing light energy. For instance, fluorescein emits green light after being excited by blue light.

Signup and view all the flashcards

Beam-Splitting Mirror

A specialized optical component in a fluorescence microscope that separates the excitation light from the emitted fluorescent light. It reflects light below a certain wavelength (e.g., blue excitation light) while transmitting light above that wavelength (e.g., green emission light).

Signup and view all the flashcards

First Barrier Filter

A filter in a fluorescence microscope that blocks all light except for a narrow band of wavelengths needed to excite the fluorescent molecules in the specimen.

Signup and view all the flashcards

Limit of Resolution

The smallest distance between two points that can be distinguished as separate entities under a microscope.

Signup and view all the flashcards

Light Microscopy

A type of microscopy that uses visible light and glass lenses to form an image.

Signup and view all the flashcards

Wavelength and Resolution

The wavelength of light used in a microscope affects its limit of resolution, meaning the smallest object it can distinguish. Shorter wavelengths provide better resolution.

Signup and view all the flashcards

Immersion Oil

Immersion oil is used in microscopy to help improve the resolution by capturing more light passing through the specimen.

Signup and view all the flashcards

Brightfield Microscopy

A type of light microscopy where the image is seen on a bright background.

Signup and view all the flashcards

Phase-contrast Microscopy

A type of light microscopy that uses a phase plate to visualize differences in refractive index, making transparent structures visible.

Signup and view all the flashcards

Differential Interference Contrast (DIC) Microscopy

A type of light microscopy that uses polarized light to highlight differences in refractive index, creating a 3D-like effect.

Signup and view all the flashcards

Transmission Electron Microscope (TEM)

A type of microscopy that uses electrons instead of light to form an image, providing much higher resolution than light microscopy.

Signup and view all the flashcards

Second barrier filter

A filter that blocks unwanted fluorescent signals, allowing only green fluorescein emission between 520 and 560 nm.

Signup and view all the flashcards

Dichroic mirror

A device that reflects light of one wavelength and transmits light of another wavelength. It directs the laser light onto the specimen and reflects the emitted fluorescent light to the detector.

Signup and view all the flashcards

Pinhole

A small opening that allows light from the focal point to pass through to the detector. It helps to eliminate out-of-focus light and improve image resolution.

Signup and view all the flashcards

Confocal microscopy

A specialized microscope that illuminates a single point of light at a specific depth in the specimen, reducing blur from other light sources.

Signup and view all the flashcards

Immunofluorescence microscopy

A method that uses fluorescent antibodies to visualize specific proteins in cells. It helps to understand the distribution and localization of proteins within the cell.

Signup and view all the flashcards

Primary antibody

An antibody that binds to an antigen, which can be a protein or another molecule. It's used in immunofluorescence microscopy.

Signup and view all the flashcards

Secondary antibody

An antibody that binds to the primary antibody to amplify the fluorescent signal. It's often conjugated with a fluorescent dye.

Signup and view all the flashcards

Fixation

A process that immobilizes proteins in their natural positions within the cell, preserving the structure and function of the cells. It uses chemicals to cross-link biomolecules.

Signup and view all the flashcards

Fluorescence-activated cell sorting (FACS)

A technique used to separate different cell types based on their surface protein expression using fluorescently labeled antibodies and electrical charges.

Signup and view all the flashcards

Passaging cells

The process of removing cells from a culture dish and transferring them to a new dish.

Signup and view all the flashcards

Primary cells

Cells that are derived from a tissue and can only be passaged a limited number of times before they stop dividing.

Signup and view all the flashcards

100% confluence

The state when cells in a culture dish have completely covered the surface.

Signup and view all the flashcards

Immortalization of cells

A process that makes primary cells immortal by artificially adding a gene that expresses telomerase, allowing them to divide indefinitely.

Signup and view all the flashcards

Transformed cell line

Cells that have the ability to grow indefinitely, often derived from cancerous tissues.

Signup and view all the flashcards

Growth media for cultured cells

A liquid medium that provides necessary nutrients and growth factors for cells to grow and thrive in a culture environment.

Signup and view all the flashcards

Serum

A non-cellular portion of clotted blood that contains essential growth factors, hormones, and other components for cell growth.

Signup and view all the flashcards

Cross-linking reagents

Chemicals like paraformaldehyde and glutaraldehyde used to fix biological samples for microscopy.

Signup and view all the flashcards

Permeabilization

The process of making cell membranes permeable to allow antibodies to enter and bind to target molecules.

Signup and view all the flashcards

Host species

The species in which an antibody is produced, for example, a rabbit antibody is produced in a rabbit.

Signup and view all the flashcards

Multi-color immunofluorescence

The process of using different fluorescent dyes or proteins to visualize multiple targets in a single cell, allowing for the simultaneous observation of different cellular structures.

Signup and view all the flashcards

Image overlay

The process of combining multiple images captured using different fluorescent dyes to create a composite image that shows the co-localization of different cellular structures.

Signup and view all the flashcards

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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Quiz Team

Related Documents

Biol 266 – Cell Biology UNIT 2 PDF

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.

More Like This

Compound Microscope: Parts and Usage
10 questions

Compound Microscope: Parts and Usage

UpbeatTheremin avatar
UpbeatTheremin
Compound Microscope: Parts and Functions
15 questions

Compound Microscope Parts and Functions Quiz and Flashcards

RiskFreeSolarSystem avatar
RiskFreeSolarSystem
Microscope Overview and Parts
10 questions

Microscope Overview and Parts

WellEstablishedMinotaur avatar
WellEstablishedMinotaur
Compound Microscopes Overview
32 questions

Compound Microscopes Overview

ProactiveDulcimer3292 avatar
ProactiveDulcimer3292
Use Quizgecko on...
Browser
Open
Browser
Browser