Lecture 2: Cells and Microscopy

Flashcards about Cells and Microscopy

What are the key differences and similarities between bacterial, plant, and animal cells in terms of their major organelles?

Bacterial, plant, and animal cells differ in terms of their major organelles. Understanding the similarities and differences requires reviewing basic cell organization.

What is the focus of cell biology in terms of object sizes?

Cell biology studies objects of different sizes, requiring a 'feel' for the relative sizes of cells, organelles, and molecules.

Why are microscopes necessary in cell biology?

To see objects smaller than approximately 0.2 mm, a microscope is needed due to the small size of most objects of interest in cell biology.

What was the primary goal of early microscopists?

The goal was to enlarge small living objects.

Who was Robert Hooke and what was his contribution to cell biology?

Robert Hooke was the first to describe 'cells' in cork in 1665 using a compound microscope with separate objective and eyepiece.

Who was Antonius van Leeuwenhoek and what did he discover?

Antonius van Leeuwenhoek was the first to describe protozoa (1674), sperm (1677), and bacteria (1683) using a 'simplex' microscope with a single lens at 275x magnification.

What are the three components of the cell theory?

1. All life forms are made from one or more cells. 2. The cell is the smallest form of life. 3. Every cell originates from a pre-existing cell.

Who was Walther Flemming and what did he contribute to cell biology?

Walther Flemming described chromosomes and mitosis in 1879.

How does a light microscope work, and what is its resolution?

The light microscope transmits light through the specimen, offering a resolution of 0.2 m (200 nm), about 1000x better than the unaided eye.

What are some ways to achieve higher contrast in light microscopy when observing live cells?

Phase contrast and differential interference contrast (DIC or Nomarski optics)

Besides phase contrast and DIC, what is another method used to achieve higher contrast in light microscopy?

Tissue fixing and staining

Which type of microscope provides a resolution of 0.2 nm, about 1,000,000x better than the unaided eye and 1,000x better than the light microscope?

Transmission Electron Microscope (TEM)

Which type of electron microscope has a resolution of 3-20 nm and provides a large depth of focus, allowing observation of the surface of a sample?

Scanning Electron Microscope (SEM)

What are the key properties of fluorescent molecules that make them useful for microscopy?

Fluorescent molecules preferentially absorb light of a specific wavelength (excitation light) and emit light at a longer wavelength (emission light).

What type of microscopy uses excitation and emission filters?

Fluorescence microscopy

What can be highlighted using fluorescent molecules inside the cell?

Actin filaments, microtubules, and intermediate filaments

What type of microscopy overcomes the 200 nm resolution limit, achieving a resolution limit of 20 nm for structures like microtubules?

Super-resolution fluorescence microscopy

What is an advanced form of fluorescence microscopy that uses a laser for excitation and allows for optical sectioning and 3-D reconstruction?

Confocal microscopy

What are the pros and cons of using conventional light microscopy (bright-field, phase, and DIC)?

Conventional light (bright-field, phase, and DIC) microscopy: + easiest to do, can look at live cells – limited ability to visualize structures – sample must be transparent, otherwise requires sectioning

What are the pros and cons of using Electron microscopy (TEM and SEM)?

TEM: (–) requires thin sectioning and contrasting treatment SEM: (+) large depth of field, but (–) can only see the surface – cells must be fixed (no live imaging)

What are the pros and cons of using Fluorescence (light) microscopy

• allows visualization of specific structures in cells + can do live cell imaging + confocal: sharp images, ideal for co-localization studies – signal may fade due to photobleaching

What does GFP allow for?

The Green Fluorescent Protein (GFP) allows for live imaging of fluorescence.

Who received the Nobel Prize in Chemistry for the discovery and development of GFP?

Roger Tsien, Osamu Shimomura, and Martin Chalfie received the Nobel Prize in Chemistry in 2008 for the discovery and development of GFP.

How can we use GFP for live imaging?

Express under a cell-type specific DNA regulatory element to allow visualization or marking of a particular type of cells and Express as a protein fusion to examine the localization of a particular protein within a cell.

In what two ways can GFP be utilized to research the activities of specific DNA regions?

GFP can be placed under a cell-type specific regulatory region to drive expression in a particular type of cell in a multicellular organism, or fused to a normal protein to examine that protein’s subcellular localization

Which type of microscopy, when combined with GFP fusion proteins, allows visualization of sub-cellular localization over time?

Time-lapse microscopy