Microscopy Techniques and Cell Structure: Light, Electron, and Cryo EM

What is the relationship between meters and millimeters?

1 meter (m) = 1000 millimeters (mm)

How many micrometers are in a millimeter?

1 millimeter (mm) = 1000 micrometers (µm)

What is the size range of eukaryotic cells?

Typically 10-100 µm, with an average size of 20 µm.

What is the size range of prokaryotic cells?

Typically 1-10 µm.

What is the size of viruses?

About 100 nm.

What is the significance of 1 nm in microscopy?

1 nm is the size of small molecules such as glucose.

What type of microscope uses visible light to magnify images?

Light microscope (LM)

What is the main advantage of light microscopes?

They are simple, inexpensive, and can examine living samples.

What is a major limitation of light microscopes?

They cannot resolve fine details of subcellular structures.

What does magnification refer to in microscopy?

The enlargement of an image under a microscope.

What is resolution in the context of microscopy?

The minimum distance two objects can be separated and still be distinguished as separate.

What is the resolution limit of a microscope determined by?

The resolving power, not the magnification.

How does the wavelength of light affect resolution?

Resolution is inversely proportional to wavelength; shorter wavelengths lead to higher resolution.

What is the primary advantage of electron microscopes (EM)?

They provide higher magnification and can see subcellular organelles.

What is a significant disadvantage of electron microscopes?

Samples cannot be living and preparation is complicated and expensive.

Who first used the term 'cell' in microscopy?

Robert Hooke in 1665.

What did Antonie van Leeuwenhoek achieve in microscopy?

He was the first to observe living cells under a light microscope in 1674.

What was proposed by Matthias Schleiden and Theodor Schwann in 1838?

The cell theory: all living things are composed of one or more cells.

What did Rudolf Virchow conclude in 1855?

Cells only arise by division of other preexisting cells.

What was a significant development in microscopy in 1931?

The invention of the electron microscope (EM).

What does cryogenic electron microscopy (cryo EM) enable?

It extends the effective resolution of electron microscopy.

What is the role of heavy metals in electron microscopy?

They are used to stain specimens to make them visible under the microscope.

What is phase contrast microscopy used for?

It is used to view unstained cells by exploiting differences in density, adding a light/dark effect.

What are the two main types of electron microscopy?

Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).

What is a key requirement for specimens in electron microscopy?

Specimens must be sliced extremely thin to allow for high magnification of cellular ultrastructure.

How can you distinguish images taken by light microscopy from those taken by TEM?

By looking at the scale bar; light microscopy uses tens of micrometers, while TEM uses 1 or 2 micrometers or lower.

What is Cryo Electron Microscopy (Cryo EM)?

A form of transmission electron microscopy that freezes samples to protect them from radiation damage.

What is the purpose of vitrification in Cryo EM?

To rapidly freeze samples in liquid ethane and nitrogen, preventing ice crystal formation that interferes with imaging.

What is tomography in the context of Cryo EM?

A technique used to combine many 2D images taken from different angles into a 3D model.

What are the advantages of Cryo EM?

It allows visualization of the inner space of cells and captures proteins in a near-native state for dynamic imaging.

What are the disadvantages of Cryo EM?

It is expensive and requires significant computing power, limiting its availability.

What recent advancements have improved Cryo EM resolution?

The development of high-quality modern electron detectors and advanced imaging processing software.

What is immunolabeling?

The use of antibodies as labels to detect specific molecules in biological experiments.

How are antibodies used in immunolabeling?

They bind to target molecules and are attached to markers for detection, such as fluorescent molecules or enzymes.

What is the difference between surface staining and intracellular staining in immunolabeling?

Surface staining can be done on living cells, while intracellular staining requires cells to be fixed and permeabilized, killing them.

What are fluorophores?

Molecules that are excited by light of one wavelength and emit light of a different wavelength, used in fluorescence microscopy.

What is fluorescence microscopy?

A type of light microscopy that visualizes the distribution of specific molecules in cells using fluorescent labels.

What is confocal microscopy?

A technique that uses a laser beam to focus on a precise point, generating sharp 2D images and reducing blurring.

What is the significance of natural fluorescent proteins like GFP?

They can be used to tag proteins in living cells, allowing visualization without the need for immunolabeling.

What is the role of recombinant DNA techniques in fluorescence microscopy?

They enable the fusion of genes for fluorescent proteins to other genes, tagging proteins for visualization in living organisms.