CB LECTURE 2 — Microscopy Techniques in Cell Biology

What is the resolution limit of the unaided eye?

Approximately 0.2 mm.

What is the resolution of a conventional light microscope?

Approximately 200 nm.

What is the resolution of a super-resolution fluorescence microscope?

Approximately 20 nm.

What is the resolution of an electron microscope?

Approximately 0.2 nm.

Put the imaging techniques in order from lowest to highest resolving power.

Unaided eye → conventional light microscope → super-resolution fluorescence microscope → electron microscope.

Why are microscopes necessary for studying cells?

Most cells and cellular structures are below the resolution limit of the unaided eye.

What is the basic light path through a conventional light microscope?

Light source → condenser → specimen → objective lenses → ocular lenses or camera.

What does the condenser do?

It focuses light onto the specimen.

What do objective lenses do?

They collect light from the specimen and help form the image.

What can a conventional light microscope visualize?

Structures such as nuclei

What is fluorescence?

A molecule absorbs a high-energy photon and emits a lower-energy photon.

What happens to an electron during fluorescence?

It absorbs energy and moves to a higher-energy state, then returns toward a lower-energy state while releasing energy as light.

What is the Stokes shift?

The difference in wavelength between the light absorbed by a molecule and the light it emits.

What are the two important filter sets in fluorescence microscopy?

Excitation filters and emission filters.

What does an excitation filter do?

It allows the wavelengths needed to excite the fluorescent dye to reach the specimen.

What does an emission filter do?

It allows the wavelengths emitted by the fluorescent dye to reach the detector.

What are the three ways fluorescent probes can specifically target cellular structures?

• Binding to specific molecules – fluorescent probes can attach directly to certain proteins or structures.

• Using antibodies – fluorescently labeled antibodies bind to a specific target protein or molecule.

• Genetically tagging proteins – cells can be engineered to produce a protein fused to a fluorescent protein, like GFP.

What is an example of chemical fluorescent labeling?

A fluorescent dye that binds with high affinity to DNA.

What is an example of immunological fluorescent labeling?

A fluorescently tagged antibody that binds specifically to tubulin.

What is genetically encoded targeting?

Using a fluorescent protein encoded by a gene to label a protein of interest.

What does GFP stand for?

Green fluorescent protein.

Where was GFP discovered?

The jellyfish Aequorea victoria.

What happens in the jellyfish's photophore?

Aequorin releases blue light after binding calcium, and GFP absorbs that blue light and emits green light.

Why did fluorescent proteins revolutionize cell biology?

They made it possible to visualize cellular structures and protein dynamics in living cells.

What is YFP?

Yellow fluorescent protein.

What is the basic structure of fluorescent proteins such as YFP?

A beta-barrel containing a chromophore.

Which amino acids form the YFP chromophore described in lecture?

Threonine 65

How can GFP be used to track a protein?

The GFP gene can be fused to the gene encoding the protein of interest, producing a GFP-tagged fusion protein.

What can GFP-tagged proteins allow scientists to observe?

The location and movement of proteins inside living cells.

Who received the 2008 Nobel Prize in Chemistry for GFP-related work?

Osamu Shimomura

What was Osamu Shimomura's contribution to GFP?

He was awarded for isolating the GFP protein from jellyfish.

What was Martin Chalfie's contribution to GFP?

He was awarded for first expressing GFP in a non-jellyfish cell, E. coli.

What was Roger Tsien's contribution to GFP?

He was awarded for developing a library of fluorescent proteins with different colors.

Who was Douglas Prasher?

The scientist who first recognized GFP's potential as a tracer molecule and cloned the GFP gene in 1987.

Why was Douglas Prasher's contribution important?

He proposed using GFP to report the localization of proteins in living cells.

What does FRAP stand for?

Fluorescence Recovery After Photobleaching.

What is FRAP used to measure?

Protein diffusion or exchange rates within a cellular region.

What are the basic steps of FRAP?

Fluorescently label the protein → select a region → use a strong laser to bleach the region → measure fluorescence over time → determine how quickly fluorescence returns.

Why does fluorescence recover after photobleaching?

Unbleached fluorescent molecules move into the bleached region.

What does rapid FRAP recovery indicate?

The protein is highly mobile and/or exchanges rapidly.

What does little or no FRAP recovery indicate?

The protein is relatively immobile or anchored.

What does a steeper FRAP recovery curve indicate?

A faster diffusion rate.

What does TEM stand for?

Transmission Electron Microscopy.

How does TEM create an image?

Electrons pass through the specimen and are focused to form an image.

Why must TEM samples be very thin?

Electrons have poor penetration power and must pass through the specimen.

What is TEM primarily used to visualize?

Internal cellular structures.

What structures can TEM reveal?

Fine details of structures such as the nucleus

What does SEM stand for?

Scanning Electron Microscopy.

How does SEM create an image?

It uses electrons reflected by the specimen.

What is SEM primarily used to visualize?

Surface structures.

Why can SEM handle larger or thicker samples than TEM?

It uses reflected electrons rather than requiring electrons to pass through the specimen.

Why do SEM images often have a 3D appearance?

The angle of the specimen's surface relative to the detector affects the reflected electrons and creates a three-dimensional appearance.

What is the main difference between TEM and SEM?

TEM uses electrons that pass through the specimen and is mainly used for internal structures