Chapter 9: Microscopy

What is resolution (resolving power) in microscopy?

The minimum distance between two points that can still be seen as separate.

What is the best resolving power of a conventional light microscope?

~0.2 μm (200 nm).

What fundamentally limits light microscope resolution?

The wavelength (λ) of visible light (diffraction limit).

What is the resolving power of the human eye (approx.)?

~0.2 mm.

In a wave, what does wavelength correspond to?

Color

In a wave, what does amplitude correspond to?

Brightness (intensity).

Why are living cells hard to see in bright-field microscopy?

They are mostly transparent (low contrast).

What two steps are commonly used to see cells better in bright-field microscopy?

Fixing and staining.

What does “fixing” do to cells?

Chemically “freezes” cellular structures in place.

Why does fixation help staining/immunostaining?

It makes cells more permeable to dyes and antibodies.

What is a common fixative mentioned?

Methanol (MeOH).

What is the main advantage of phase-contrast or DIC microscopy?

Better contrast in unstained, living cells.

What does phase-contrast microscopy convert into brightness differences?

Phase differences → amplitude (brightness) differences.

What type of cells/processes are phase-contrast/DIC best for?

Watching internal structures and processes in living cells without staining.

What is fluorescence?

A molecule absorbs light at one wavelength and emits light at a longer wavelength.

Why is emitted light longer wavelength than absorbed light?

Some energy is lost before emission (so emitted photons have less energy).

What is the key advantage of fluorescence microscopy?

Only fluorescently labeled structures light up → high contrast on a dark background.

What are the two key filter types in a fluorescence microscope?

Excitation filter and emission filter.

What does the excitation filter do?

Lets through the wavelength that excites the fluorophore.

What does the emission filter do?

Lets through only the emitted (longer) wavelength to the detector/eyes.

What does a dichroic (beam-splitting) mirror do?

Reflects excitation light and transmits emitted light (separates them).

What are the approximate GFP wavelengths?

Excites ~460 nm, emits ~520 nm.

What is an antibody?

An immune protein that binds specifically to an antigen.

What is an antigen?

A target molecule (often a protein) recognized by an antibody.

What is a primary antibody?

The antibody that binds directly to the protein (antigen) of interest.

What is a secondary antibody?

An antibody that binds the constant region of the primary antibody.

Why are secondary antibodies “species-specific”?

They recognize antibodies made by a specific species (e.g., anti-rabbit IgG).

What is indirect immunofluorescence?

Primary antibody binds antigen; fluorescent secondary antibody binds the primary.

Why use a secondary antibody instead of labeling every primary antibody?

Convenience and amplification (multiple secondaries can bind one primary).

What is GFP used for in cell biology?

Tagging proteins to visualize their location in living cells.

How do you create a GFP-tagged protein?

Fuse the gene of interest to the GFP gene → express a fusion protein

Why is GFP-tagging useful compared to antibody staining?

It can track proteins in living cells (antibody staining usually requires fixation).

What is the key idea of confocal microscopy?

Produces optical sections by excluding out-of-focus light.

What kind of light source does confocal microscopy use?

A laser of a specific wavelength.

What is the purpose of confocal pinholes?

Block out-of-focus light.

What is a “Z-stack”?

A series of images taken at different focal depths through the sample.

What kind of image does confocal microscopy help create?

3D reconstructions from optical slices.

What is emphasized about detection in confocal?

Uses a detector (not eyepieces).

What is deconvolution microscopy?

A computational method that improves fluorescence images by removing out-of-focus blur.

What does deconvolution microscopy require you to collect?

A Z-stack.

What’s the big difference: confocal vs deconvolution?

Confocal blocks out-of-focus light optically; deconvolution removes it computationally.

What is the source of illumination in electron microscopy (EM)?

An electron beam.

Why does EM have much better resolution than light microscopy?

Electrons have a much shorter wavelength than visible light.

Can EM be used to view living cells?

No (samples must be fixed; prep is harsh and vacuum is required).

What is a major disadvantage of EM?

Sample preparation is laborious and cells cannot be alive.

What focuses the electron beam in EM?

Magnetic coils (electromagnetic lenses).

What is fixation in EM prep?

chemically preserved to stop biological activity, prevent decay, and stabilize cellular structures as close to their living state as possible for imaging.

Why are heavy metals used in EM staining?

They absorb electrons, creating contrast.

Name a heavy metal stain that binds membranes.

Osmium tetroxide.

Why must samples be thin-sectioned for TEM?

Electrons must pass through the specimen.

What are TEM sections mounted on?

Electrons transmitted through the sample that reach the detector/screen.

You want to watch organelles moving in a living cell without staining. Best microscope?

Phase-contrast or DIC.

You want to locate a specific protein in fixed cells using antibodies. Best microscope?

Fluorescence microscopy (immunofluorescence).

You want to track a protein’s location in living cells over time. Best approach?

GFP-tagging + fluorescence microscopy.

You want crisp 3D optical slices of a thick sample. Best microscope?

Confocal microscopy (Z-stacks + optical sections).

You have a fluorescence Z-stack but it’s blurry; you want to computationally sharpen it. Best method?

Deconvolution microscopy.