Microscopy Study Guide - Practice Flashcards

Flashcards covering key concepts from the Microscopy study guide.

What is the approximate resolution range for light microscopy, super-resolution microscopy, and electron microscopy?

Light microscopy ~200 nm; super-resolution ~20–100 nm; electron microscopy ~0.1–1 nm.

Why is contrast important in microscopy, especially for examining cells?

Because most cells are largely transparent; contrast (via staining, phase contrast, etc.) highlights structures and makes them visible.

What do stains accomplish in microscopy?

Stains increase image contrast by selectively binding to cellular components, making specific structures visible.

Which type of light microscopy is particularly useful for observing living cells?

Phase-contrast (and differential interference contrast, DIC) microscopy, which allows visualization of living cells with minimal disruption.

What is the basic principle behind fluorescence?

A fluorophore absorbs light at a short wavelength and emits light at a longer wavelength, producing fluorescence.

How do immunofluorescence experiments allow you to determine the distribution of two proteins within the same cell?

Label two proteins with distinct fluorophores (e.g., different antibodies or labeled secondary antibodies) and image in separate channels to assess distribution and co-localization.

What is fluorescent protein technology and what capability does it provide beyond traditional immunofluorescence?

Genetically encoded fluorescent proteins (e.g., GFP) fused to proteins of interest enable live-cell imaging and tracking of dynamics, not possible with fixed immunofluorescence.

Why do images from fluorescent microscopes often appear fuzzy when imaging thick sections?

Fluorescence from out-of-focus planes adds blur; thicker sections reduce z-resolution and blur signals.

How is GFP used to track the behavior of specific proteins in living cells?

GFP is genetically fused to the protein of interest, allowing real-time visualization of its localization, movement, and dynamics in living cells.

Name two methods to remove out-of-focus light from images of thick samples and briefly describe how they work.

Confocal laser scanning microscopy uses a pinhole to reject out-of-focus light; two-photon (multiphoton) excitation confines excitation to the focal volume, reducing out-of-focus emission.

Why does a 1 nm diameter fluorescent molecule appear about 200 nm in diameter under a light microscope?

Because of the diffraction limit and the point-spread function of light, features smaller than ~200 nm cannot be resolved and appear blurred as larger spots.

What is the Raleigh criterion or Abbe equation, and how does the wavelength of light affect ultimate resolution?

Resolution d ≈ λ/(2NA) (Abbe). Shorter wavelengths and higher numerical aperture yield better (smaller) resolution; visible-light limits are about ~200 nm.

How does PALM microscopy work and why is Photoactivatable GFP used in the examples?

PALM localizes individual photoactivated fluorophores sequentially to reconstruct a high-resolution image; PA-GFP is used because it can be switched on with light, enabling sparse activation for precise localization.