What is fluorescence microscopy?
A technique that uses fluorophores to emit light after excitation, enabling visualization of specific structures.
What is the Stokes shift?
The difference between excitation and emission peak wavelengths in fluorescence.
What are two common fluorescence labeling strategies?
Direct labeling (primary antibody with fluorophore) and indirect labeling (secondary antibody with fluorophore).
What is a fluorescence microscope?
A microscope that uses excitation light and detects emitted fluorescence, often via epi-illumination.
What is epi-fluorescence?
Excitation and emission light pass through the same objective lens (epi = same).
What is confocal microscopy?
Advanced microscopy using a pinhole to eliminate out-of-focus light, improving optical sectioning.
What is magnification in optical microscopy?
The ratio of image size to object size (v/u).
What is the eyepiece used for?
Magnifies the intermediate image, typically 5x–15x.
What does the condenser do?
Focuses and aligns light onto the specimen.
What is an objective lens?
The primary imaging lens that determines resolution, magnification, and numerical aperture.
What is numerical aperture (NA)?
A measure of an objective’s ability to gather light: NA = n × sin(µ).
How does NA affect resolution?
Higher NA improves resolution; resolution R = λ/(2 × NA).
What is refractive index?
A number describing how much light bends when entering a medium.
Why use immersion objectives?
Oil, glycerol, or water immersion increases refractive index → improves NA and resolution.
What is resolution in microscopy?
The smallest distance at which two points can be distinguished as separate.
What overcame the Abbe diffraction limit?
Super‑resolution microscopy techniques (e.g., STED, PALM, STORM).
What are digital images made of?
Pixels—each pixel represents a numerical value related to detected light.
What is an 8‑bit image?
An image with 256 possible pixel values (0–255).
What is a 16‑bit image?
An image with 65,536 possible pixel values.
How are colors represented in RGB images?
Three 8‑bit integer values representing red, green, and blue intensities.
Why are RGB images poor for quantitative analysis?
RGB mixes signals; multichannel images are better for separate measurement of markers.
What steps are required before image analysis?
Image acquisition → image processing → image analysis.
What is image filtering used for?
To clean noise and improve signal‑to‑noise ratio (SNR).
What is thresholding?
Dividing an image into foreground and background based on pixel intensity.
