Histology Certification Microscopy Techniques 3 — Questions and Answers
Question 1: In fluorescence microscopy, what is the role of the dichroic mirror?
- It focuses the excitation light onto the specimen
- It reflects excitation light toward the specimen while transmitting emission light to the detector (Correct answer)
- It filters out background fluorescence
- It splits white light into its component wavelengths
Correct answer: It reflects excitation light toward the specimen while transmitting emission light to the detector
The dichroic mirror reflects shorter-wavelength excitation light onto the specimen while transmitting longer-wavelength emission light through to the detector, separating illumination from imaging pathways.
Question 2: What is the primary difference between epifluorescence and transmitted fluorescence microscopy?
- Only epifluorescence uses fluorescent dyes
- Epifluorescence illuminates through the objective, while transmitted fluorescence illuminates from below (Correct answer)
- Only transmitted fluorescence achieves super-resolution
- Epifluorescence cannot image living cells
Correct answer: Epifluorescence illuminates through the objective, while transmitted fluorescence illuminates from below
In epifluorescence microscopy, the objective serves as both the condenser and imaging lens, with excitation light passing through the objective downward onto the specimen.
Question 3: Which super-resolution technique uses targeted depletion of excited fluorophores around a focal spot to achieve sub-diffraction resolution?
- STORM
- PALM
- STED (Correct answer)
- SIM
Correct answer: STED
Stimulated Emission Depletion (STED) microscopy uses a donut-shaped depletion laser to suppress fluorescence at the periphery of the excitation spot, shrinking the effective focal volume.
Question 4: What is the purpose of antifade mounting medium in fluorescence microscopy?
- It increases fluorophore brightness
- It prevents photobleaching by scavenging reactive oxygen species (Correct answer)
- It improves the refractive index match for oil immersion
- It acts as a fixative for the cover glass
Correct answer: It prevents photobleaching by scavenging reactive oxygen species
Antifade mounting media contain antioxidants such as DABCO or ProLong Gold that quench reactive oxygen species, significantly slowing the photobleaching of fluorescent dyes.
Question 5: In differential interference contrast (DIC) microscopy, what optical element creates the two laterally sheared beams of polarized light?
- The analyzer
- The Wollaston prism (Correct answer)
- The condenser iris
- The objective back focal plane filter
Correct answer: The Wollaston prism
The Wollaston (or Nomarski) prism splits polarized light into two closely spaced, laterally sheared beams that travel through slightly different regions of the specimen.
Question 6: What does numerical aperture (NA) physically represent for a microscope objective?
- The magnification power of the objective
- The sine of the half-angle of the light cone entering the objective multiplied by the refractive index of the medium (Correct answer)
- The working distance between the objective and specimen
- The diameter of the front lens element
Correct answer: The sine of the half-angle of the light cone entering the objective multiplied by the refractive index of the medium
NA = n × sin(θ), where n is the refractive index of the medium between lens and specimen, and θ is the half-angle of the maximum cone of light collected by the objective.
Question 7: Which artifact is most commonly associated with improper use of immersion oil in high-magnification microscopy?
- Chromatic aberration
- Spherical aberration
- Air bubble formation causing uneven illumination (Correct answer)
- Increased phototoxicity
Correct answer: Air bubble formation causing uneven illumination
Air bubbles trapped in immersion oil create regions of mismatched refractive index that scatter light, producing dark or bright patches and degrading image quality.
Histology Certification Microscopy Techniques 3
In fluorescence microscopy, what is the role of the dichroic mirror?