HT Microscopy & Image Analysis 2 — Questions and Answers
Question 1: What is the purpose of using immersion oil with a 100x objective lens?
- To stain the specimen more evenly
- To increase the refractive index and improve resolution (Correct answer)
- To reduce chromatic aberration in the eyepiece
- To cool the specimen during examination
Correct answer: To increase the refractive index and improve resolution
Immersion oil matches the refractive index of glass, reducing light refraction loss and improving resolution at high magnifications.
Question 2: Which microscopy technique uses evanescent waves to illuminate only the region immediately adjacent to the coverslip?
- Confocal microscopy
- Phase contrast microscopy
- Total internal reflection fluorescence (TIRF) microscopy (Correct answer)
- Dark-field microscopy
Correct answer: Total internal reflection fluorescence (TIRF) microscopy
TIRF microscopy excites fluorophores only within ~100-200 nm of the coverslip surface using evanescent waves, giving excellent signal-to-noise for surface imaging.
Question 3: In digital pathology, what does the term 'WSI' stand for?
- Wide-spectrum imaging
- Whole slide imaging (Correct answer)
- Wavelength-specific illumination
- White-source illumination
Correct answer: Whole slide imaging
Whole slide imaging (WSI) refers to digitizing an entire glass slide at high resolution for remote viewing and analysis.
Question 4: A histotechnician notices that cells appear artificially elongated on a photomicrograph taken at low magnification. This artifact is most likely due to:
- Overexposure of the camera sensor
- Incorrect aspect ratio or pixel mapping settings (Correct answer)
- Use of a plan-achromat objective
- Insufficient condenser aperture
Correct answer: Incorrect aspect ratio or pixel mapping settings
Incorrect aspect ratio settings or non-square pixel mapping in the imaging software can cause artificial stretching or compression of the captured image.
Question 5: What does numerical aperture (NA) determine in light microscopy?
- The total magnification of the system
- The light-gathering ability and resolving power of the objective (Correct answer)
- The working distance between objective and specimen
- The field of view diameter
Correct answer: The light-gathering ability and resolving power of the objective
Numerical aperture (NA = n·sin θ) determines both the light-gathering ability and the maximum resolution an objective can achieve.
Question 6: Which condenser type is required for optimal dark-field microscopy at high magnifications?
- Abbe condenser
- Cardioid or paraboloid dark-field condenser (Correct answer)
- Phase annulus condenser
- DIC Nomarski prism condenser
Correct answer: Cardioid or paraboloid dark-field condenser
Dark-field microscopy at high magnification requires a specialized cardioid or paraboloid condenser to block direct light and illuminate specimens obliquely.
Question 7: When calibrating a microscope's eyepiece micrometer, a stage micrometer reads 10 divisions equal to 100 µm, and the eyepiece micrometer reads 20 divisions for the same length. What is the value of each eyepiece division?
- 10 µm
- 5 µm (Correct answer)
- 20 µm
- 2 µm
Correct answer: 5 µm
100 µm ÷ 20 eyepiece divisions = 5 µm per eyepiece division, which is used for all subsequent measurements at that magnification.
HT Microscopy & Image Analysis 2
What is the purpose of using immersion oil with a 100x objective lens?