HT Microscopy & Image Analysis 3 — Questions and Answers
Question 1: Which artifact in fluorescence microscopy causes the signal from a structure to appear in a different focal plane than its true location?
- Spherical aberration
- Chromatic aberration (Correct answer)
- Optical astigmatism
- Photobleaching
Correct answer: Chromatic aberration
Chromatic aberration causes different wavelengths to focus at different planes, making multicolor fluorescence images appear misregistered in Z.
Question 2: In image analysis, what does the term 'thresholding' refer to?
- Setting the maximum exposure time for image capture
- Separating objects from background based on pixel intensity values (Correct answer)
- Adjusting the white balance of the camera
- Defining the color profile for CMYK printing
Correct answer: Separating objects from background based on pixel intensity values
Thresholding converts a grayscale image to binary by classifying pixels above or below a set intensity value as foreground or background.
Question 3: A 40x objective with NA 0.65 is replaced with a 40x objective with NA 0.95. What is the primary benefit?
- Greater working distance for thick specimens
- Improved resolution and light collection (Correct answer)
- Reduced photobleaching of fluorescent dyes
- Wider field of view
Correct answer: Improved resolution and light collection
Higher NA improves resolution (d = 0.61λ/NA) and gathers more light, resulting in brighter, sharper images at the same magnification.
Question 4: What is the primary disadvantage of laser scanning confocal microscopy compared to widefield fluorescence?
- Lower spatial resolution in XY
- Inability to image thick specimens
- Slower image acquisition and increased photobleaching (Correct answer)
- Incompatibility with immunofluorescence staining
Correct answer: Slower image acquisition and increased photobleaching
Confocal microscopy scans point by point, making it slower than widefield and concentrating laser energy that accelerates photobleaching of fluorescent labels.
Question 5: In H&E photomicrography, which white balance setting best reproduces the characteristic pink-purple staining accurately?
- Auto white balance under tungsten illumination
- Calibration against a neutral gray card under the same light source (Correct answer)
- Fixed daylight (5500K) regardless of illumination
- Maximum color temperature setting
Correct answer: Calibration against a neutral gray card under the same light source
White balance should be calibrated against a neutral reference (gray card or blank slide) under the actual microscope illumination to faithfully reproduce stain colors.
Question 6: Köhler illumination in a brightfield microscope ensures that:
- The lamp filament is visible in the image plane
- The specimen is evenly illuminated with no filament image in the field (Correct answer)
- Fluorescent excitation is maximized
- The condenser is bypassed for direct illumination
Correct answer: The specimen is evenly illuminated with no filament image in the field
Köhler illumination focuses the lamp filament at the condenser aperture plane, producing uniform, glare-free illumination at the specimen without imaging the source.
Question 7: Which image file format is most appropriate for archiving uncompressed high-resolution histology images without quality loss?
- JPEG
- TIFF (Correct answer)
- PNG (8-bit)
- BMP
Correct answer: TIFF
TIFF supports lossless compression and high bit-depth images, making it the standard archival format for high-quality histological photomicrographs.
HT Microscopy & Image Analysis 3
Which artifact in fluorescence microscopy causes the signal from a structure to appear in a different focal plane than its true location?