HT Microscopy & Image Analysis 3

Question 1

Which artifact in fluorescence microscopy causes the signal from a structure to appear in a different focal plane than its true location?

Accepted Answer

Chromatic aberration

Answer

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?

Accepted Answer

Separating objects from background based on pixel intensity values

Answer

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?

Accepted Answer

Improved resolution and light collection

Answer

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?

Accepted Answer

Slower image acquisition and increased photobleaching

Answer

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?

Accepted Answer

Calibration against a neutral gray card under the same light source

Answer

White balance should be calibrated against a neutral reference (gray card or blank slide) under the actual microscope illumination to faithfully reproduce stain colors.

(HT) Histotechnician Certification Practice Test

(HT) Histotechnician Certification Practice Test

HT Microscopy & Image Analysis 3