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AUBF > M2: Microscopic Examination (Part 3: Microscopy) > Flashcards

M2: Microscopic Examination (Part 3: Microscopy) Flashcards

1
Q

is the most common type of microscopy performed in the urinalysis laboratory.

A

Bright-field microscopy

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2
Q

The type of microscopy used depends on what 3 factors

A
  1. specimen type
  2. refractive index of the object
  3. ability to image unstained living cells.
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3
Q

Identify what system the following parts of microscope belong to:

oculars, objectives (coarse & fine adjustment
knobs)

A

Lens system

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4
Q

Identify what system the following parts of microscope belong to:

light source, condenser, field diaphragm

A

Illumination system

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5
Q

Identify what system the following parts of microscope belong to:

base, body tube, and nosepiece

A

Body

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6
Q

Identify what system of the microscope:

holds the slide on place

A

Mechanical stage

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7
Q

EYEPIECE

Clinical laboratory microscopes are?

A

binocular

allowing the examination to be performed using both eyes to provide
more complete visualization

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8
Q

EYEPIECE

can be rotated to compensate for variations in vision between the operators’ eyes

A

diopter adjustment knob

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9
Q

EYEPIECE

can be adjusted horizontally to adapt to differences in interpupillary distance between operators.

A

oculars

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10
Q

EYEPIECE

Laboratory microscopes normally contain oculars capable of increasing the magnification to how many times.

A

10 times (10x)

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11
Q

EYEPIECE

T or F

The field of view varies with the field number engraved
on the eyepiece and the magnification of the objective

A

T

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12
Q

EYEPIECE

T or F

The higher the magnification, the higher the field of view
will be

A

F (The higher the magnification, the smaller the field of view
will be)

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13
Q

EYEPIECE

Formula for field of view?

A

Field no. ( diameter in mm) / M (magnification of objective)

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14
Q
  • are adjusted to be near the specimen and perform
    the initial magnification of the object on the mechanical
    stage
  • image then passes to the oculars for further
    resolution (ability to visualize fine details)
A

Objectives

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15
Q

2 features of objective lenses?

A
  1. Parcentered
  2. Parfocal
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16
Q

OBJECTIVES

ability to retain the central FOV ( (when the
user switches from one objective to another)

2 features of objective lenses

A

Parcentered

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17
Q

OBJECTIVES

ability of the objective to remain in focus regardless of the objective used

2 features of objective lenses

A

Parfocal

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18
Q

OBJECTIVES

is the ability of the lens to distinguish two small
objects that are a specific distance apart

A

Resolution

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19
Q

OBJECTIVES

  • is best when the distance between the two objects is small
  • dependent on the wavelength of light and the numerical aperture of the lens
A

Resolving power

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20
Q

OBJECTIVES

T or F

The shorter the wavelength of light, the greater the resolving power of the microscope will be.

A

T

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21
Q

OBJECTIVES

Routinely used objectives in the clinical laboratory and their magnification

A

10× (low power, dry), 40× (high power, dry),
and 100× (oil immersion)

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22
Q

OBJECTIVES

objectives used for examination of urine
sediment

A

10× and 40×

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23
Q

OBJECTIVES

The distance between the slide and the objective is controlled by the?

A

coarse- and fine-focusing knobs

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24
Q

OBJECTIVES

  • Initial focusing is performed using this
  • moves the mechanical stage noticeably up and down until the object comes into view
A

coarse knob

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25
Q

OBJECTIVES

sharpen the image

A

Fine-focusing knob

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26
Q

OBJECTIVES

T or F

When using a parfocal microscope, the coarse and fine knob should be used for adjustment when changing
magnifications

A

F ( only the fine knob should be used for adjustment when changing magnifications)

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27
Q

OBJECTIVES

  • is the distance between the objective & the coverslip on the slide
  • decreases as magnification of the objective increases
A

Working distance

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28
Q

OBJECTIVES

T or F

Working distance increases as magnification of the objective increases

A

F (Decreases as magnification of the objective increases)

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29
Q

OBJECTIVES

Identify what objective:
Magnification - 4x
Color- red

A

Scanner

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30
Q

OBJECTIVES

Identify what objective:
Magnification - 10x
Color- yellow

A

LPO

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31
Q

OBJECTIVES

Identify what objective:
Magnification - 40x
Color- Blue

A

HPO

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32
Q

OBJECTIVES

Identify what objective:
Magnification - 100x
Color- White

A

OIO

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33
Q

ILLUMINATION

light source located in the base of the microscope

A

Illuminator

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34
Q

ILLUMINATION

Equipped in light source that regulate the intensity of the light

A

rheostat

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35
Q

ILLUMINATION

may also be placed on the light source to vary the illumination and wavelengths of the emitted
light

A

Filters

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36
Q

ILLUMINATION

contained in the light source controls the diameter
of the light beam reaching the slide and is adjusted for optimal illumination

A

field diaphragm

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37
Q

ILLUMINATION

located below the stage then focuses the light on the specimen and controls the light for uniform illumination

A

condenser

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38
Q

ILLUMINATION

this specific component in the condenser controls the amount of light and the angle of light rays that pass to the specimen and lens, which affects resolution, contrast, and depth of the field of image

A

aperture diaphragm

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39
Q

ILLUMINATION

moves the condenser up and down to focus light on the object

A

condenser adjustment (focus) knob

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40
Q

ILLUMINATION

Maximum resolution is achieved by adjusting aperture diaphragm to what percent?

A

75% of the
numerical aperture of the objective

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41
Q

ILLUMINATION

T or F
The aperture diaphragm can be used to reduce light intensity because it increases resolution

A

F ( The aperture diaphragm should
not be used to reduce light intensity because it decreases resolution)

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42
Q

ILLUMINATION

What is used to reduce light but retain resolution

A

microscope lamp rheostat

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43
Q

CENTERING THE CONDENSER AND KÖHLER

ILLUMINATION

Two adjustments to the condenser that provides optimal viewing of the illuminated field

A

centering and Köhler
illumination

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44
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

These (2) must be adjusted each time the microscope is used and each time the objective is changed.

A

condenser and field diaphragms

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45
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

Familiarize the steps in centering the condenser and kohler illumination

A
  1. Place a slide on the stage and focus the object using the low-power objective with the condenser raised
  2. Close the field diaphragm
  3. Lower the condenser until the edges of the field diaphragm are sharply focused
  4. Center the image of the field diaphragm with the condenser centering screws
  5. Open the field diaphragm until its image is at the edge of the field
  6. Adjust the aperture diaphragm until approximately 75% of the field is visible (OR Remove an eyepiece and look down through the eyepiece tube; OR replace eyepiece)
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46
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

T or F

The microscope should always be covered when not in use to protect it from dust

A

T

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47
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

If any optical surface becomes coated with dust, it should be carefully removed with a?

A

camel-hair brush

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48
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

Optical surfaces should be cleaned with ?

A

lens paper

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49
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

Clean any contaminated lens immediately with a?

A

commercial lens cleaner

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50
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

T or F

An oil immersion lens must be wiped free of oil and cleaned after each use

A

T

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51
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

T or F

Light sources are replaced as necessary

A

T

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52
Q

CENTERING THE CONDENSER AND KÖHLER ILLUMINATION

T or F

A monthly professional cleaning for the microscope is recommended

A

F (annual professional cleaning)

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53
Q

TYPES OF MICROSCOPY

  • Most frequently used in the clinical laboratory routine urinalysis
  • Objects appear dark against a light background
  • light source emitting light in the visible wavelength range
A

Bright-Field Microscopy

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54
Q

TYPES OF MICROSCOPY

T or F
Use of bright-field microscopy for the examination of urine sediment can present problems when the amount of light reaching the specimen is not properly controlled

A

T

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55
Q

T or F

Sediment constituents with a low refractive index will
be observed properly when subjected to light of high intensity

A

F (Sediment constituents with a low refractive index will be overlooked when subjected to light of high intensity)

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56
Q

Subdued light is needed to see the more translucent
formed elements of the urine such as?

A

hyaline casts, crystals and mucus threads

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57
Q

T or F

In bright field microscope, light is controlled by adjusting the rheostat on the light source, not by lowering the condenser

A

T

58
Q

Bright field microscope point of reference?

A

Epithelial cell

59
Q

T or F

Staining of the sediment blurs the visualization of these elements when using bright-field microscopy

A

F (Staining of the sediment also increases the visualization of these elements when using bright-field microscopy)

60
Q

T or F

One should avoid focusing on artifacts and should not examine objects in wrong plane

A

T

61
Q
  • light that does not pass through the specimen is shifted one quarter of a wavelength and compared with the phase difference of the specimen
  • detection of more translucent or low-refractile formed elements and living cells (hyaline casts, mucous threads, mixed cellular casts and Trichomonas)
  • Hardens the outlines of even the most transparent formed elements
A

Phase-Contrast Microscopy

62
Q

Phase-Contrast Microscopy

2 components of phase-contrast microscopy which enhances contrast and improve the visibility and definition of structures having a refractive index like that of the urine

A

phase-contrast objective
lens and a matching condenser

63
Q

Phase-Contrast Microscopy

appear as “targets” are placed in the
condenser and the objective

A

Two phase rings

64
Q

Phase-Contrast Microscopy

is placed in the condenser or below it, permitting light to only pass through the central clear circular area

A

One phase ring

65
Q

Phase-Contrast Microscopy

with a central circular area that retards the light by one
quarter wavelength is placed in the objective

A

second phase-shifting ring

66
Q
A
67
Q

Phase-Contrast Microscopy

What are the two phase rings used?

A

Phase objective ring (Phase plate or dark annulus), Condenser ring (light annulus)

68
Q

Phase-Contrast Microscopy

Phase plate or dark annulus

A

Phase objective ring

69
Q

Phase-Contrast Microscopy

light annulus

A

Condenser ring

70
Q

Phase-Contrast Microscopy

Light passes to the specimen through the clear circle in the phase ring in the condenser, forming what?

A

halo of light around
the specimen

71
Q

Phase-Contrast Microscopy

when light rays pass through an object, they are slowed compared to the light passing through the air (media) so the intensity of light is decreased producing a contrast

A

Phase difference

72
Q

Phase-Contrast Microscopy

T or F

Phase difference is when light rays pass through an object, they are faster compared to the light passing through the air (media) so the intensity of light is increased producing a contrast

A

F (when light rays pass through an object, they are slowed compared to the light passing through the air (media) so the intensity of light is decreased producing a contrast)

73
Q

Phase-Contrast Microscopy

when the light that does not pass through the specimen is shifted one quarter of a wavelength and compared with the phase difference of the specimen

A

Best contrast

74
Q

Phase-Contrast Microscopy

T or F

Best contrast is when the light that does not pass through the specimen is shifted one quarter of a wavelength and compared with the phase difference of the specimen

A

T

75
Q

Phase-Contrast Microscopy

T or F

Phase rings must be different

A

F (Phase rings must match)

it is important to check that the objective and condenser mode are the same

76
Q

Phase-Contrast Microscopy

T or F

The diameter of the rings varies with the magnification

A

T

77
Q

Phase-Contrast Microscopy

The image has the best contrast when the background is ?

A

darkest

78
Q

Phase-Contrast Microscopy

must be adjusted to have maximum
contrast and make them concentric

A

Phase-contrast rings

79
Q

Phase-Contrast Microscopy

enters the central circle of the phase-shifting ring, and all other light is moved one quarter of a wavelength out of phase

A

diffracted light

80
Q
  • aids in the identification of crystals and lipids
A

Polarizing Microscopy

81
Q

Polarizing Microscopy

These (2) substances have the ability to rotate the path of the unidirectional polarized light beam to produce
characteristic colors in crystals and Maltese cross
formation in lipids

A

Crystals and Lipids

82
Q

Polarizing Microscopy

Bidirectional polarized light

a. polarizing microscopy
b. phase-contrast microscopy
c. both
d. neither

A

d. neither

polarizing microscopy rotates path of unidirectional polarized light beam

83
Q

Polarizing Microscopy

identify what substance:
produce characteristic colors

A

crystals

84
Q

Polarizing Microscopy

identify what substance:
Maltese cross formation

A

lipids

85
Q

Polarizing Microscopy

What specific fat substance produces maltese cross formation

A

Cholesterol (droplets)

86
Q

Polarizing Microscopy

  • Circles divided into 4 quadrants by a bright Maltese style cross against a black background
  • Differentiates crystals and fibers from cellular or protein cast materials
  • seen under polarized light microscopy
    are birefringent
A

Cholesterol (droplets)

87
Q

Polarizing Microscopy

These granules also produce Maltese-cross pattern

A

Starch granules

88
Q

Polarizing Microscopy

Maltese corss formation of triglycerides are seen or not seen?

A

not seen

89
Q

Polarizing Microscopy

A property of fat which indicates hat the element can refract light in two dimensions at 90 degrees to each
other.

A

birefringent

90
Q

Polarizing Microscopy

Light source in polarizing microscopy that produces light
rays of many different waves. Each wave has a distinct
direction and a vibration perpendicular to its direction

A

halogen quartz lamp

91
Q

Polarizing Microscopy

Aside from halogen quartz lamp, what are the other (2) components in polarizing microscopy?

A

polarizer and an analyzer

92
Q

Polarizing Microscopy

Where is the analyzer located

A

between the objectives and the ocular

OO

93
Q

Polarizing Microscopy

where is the polarizing filter located?

A

condenser filter holder

94
Q

Polarizing Microscopy

T or F

Polarized light vibrates in the same plane or direction

A

T

95
Q

Polarizing Microscopy

T or F

Polarizing microscopy is when light passes through a birefringent substance, it splits into two beams, one beam rotated 90 degrees to the other

A

T

96
Q

Polarizing Microscopy

A substance that rotates the plane of polarized light 90
degrees in a clockwise direction is said to have? (positive or negative birefringence)

A

positive birefringence

97
Q

Polarizing Microscopy

a substance that rotates the plane in a
counterclockwise direction has? (positive or negative birefringence)?

A

negative birefringence

No light will reach the analyzer filter; object appears black.

98
Q

Polarizing Microscopy

T or F

Polarized light is obtained by using two polarizing filters

A

T

99
Q

Polarizing Microscopy

T or F

The light emerging from one filter vibrates in one plane, and a second filter placed at a 90-degree angle allows all light to pass through

A

F (light emerging from one filter vibrates in one plane, and a second filter placed at a 90-degree angle blocks all incoming light, except that rotated by the birefringent substance)

100
Q

Polarizing Microscopy

filters are in opposite directions

A

crossed configuration

101
Q

Polarizing Microscopy

An additional filter can be added which divides the
light entering the microscope into slow and fast vibrations

A

red compensated polarizing filter

102
Q

Polarizing Microscopy

These sediments can be more easily identified by aligning them with the slow vibration and observing the blue or yellow color they produce

A

Crystals

103
Q

Polarizing Microscopy

properties of a material are the same in all
directions

A

Isotropic

104
Q

Polarizing Microscopy

when the properties of a material vary with
different orientation

A

Anisotropic

105
Q

Polarizing Microscopy

T or F
Polarizing microscopy is used in urinalysis to confirm the
identification of fat droplets, oval fat bodies, and fatty
casts that produce a characteristic Maltese cross pattern

A

T

106
Q

Polarizing Microscopy

Identify if seen or not seen in polarizing microscopy:
CaOx, Fibers, Amorphous crystals, Cholesterol,
Starch granules, Uric acid

A

Seen

107
Q

Polarizing Microscopy

Identify if seen or not seen in polarizing microscopy:
Cells (RBC, WBC), Casts, Bacteria, Triglycerides

A

not seen

108
Q

Polarizing Microscopy

  • Provides a three-dimensional image showing very fine structural detail by splitting the light ray so that the beams pass through different areas of the specimen
  • layer-by-layer imaging of a specimen and
    enhanced detail for specimens with either a low or high
    refractive index.
A

Interference-Contrast Microscopy

109
Q
  • object appears bright against a dark background but without the diffraction halo associated with phase contrast microscopy
  • More extensive modifications to the bright-field microscope are required to perform this technique, not routinely used in the urinalysis laboratory
A

Interference-Contrast Microscopy

110
Q

Interference-Contrast Microscopy

Two types of interference-contrast miscroscopy?

A
  1. Modulation contrast (Hoffman)
  2. Differential-interference contrast (Nomarski)
111
Q

Interference-Contrast Microscopy

Uses split aperture, polarizer, filter

Two types of interference-contrast miscroscopy

A

Modulation contrast (Hoffman)

112
Q

Interference-Contrast Microscopy

Familiarize concept of Modulation contrast (hoffman)

Two types of interference-contrast miscroscopy

A
  1. Split aperture is placed below the condenser
  2. Polarizer is placed below the split aperture
  3. Amplitude filter is placed in back of each objective.
113
Q

Interference-Contrast Microscopy

three zones of light
transmission of Modulation contrast (Hoffman)

Two types of interference-contrast miscroscopy

A

Dark zone, Gray zone, Clear zone

114
Q

Interference-Contrast Microscopy

transmits 1% of light

three zones of light transmission of Modulation contrast (Hoffman)

A

dark zone

115
Q

Interference-Contrast Microscopy

transmits 15% of light

three zones of light transmission of Modulation contrast (Hoffman)

A

gray zone

116
Q

Interference-Contrast Microscopy

transmits 100% of light

three zones of light transmission of Modulation contrast (Hoffman)

A

clear zone

117
Q

Interference-Contrast Microscopy

Uses Wollaston prism and Polarizing filter

A

Differential-interference contrast (Nomarski)

118
Q

Interference-Contrast Microscopy

Familiarize concept of Differential-interference contrast (Nomarski)

A
  1. A polarizing filter to output plane-polarized light is placed between the light source and the condenser
  2. A two-layered Nomarski-modified Wollaston prism that separates individual rays of light into ray pairs is required
  3. The lower Wollaston prism is built into the condenser of the microscope
  4. The upper prism is placed between the objective and the eyepiece and recombines the rays
  5. Above the top Wollaston prism, another polarizing filter is placed that causes wave interference to occur and produce the three-dimensional
119
Q
  • enhance visualization of specimens that cannot be seen easily viewed with a bright-field microscope
  • often used for unstained specimens, and, in particular, to identify the spirochete Treponema pallidum
  • Indirect light is reflected off the object
A

Dark-field Microscopy

120
Q

Dark-field Microscopy

The condenser of bright-field microscope is replaced with?

A

dark-field condenser that contains an opaque disk

121
Q
  • used to detect bacteria and viruses within cells and
    tissues through a technique called immunofluorescence
  • allows the visualization of naturally fluorescent substances or those that have been stained with a fluorochrome or fluorophore (fluorescent dyes) to produce an image
A

Fluorescence Microscopy

122
Q

Fluorescence Microscopy

  • is the property by which some atoms absorb light at a particular wavelength and subsequently emit light of a longer wavelength, termed fluorescence lifetime
  • Detects specific wavelengths of light emitted from
    objects
A

Fluorescence

123
Q

Fluorescence Microscopy

Fluorescent substances absorb the energy and emit a longer wavelength of light that is visualized with the use of special filters called the?

A

excitation filter and the emission filter

124
Q

Fluorescence Microscopy

(excitation filter or emission filter)

selects the excitation wavelength of
light from a light source.

A

excitation filter

125
Q

Fluorescence Microscopy

(excitation filter or emission filter)

selects a specific wavelength of
emitted light from the specimen to become visible

A

emission filter

126
Q

Fluorescence Microscopy

T or F

The filters are chosen to be different from the excitation and emission wavelengths of the fluorophore used to label the specimen

A

F (The filters are chosen to match the excitation and
emission wavelengths of the fluorophore used to label the specimen)

127
Q

Fluorescence Microscopy

reflects the excitation light to the specimen
and transmits the emitted light to the emission filter, which is collected with the objective and imaged by the detector

A

dichroic mirror

128
Q

Fluorescence Microscopy

T or F

The fluorescent substancecan be observed in the fluorescent microscope as a bright object against a dark background with high contrast when ultraviolet light source is used.

A

T

129
Q

Fluorescence Microscopy

Powerful light sources required for this

A

mercury or xenon arc lamps

130
Q
A
131
Q

Identify what type of microscope based from function and features
Function: Routine urinalysis

A

Bright-field

132
Q

Identify what type of microscope based from function and features

Function: Elements w/ low RI Transparent
Features: Phase objective ring, Condenser ring

A

Phase-contrast

133
Q

Identify what type of microscope based from function and features

Function: Anisotropic elements, Birefringent crystals, Lipids
Features: Phase objective ring, Condenser ring

A

Polarizing

134
Q

Identify what type of microscope based from function and features

Function: T. pallidum
Features: Dark-field condenser

A

Dark-field

135
Q

Identify what type of microscope based from function and features

Function: Fluorescent microorganisms
Features: Fluorescent dye, Special filters

A

Fluorescence

136
Q

Identify what type of microscope based from function and features

Function: 3D image, Layer by layer image
Features: Wollaston prism, Polarizing filter

A

Differential-interference contrast

137
Q
  • estimate of the formed elements (RBC, WBC, epithelial cells and cast) and protein content of urine specimen
  • First procedure to standardize the quantitation of formed elements in urine microscopic analysis
A

Addis count

138
Q

ADDIS COUNT

Specimen in addis count

A

12-hour urine

139
Q

ADDIS COUNT

Reference value for RBC

A

0 to 500,000

140
Q

ADDIS COUNT

Reference value for WBCs and Epithelial Cells

A

0 to 1,800,000

141
Q

ADDIS COUNT

Reference value for hyaline casts

A

0 to 5,000

AUBF (45 decks)

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