M2: Microscopic Examination (Part 3: Microscopy) Flashcards by Geno Alinsub

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

Bright-field microscopy

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The type of microscopy used depends on what 3 factors

specimen type
refractive index of the object
ability to image unstained living cells.

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Identify what system the following parts of microscope belong to:

oculars, objectives (coarse & fine adjustment
knobs)

Lens system

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Identify what system the following parts of microscope belong to:

light source, condenser, field diaphragm

Illumination system

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Identify what system the following parts of microscope belong to:

base, body tube, and nosepiece

Body

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Identify what system of the microscope:

holds the slide on place

Mechanical stage

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EYEPIECE

Clinical laboratory microscopes are?

binocular

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

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EYEPIECE

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

diopter adjustment knob

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EYEPIECE

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

oculars

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EYEPIECE

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

10 times (10x)

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EYEPIECE

T or F

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

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EYEPIECE

T or F

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

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

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EYEPIECE

Formula for field of view?

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

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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)

Objectives

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2 features of objective lenses?

Parcentered
Parfocal

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OBJECTIVES

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

2 features of objective lenses

Parcentered

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OBJECTIVES

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

2 features of objective lenses

Parfocal

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OBJECTIVES

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

Resolution

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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

Resolving power

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OBJECTIVES

T or F

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

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OBJECTIVES

Routinely used objectives in the clinical laboratory and their magnification

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

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OBJECTIVES

objectives used for examination of urine
sediment

10× and 40×

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OBJECTIVES

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

coarse- and fine-focusing knobs

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OBJECTIVES

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

coarse knob

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# OBJECTIVES sharpen the image

Fine-focusing knob

# OBJECTIVES T or F When using a parfocal microscope, the coarse and fine knob should be used for adjustment when changing magnifications

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

# OBJECTIVES * is the distance between the objective & the coverslip on the slide * decreases as magnification of the objective increases

Working distance

# OBJECTIVES T or F Working distance increases as magnification of the objective increases

F (**Decreases** as magnification of the objective increases)

# OBJECTIVES Identify what objective: Magnification - 4x Color- red

Scanner

# OBJECTIVES Identify what objective: Magnification - 10x Color- yellow

LPO

# OBJECTIVES Identify what objective: Magnification - 40x Color- Blue

HPO

# OBJECTIVES Identify what objective: Magnification - 100x Color- White

OIO

# ILLUMINATION light source located in the base of the microscope

Illuminator

# ILLUMINATION Equipped in light source that regulate the intensity of the light

rheostat

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

Filters

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

field diaphragm

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

condenser

# 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

aperture diaphragm

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

condenser adjustment (focus) knob

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

75% of the numerical aperture of the objective

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

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

# ILLUMINATION What is used to reduce light but retain resolution

microscope lamp rheostat

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION Two adjustments to the condenser that provides optimal viewing of the illuminated field

centering and Köhler illumination

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION These (2) must be adjusted each time the microscope is used and each time the objective is changed.

condenser and field diaphragms

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION Familiarize the steps in centering the condenser and kohler illumination

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)

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION T or F The microscope should always be covered when not in use to protect it from dust

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION If any optical surface becomes coated with dust, it should be carefully removed with a?

camel-hair brush

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION Optical surfaces should be cleaned with ?

lens paper

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION Clean any contaminated lens immediately with a?

commercial lens cleaner

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION T or F An oil immersion lens must be wiped free of oil and cleaned after each use

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION T or F Light sources are replaced as necessary

# CENTERING THE CONDENSER AND KÖHLER ILLUMINATION T or F A monthly professional cleaning for the microscope is recommended

F (**annual** professional cleaning)

# 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

Bright-Field Microscopy

# 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

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

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

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

hyaline casts, crystals and mucus threads

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

Bright field microscope point of reference?

Epithelial cell

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

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

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

* 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

Phase-Contrast Microscopy

# 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

phase-contrast objective lens and a matching condenser

# Phase-Contrast Microscopy appear as “targets” are placed in the condenser and the objective

Two phase rings

# Phase-Contrast Microscopy is placed in the condenser or below it, permitting light to only pass through the central clear circular area

One phase ring

# Phase-Contrast Microscopy with a central circular area that retards the light by one quarter wavelength is placed in the objective

second phase-shifting ring

# Phase-Contrast Microscopy What are the two phase rings used?

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

# Phase-Contrast Microscopy Phase plate or dark annulus

Phase objective ring

# Phase-Contrast Microscopy light annulus

Condenser ring

# Phase-Contrast Microscopy Light passes to the specimen through the clear circle in the phase ring in the condenser, forming what?

halo of light around the specimen

# 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

Phase difference

# 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

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)

# 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

Best contrast

# 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

# Phase-Contrast Microscopy T or F Phase rings must be different

F (Phase rings must match) ## Footnote it is important to check that the objective and condenser mode are the same

# Phase-Contrast Microscopy T or F The diameter of the rings varies with the magnification

# Phase-Contrast Microscopy The image has the best contrast when the background is ?

darkest

# Phase-Contrast Microscopy must be adjusted to have maximum contrast and make them concentric

Phase-contrast rings

# 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

diffracted light

* aids in the identification of crystals and lipids

Polarizing Microscopy

# 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

Crystals and Lipids

# Polarizing Microscopy Bidirectional polarized light a. polarizing microscopy b. phase-contrast microscopy c. both d. neither

d. neither ## Footnote polarizing microscopy rotates path of unidirectional polarized light beam

# Polarizing Microscopy identify what substance: produce characteristic colors

crystals

# Polarizing Microscopy identify what substance: Maltese cross formation

lipids

# Polarizing Microscopy What specific fat substance produces maltese cross formation

Cholesterol (droplets)

# 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

Cholesterol (droplets)

# Polarizing Microscopy These granules also produce Maltese-cross pattern

Starch granules

# Polarizing Microscopy Maltese corss formation of triglycerides are seen or not seen?

not seen

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

birefringent

# 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

halogen quartz lamp

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

polarizer and an analyzer

# Polarizing Microscopy Where is the analyzer located

between the objectives and the ocular | OO

# Polarizing Microscopy where is the polarizing filter located?

condenser filter holder

# Polarizing Microscopy T or F Polarized light vibrates in the same plane or direction

# 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

# 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)

positive birefringence

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

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

# Polarizing Microscopy T or F Polarized light is obtained by using two polarizing filters

# 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

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)

# Polarizing Microscopy filters are in opposite directions

crossed configuration

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

red compensated polarizing filter

# 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

Crystals

# Polarizing Microscopy properties of a material are the same in all directions

Isotropic

# Polarizing Microscopy when the properties of a material vary with different orientation

Anisotropic

# 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

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

Seen

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

not seen

# 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.

Interference-Contrast Microscopy

* 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

Interference-Contrast Microscopy

# Interference-Contrast Microscopy Two types of interference-contrast miscroscopy?

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

# Interference-Contrast Microscopy Uses split aperture, polarizer, filter | Two types of interference-contrast miscroscopy

Modulation contrast (Hoffman)

# Interference-Contrast Microscopy Familiarize concept of Modulation contrast (hoffman) | Two types of interference-contrast miscroscopy

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.

# Interference-Contrast Microscopy three zones of light transmission of Modulation contrast (Hoffman) | Two types of interference-contrast miscroscopy

Dark zone, Gray zone, Clear zone

# Interference-Contrast Microscopy transmits 1% of light | three zones of light transmission of Modulation contrast (Hoffman)

dark zone

# Interference-Contrast Microscopy transmits 15% of light | three zones of light transmission of Modulation contrast (Hoffman)

gray zone

# Interference-Contrast Microscopy transmits 100% of light | three zones of light transmission of Modulation contrast (Hoffman)

clear zone

# Interference-Contrast Microscopy Uses Wollaston prism and Polarizing filter

Differential-interference contrast (Nomarski)

# Interference-Contrast Microscopy Familiarize concept of Differential-interference contrast (Nomarski)

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

* 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

Dark-field Microscopy

# Dark-field Microscopy The condenser of bright-field microscope is replaced with?

dark-field condenser that contains an opaque disk

* 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

Fluorescence Microscopy

# 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

Fluorescence

# 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?

excitation filter and the emission filter

# Fluorescence Microscopy (excitation filter or emission filter) selects the excitation wavelength of light from a light source.

excitation filter

# Fluorescence Microscopy (excitation filter or emission filter) selects a specific wavelength of emitted light from the specimen to become visible

emission filter

# 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

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

# 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

dichroic mirror

# 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.

# Fluorescence Microscopy Powerful light sources required for this

mercury or xenon arc lamps

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

Bright-field

Identify what type of microscope based from function and features Function: Elements w/ low RI Transparent Features: Phase objective ring, Condenser ring

Phase-contrast

Identify what type of microscope based from function and features Function: Anisotropic elements, Birefringent crystals, Lipids Features: Phase objective ring, Condenser ring

Polarizing

Identify what type of microscope based from function and features Function: T. pallidum Features: Dark-field condenser

Dark-field

Identify what type of microscope based from function and features Function: Fluorescent microorganisms Features: Fluorescent dye, Special filters

Fluorescence

Identify what type of microscope based from function and features Function: 3D image, Layer by layer image Features: Wollaston prism, Polarizing filter

Differential-interference contrast

* 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

Addis count

# ADDIS COUNT Specimen in addis count

12-hour urine

# ADDIS COUNT Reference value for RBC

0 to 500,000

# ADDIS COUNT Reference value for WBCs and Epithelial Cells

0 to 1,800,000

# ADDIS COUNT Reference value for hyaline casts

0 to 5,000