MICROSCOPY

CM LEC 5

• lens system

• illumination system

• mechanical stage

The microscope contains the following:

• Oculars

• the objectives

• coarse and fine adjustment knob

lens system consists of:

• Light source

• condenser

• field

• iris diaphragms

illumination system consists of:

• brightfield microscopy

• phase-contrast microscopy

• polarizing microscopy

• dark-field microscopy

• fluorescence microscopy

• interference-contrast microscopy

URINALYSIS MICROSCOPY TECHNIQUES: 

brightfield microscopy

• Most common type of microscopy used in urinalysis

• Objects appear dark against a light background

• Sediments for urinalysis must be examined using decreased light controlled by adjusting the rheostat on the light source, not by lowering the condenser.

• Sediment constituents with a low refractive index will be overlooked when subjected to light of high intensity.

Phase-contrast microscopy

• Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas

• Advantage: identifying low refractive hyaline casts or mixed cellular casts, mucous threads, and Trichomonas

• Eliminates the need to fix or stain living cells

• The light rays are slowed when passing through an object compared to rays passing through air, decreasing the intensity of light and producing contrast. This is called phase difference.

• Phase difference is affected by thickness of the object, refractive index, and other light-absorbance properties

• Best contrast is obtained 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.

• Light passes to the specimen through the clear circle in the phase ring in the condenser, forming a halo of light around the specimen.

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

phase difference

The light rays are slowed when passing through an object compared to rays passing through air, decreasing the intensity of light and producing contrast.

Polarizing microscopy

• Aids in identification of cholesterol in oval fat bodies, fatty casts (Maltese cross pattern), and crystals

• Uses polarized light in the identification of crystals and lipids

• Both crystals and lipids have the ability to rotate the path of the unidirectional polarized lightbeam to produce characteristic colors in crystals and Maltese cross formation in lipids.

• Polarized light is obtained by using 2 polarizing filters.

• Light emerging from one filter vibrates in one plane, and the second filter placed at a 90 deg angle blocks all incoming light except that rotated by the birefringent substance.

Polarizing microscopy

• Normal or unpolarized light vibrates in equal intensity in all directions.

• Cross-configuration uses filters that are in opposite direction.

• Birefringent is a property indicating that the element can refract light in 2 dimensions at 90 deg angle to each other

• As the light passes through a birefringent substance, it splits into 2 beams. One beam rotated 90 degrees to the other.

Polarizing microscopy

• Halogen quartz lamp produces light rays of many different waves —> each wave has a distinct direction and a vibration perpendicular to its direction.

• The term causative birefringents is used if the substance rotates the plane of polarazing light 90 degrees in a clockwise direction.

• Negative birefringent - if the substance rotates the plane in a counter clockwise direction.

• Isotropic substances (ex.: blood cells) do not have refractive property, the light passes through unchanged.

❖RBCs

❖Casts, mucus

❖Bacteria

❖Cells, cellular debris

Do NOT polarize light:

❖Monohydrate calcium oxalate crystals

❖Fibers (clothing, diapers), plastic fragments

❖Amorphous crystals (urates: strongly; phosphates: very weakly)

❖Cholesterol globules, starch granules

DO polarize light:

Dark-field microscopy

Aids in identification of Treponema pallidum

Fluorescence microscopy

Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye

Interference-contrast microscopy

Produces a 3-dimensional microscopy image and layer-by-layer imaging of specimen

Interference-contrast microscopy

• Produces a 3-dimensional microscopy image and layer-by-layer imaging of specimen

• Provides a 3-dimensional image showing very fine structural detail by splitting the light ray so that the beams pass through different areas of the specimen

• Advantage: an object appears bright against a dark background but without the diffraction halo associated with phase-contrast microscopy

• However, Interference-Contrast Microscopy is not routinely used in Urinalysis

• modulation contrast microscope (hoffman)

• differential-interference contrast (nomarski) microscope

TYPES OF INTERFERENCE-CONTRAST MICROSCOPY

Modulation Contrast Microscope (Hoffman)

• Split aperture is placed below the condenser and an amplitude filter is placed below the split aperture.

• An amplitude filter is placed on the back of each objective.

• dark zone

• gray zone

• clear zone

Modulation contrast microscope has 3 zones of light transmission:

Dark zone

Transmits 1% of light

Gray zone

Transmits 15% of light

Clear zone

Transmits 100% of light

Differential-Interference Contrast (Nomarski) Microscope

• Uses prisms

• A polarizing filter is placed between the light source and the condenser

• Two-layered Nomarski Modified Wollaston Prism which separates individual rays of light into ray of pairs

• A polarizing filter is placed above the Wollaston Prism which cause wave interference to occur and to produce a three-dimensional image.

• Provide layer by layer imaging of specimen and enhance detail for specimens with either a low or high refractive index

Dark-field microscopy

• Used to enhance the visualization of specimens that cannot be seen easily with a bright-field microscope.

• Often used for unstained specimen

• Used to identify the spirochete bacteria, Treponema pallidum

treponema pallidum

what spirochete bacteria is identified in dark-field microscopy

Fluorescence microscopy

• Used to detects the bacteria and viruses within cells and tissues through a technique called immunofluorescence with the help of fluorescence property of the dye.

• It is used to visualize naturally fluorescent substances or those that have been stained by a fluorochrome/fluorophore (fluorescent dyes) to produce an image.

• 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 emission filter.

excitation filter and emission filter

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

Excitation filter

(FLUORESCENCE MICROSCOPY) Selects excitation wavelengths of light from a light source.

Emission Filter

(FLUORESCENCE MICROSCOPY) Selects a specific wavelength of emitted light to become visible.