Test 2 - Specialty Microscopy

darkfield, phase, polarizing, electron, fluorescent, inverted, & stereoscopic microscopes

In an inverted microscope, from which surface are specimens examined?

The under surface.

What is unique about the optical pathway of an inverted microscope?

The entire optical pathway is flipped (inverted) compared to a brightfield microscope.

What is a primary clinical use for the inverted microscope?

Examination of cell suspensions and red cell agglutinates.

In cytogenetics, what specimens are routinely examined using an inverted microscope?

Cell and tissue cultures.

Why is an inverted microscope preferred for cells suspended in liquid?

It prevents the resolution loss caused by looking through varying depths of fluid.

Where are the light source and condenser located in an inverted microscope?

Above the plane of the specimen.

Where are the objectives located in an inverted microscope?

Below the plane of the specimen.

How are image-forming rays reflected to the observer in an inverted microscope?

Through a combination of two mirrors or prism systems.

What is a physical requirement for containers used in inverted microscopy?

They must be transparent and have a thin base.

Why are oil immersion objectives rarely used with inverted microscopes?

High numerical apertures are limited by the thickness of the specimen container base.

What is the common alternative name for the stereoscopic microscope?

The dissecting microscope.

What is the primary visual advantage of a stereoscopic microscope?

It provides a three-dimensional (3D) perspective of the specimen.

How does the image orientation in a stereoscopic microscope differ from a compound microscope?

The image is "right side up" (not reversed or inverted).

Which light rays form the image in a stereoscopic microscope?

Light rays reflected from the surface of the specimen (incident light).

How many separate compound microscopes comprise a stereoscopic system?

Two separate lens systems.

At what angle are the lens systems in a stereoscopic microscope mounted to each other?

15 to 16 degrees.

Why is a stereoscopic image seen in 3D?

The two lens systems are non-parallel, providing a separate image for each eye.

How are both lens systems in a stereoscopic microscope adjusted?

By a single coarse focusing control.

What feature allows for infinitely variable magnifications in a stereoscopic microscope?

A system of "zoom" lenses that move in relation to each other.

What is the typical magnification range of a stereoscopic microscope?

1X to 100X.

In stereoscopic microscopy, what is magnification in excess of 100X called?

Empty magnification.

What is the primary limitation of the field of vision in stereoscopic microscopy?

Only the central area is sharply focused because the lens systems are inclined at an angle.

Why does the stereoscopic microscope have a very large depth of field?

To allow for the viewing of thick objects like gemstones or surgical sites.

What clinical surgical application uses stereoscopic principles?

Microsurgery (repair of nerves and tiny blood vessels).

How is a brightfield microscope modified for darkfield microscopy?

By installing a darkfield condenser.

What structural modification creates the hollow cone of light in a darkfield condenser?

A black patch stop (annular stop) or a reflective hemisphere.

At what angle do light rays strike the specimen in darkfield microscopy?

An oblique angle.

Why is the background dark in darkfield microscopy?

Light rays strike the specimen at an angle that does not allow non-diffracted rays to enter the objective.

What physical process allows light to enter the objective in darkfield microscopy?

Diffraction of light rays by the specimen.

How do images appear in darkfield microscopy?

Brilliantly white (bright) against a black background.

What type of darkfield condenser is used for high-magnification work?

A Cardoid condenser (utilizes a silver hemisphere).

Which darkfield condenser uses a black patch stop on the central lens?

The Paraboloid condenser.

Name one clinical structure best viewed with darkfield microscopy.

Spirochetes (e.g., Treponema pallidum).

Why is the vertical position of the condenser critical in darkfield microscopy?

The oblique rays must focus exactly at the object plane to be diffracted into the objective.

What is the resolution limit of structures detected by darkfield microscopy?

Structures less than 0.25 µm.

What property allows a substance to emit longer wavelength radiation after being excited by shorter wavelengths?

Luminescence.

How does phosphorescence differ from fluorescence?

Phosphorescence continues emitting light after the excitation source is removed; fluorescence stops immediately.

In fluorescence microscopy, what is a "fluorochrome"?

A substance that produces secondary light emission after reacting with short-wavelength light.

What is the term for natural fluorescence found in tissue components like collagen?

Autofluorescence.

How does an excited electron produce visible light in a fluorochrome?

It releases extra energy as it returns from an outer shell to its normal valence shell.

What range of light is typically used as the excitation source in fluorescence microscopy?

300 nm to 550 nm (UV, violet, or blue light).

What is the purpose of the heat filter in a fluorescence microscope?

To protect the exciter filter from the intense heat produced by mercury vapor or halogen lamps.

What is the function of the exciter filter?

It transmits only the specific narrow-band wavelengths required to excite the fluorochrome.

What is the function of the barrier filter?

It blocks all UV radiation from reaching the observer’s eyes.

Why is a barrier filter essential for safety?

UV radiation can cause severe corneal burns.

Where is the barrier filter located?

In the light path after the specimen but before the oculars.

Why are fluorite objectives recommended for fluorescence microscopy?

They have a high numerical aperture, which is directly proportional to the intensity of excitation.

What is another name for incident light fluorescence microscopy?

Epi-fluorescence or Ploem illumination.

In epi-fluorescence, which component acts as both the condenser and the objective?

The objective lens.

What component is required in epi-fluorescence to redirect light into the optical path?

A dichroic beam splitter.

Why is epi-fluorescence brighter than transmitted fluorescence?

No light is lost to non-specific scattering because illumination is directed from above.

Why must fluorescent preparations be examined in a darkened room?

To prevent them from appearing pale due to ambient light.

Why are fluorescent slides often non-permanent?

The fluorescence tends to fade (quench) within a few hours.

What is "birefringence"?

The phenomenon where a crystalline substance refracts light into two separate images (double refraction).

In polarized light, how do individual component rays vibrate?

In a single plane (vertically, horizontally, or diagonally).

In a polarized light microscope, where is the "polarizer" located?

On top of the field lens at the base (below the condenser).

In a polarized light microscope, where is the "analyzer" located?

In the microscope tube (above or below the ocular).

What occurs when the polarizer and analyzer are in the "crossed" position?

Their crystal patterns are at right angles, and the field becomes dark.

How does a birefringent substance appear under crossed polarizers?

As a bright color against a dark background.

What defines an "isotropic" substance?

It has only one refractive index and transmits light in one plane.

What defines an "anisotropic" substance?

It splits a light beam and transmits light unequally in different directions (multiple refractive indices).

Name a clinical example of a birefringent crystal found in joint aspirates.

Uric acid crystals.

What is the composition of high-quality polarizing filters?

Herapathite crystals embedded in nitrocellulose.

How are polarizer/analyzer filters protected?

They are plastic-coated and mounted in metal rings.

Who won the Nobel Prize for inventing phase contrast microscopy?

Frederick Zernicke.

What is the primary purpose of phase contrast microscopy?

To observe unstained living cells and active processes (like mitosis).

How does phase contrast make transparent structures visible?

It converts minute differences in refractive index into visible differences in amplitude (brightness).

What is an "amplitude object"?

A specimen that absorbs light, resulting in areas of varying brightness or color.

What is a "phase object"?

A transparent object that changes the synchrony (phase) of light waves without absorbing them.

What occurs during "positive interference" of light?

Two coherent waves combine to sum their amplitudes, resulting in a point twice as bright.

What occurs during "negative interference"?

Two waves half a wavelength out of phase cancel each other out, resulting in darkness.

In phase contrast, what are "direct rays"?

Light rays that pass through the specimen undeviated to form the background.

In phase contrast, what are "indirect rays"?

Light rays that are diffracted or reflected by the specimen components.

What component in a phase contrast condenser produces a hollow cone of light?

The annulus (opaque shield).

Where is the "phase plate" located in a phase contrast microscope?

Inside the objective lens.

By how much does the phase plate deliberately retard one group of light rays?

By one-quarter (1/4) wavelength.

What is the role of an "Auxiliary microscope" in phase contrast?

To align the image of the condenser annulus with the phase plate in the objective.

How does a positive phase plate show high refractive index structures?

As dark structures on a light background.

How does a negative phase plate show high refractive index structures?

As light structures on a dark background.

Why are the eye and retina unable to see phase objects without specialized optics?

The human eye is sensitive to changes in amplitude (brightness) but not changes in phase.

What is the primary imaging source for an electron microscope?

A beam of free electrons.

What generates the electron beam in an electron microscope?

A heated tungsten filament (the "electron gun").

Why is the entire pathway of an electron microscope a vacuum?

To prevent gas molecules from colliding with and scattering the electron beam.

What type of lenses focus the electron beam?

Electromagnetic lenses.

What is the resolution range of an electron microscope?

0.2 nm to 0.07 nm.

How many times greater is electron microscope resolution compared to light microscopy?

Up to 1,000 times greater.

In Transmission Electron Microscopy (TEM), what creates the image?

Transmitted electrons that pass through the specimen without scattering.

How do dense regions appear in a TEM image?

They appear darker because they scatter more electrons.

What is the standard specimen thickness for TEM?

50 nm to 300 nm.

Why are TEM specimens mounted on tiny grids instead of glass slides?

Glass does not allow electrons to pass through.

How does a Scanning Electron Microscope (SEM) create an image?

By using electrons reflected from the surface of a specimen.

What visual effect does SEM produce?

A three-dimensional (3D) surface effect.

What must be done to SEM specimens to allow image production?

They must be coated with a vacuum-evaporated metal (gold, palladium, or chromium).

What device converts photons into electrical current in an SEM system?

A Photo multiplier tube.

What is the role of osmium tetroxide in electron microscopy?

It acts as both a fixative and a stain.

Which type of electron microscopy is most commonly used in virology research?

Transmission Electron Microscopy (TEM).

What determines the magnification of a TEM image?

The intermediate lens system and the projector lens.

What determines the resolution of a TEM image?

The objective lens.

What is "Confocal microscopy"?

A scanning laser microscope that creates a 3D image.

How does a Scanning Probe microscope function?

A sharp probe moves over a surface while maintaining a steady tunneling current.