Microscopy Final

Microscopy (FIS 312)

What microscope deals primarily with reflected light, versus transmitted light?

Stereomicroscope

Positive lenses vs Negative lenses

Positive- Convex, Convergent

Negative- Concave, Divergent

What is the focal point of a lens?

Where light rays converge through a lens

Spherical Aberrations occur when?

Light comes to focus at different locations depending on where in the lens it passes through

In the equation for Numerical Aperture (NA), what does 0 stand for?

½ the angle of cone of light reaching the objective

What is the Working Distance of an objective?

The distance between the bottom of the objective lens and specimen

Above a Numerical Aperture of 1, what special technique must we use to properly visualize a sample?

Oil-immersion

Total internal reflection occurs when?

The incident ray reflects back into the original medium when striking the interface

There are three diaphragms located within our microscopes. One is a fixed diaphragm that we cannot adjust. Where is this located?

Eyepiece

Higher Numerical Aperture objectives typically result in what?

Smaller Airy Disks; higher Resolving Power

Birefringence is also referred to as?

Double Refraction

Which filter is placed between the light source and specimen?

Polarizer

What are Conjugate Planes?

Series of locations in a microscope system that are all in focus at the same time

Kohler Illumination is attempting to find optimum balance between?

Resolution and Contrast

What are the four components required for Kohler Illumination?

1. Field Diaphragm

2. Adjustable Substage Condenser

3. Aperture Diaphragm

4. Collector lens (Bertrand Lens)

When light is concentrated and focused passing through the condenser, it travels in parallel rays. Why is this important?

This produces grainless light with uniform illumination

T or F: The first modern application of optics was the telescope?

False; Camera

T or F: Stereomicroscopes have a larger working distance than Compound microscopes

True

T or F: Empty Magnification is when we have decreased magnification with increased resolution

False; increased magnification with decreased resolution

T or F: Depth of field is the portion/amount of our specimen that is in focus at a given time

True

T or F: When looking through a Bertrand Lens you are viewing the Aperture Diaphragm

False; Field Diaphragm

T or F: A compensator plate is one of the discussed required components for Polarizing Light Microscopy

True

T or F: A containment that shows as clearly in focus when viewing a specimen could be found within the Illumination Series Conjugate Plane

False; Image Forming Series

Dispersion

Separation of light into components based upon their wavelengths

Absorption

Certain wavelengths of light that pass through an object do not emerge

Diffraction

Scattering of light when passing through an object or irregularity

Interference

Superposition of two or more light waves passing through an object

Astigmatism

Focal points vary across the plane

Coma

conical, comet-shaped blur

Distortion

Magnification of lens varies from center to periphery

Curvature of Field

Cannot focus center and periphery simultaneously

Apochromat

Highest level of correction

Plan

Correction for curvature of field

Achromat

Most common objective lens type

Fluorite

Manufactured with a different glass formulation

What are the patterns produced when diffracted light is reconstituted

Airy disks

Light traveling through a medium with a lower Refracive Index (RI) will travel what compared to light traveling through a medium with a higher RI?

faster

Who is credited with the creation/foundation of the Modern Theory of Imaging/Image Formation?

Ernest Abbe- Abbe Theory

What interference occurs when in-phase waves reinforce one another?

Constructive

What interference occurs when out-of-phase waves attenuate each other?

Destructive

During Kohler Illumination, when we are adjusting our field diaphragm and focusing the condenser, we are viewing the what conjugate plane?

Image-forming Series

What set-up is used to measure Refractive Index, Dispersion, and Dichroism?

Single Polar

What chart can help determine a material’s Birefringence by utilizing both Specimen Thickness and Retardation?

Michael-Levy Chart

For the most efficient set-up, you must match the Numerical Aperture of the what with the what?

Objective Lens

Aperture Diaphragm

What are the five steps of Image Formation?

1. Condensor concentrates light

2. Light passes through Object Plane

3. Objective forms Intermediate Image

4. Image magnification magnified by Eyepiece

5. Final Image on Retina

*Hint- look at the last word of each phrase (starts the next phrase)

Four planes of Image Forming/Orthoscopic Series?

1. Field Diaphragm

2. Object/Specimen Plane

3. Intermediate image plane

4. Retina

Four planes of Illumination/Conoscopic Series?

1. Light Source

2. Condenser/Aperture Diaphragm

3. Objective Back Focal Plane

4. Eyepoint of Eyepiece

What is the difference of reflection and refraction of light?

Reflection is the light ray htting a surface and reflecting off of it. The angle of light remains constant.

Refracted light rays hit a surface and the angle of light decreases because the light bends and changes direction.

Refractive Index

Light bending ability of a medium

In the process of Image Formation, where in the microscope is a Real Image formed? A Virtual Image? Explain the difference between a Real and Virtual Image?

A real image is formed through a positive lens which can be convergent/convex ad is inverted, while a virtual image is formed through a negative lens which can be concave/divergent and is erect. A real image occurs when light is actually converging through the lens while a virtual image appears to have converged through a lens.

A real image is formed in the objective lens

A virtual image is formed through the eyepiece lens

Numerical Aperture

The ability to gather light and resolve grainy details

How does Numerical Aperture impact resolution and the formation of airy disks?

Higher NA = Smaller Airy Disks = Better Resolution

The better the NA is, the better the ability to gather light which produces smaller airy disks, which are good because they give the viewer better resolution when viewing the specimen

Explain the Modern Theory of Imaging/Image Formation. What does this describe?

The Modern Theory of Imaging describes the relationship between diffraction and image formation. It also discusses how objective aperture impacts resolving power. The theory says that the objective lens should capture as much diffracted light as possible. A wide aperture creates a wide angle of the light cone and helps the objective capture more light. The idea is that a high NA value and a wide aperture will increase the resolving power and form an image that is able to be seen with more detail.

What is Resolving Distance “r”? If the value of “r” decreases, how does that impact the overall Resolving Power of the system?

Shortest distance between two points that can be distinguished

When resolving distance decreases, resolving power increases, making more details visible.

Explain the difference between Unpolarized and Polarized light. How do we get Polarized light? Why is this useful in microscopy/ what can this be used for?

Unpolarized light- when the field oscillates in all different directions

Polarized light- when the field oscillates in the same direction

Polarized light is useful in microscopy because it can help determine the optical properties of Anisotropic materials. We get Polarized light by placing unpolarized light through a filter that blocks out all planes of vibration except one.

Isotropic vs Anisotropic materials

How do they differ from one another? How do they relate to Birefringence? Define and Discuss Birefringence and how is it measured?

Isotropic- have the same optical properties throughout, so the RI value is consistent across the whole sample. They have a uniform structure, ex. table salt

Anisotropic- have different optical properties throughout the material, so the RI value varies depending on the area of the sample. They are not uniform in structure, ex. quartz.

Birefringence- aka double refraction- involves a ray of light that is split into 2 rays of light. It is measured using the Michael Levy Chart using the highest interference colors from the specimen, then a vertical line which is followed to a horizontal line, and a diagonal line that intersects that point is the Birefringence value.

Which conjugate plane is considered the Orthoscopic mode? Why? How does this differ from the Conoscopic mode?

The conjugate plane considered the Orthoscopic mode is the object/specimen plane because that’s what this mode shows us. It differs from the Conoscopic mode because that shows the Objective Back Focal Plane. The Orthoscopic mode is aka the Image Forming Series because it shows what the viewer’s eyes actually see, while the Conoscopic mode is aka the Illumination Series because it shows what’s being illuminated.

What microscope component has a listed Numerical Aperture value?

Objective Lens

What microscope component can adjust the angle of the cone of light passing through the condenser?

Aperture Diaphragm

Mechanical vs Optical components of Microscope

Mechanical- stage, coarse and fine adjustments

Optical- field diaphragm, condenser, objective lenses, eyepieces

Once properly aligned, the best way to adjust the brightness of the microscope is to open the what?

Field Diaphragm

What is the most common natural fiber type?

Cotton

Crossed-polars are used to measure this optical property?

Birefringence

Delustrant Particle

Synthetic material used to dull manufactured fiber by scattering and absorbing light. Increases opacity of synthetic fiber.

Green dye in Christmas tree staining?

Red Dye in Christmas Tree Staining?

Green- Picroindigocarmine (PIC)

Red- Nuclear fast red

This theory describes the need for objective lenses to capture as much divergent light as possible for best resolution

Abbes Theory of Imaging

This objective lens type has the highest level of correction

Apochromat

Human hair typically exhibits what scale structure?

Imbricate

Man-made fiber type from naturally occurring polymers

Regenerated Fibers

Fibers with continuous length vs fibers cut to specific short strands during manufacturing

Filament fibers vs staple fibers

The three primary components of glass

1. sand

2. limestone

3. Troba

Hair types with double medullas are found where?

Beard/facial hair

The direction of strands in a helix about the rope or twine axis?

Lay

The resting stage of hair growth

Telogen

Fractures extending outwards from point of impact

Radial fractures

Most common measured property of glass

Refractive Index

NIBIN vs IBIS

National Integrated Ballistics Information Network (local)

Integrated Ballistics Identification System (National)

What strikes the primer in a gun?

Firing pin

Confocal Microscopy

Method of microscopy that forms 3D images via optical sectioning

Collection of a known source material, taking into account all possible variation

Representative Sampling

Retardation

When one light ray travels faster through an object and emerges before a second ray due to a phase shift