Lecture 1 & 2: Background And History

Question 1

Define Histology.

Define Histopathology.

Answer 1

1. Histology is the study of the microscopic anatomy of cells and tissues of plants and animals.
2. Histopathology is “The science or study dealing with the cytologic and histologic structure of abnormal or diseased tissue.”

Question 2

Be able to recognize the distinct slides of tissues from lecture 1.

Answer 2

Do it. Check those pages.

Question 3

What contributions did Bichat make to the field of histology?

Answer 3

French guy who was proclaimed the father of modern histology; believed that each organ contained specific tissues or membranes. He identified over 21 of these. He hypothesized that diseases attacked specific tissues, not the whole body.

• Less acurately, he divided the body into the organic life (heart, intestines), and Animal LIfe (Ears, brain, eyes)

Question 4

Describe the contributions of Virchow to histology?

Answer 4

German guy who suspected from early on that the Cell Theory (All life is made of cells, and all cells came from existing cells) was correct.

Question 5

What contribution did Kolliker make to histology

Answer 5

Applied Schwann’s theory to embryonic animal development and wrote what’s widely considered the first official textbook on histology. (And another one on embryology)

Question 6

Describe the contributions of Schleiden

Answer 6

Botanist who correctly came up with the idea that all life is composed of cells, but incorrectly thought that cells arise from budding from the nucleus. (Called the Free Cell Theory)

Question 7

What contributions did Schwann make to histology

Answer 7

Animal zoologist who noticed similarities between plant tissues and animal tissues - Identified cartilage, eggs, and 5 types of tissues

Question 8

What’d Janssen do?

Answer 8

Produced the first operational compound microscope with a magnification of 30x

Question 9

What’d Hooke do?

Answer 9

Described cork and other cells and introduced the term “cell.”

Question 10

What’d Leeuwenhoek do?

Answer 10

Made over 247 simple microscopes capable of magnifications of around 100x. Sent 26 of them to the Royal Society. Among his observations: RBCs, protozoa, striations of skeletal muscle, sperm cells, bacteria.

Question 11

1. Define Refraction of light
2. What is the Refractive Index Formula?
3. What is the refractive index of air?

Answer 11

1. When light waves travel through a vaccum, they travel at a fixed velocity. However it is slower when traveling through air or water or stuff and the Refractive Index defines it’s fixed velocity through that medium.
2. Refractive Index = (Velocity of Light in a Vaccum)/(Velocity of light in This Medium)
3. 1ish

Question 12

What happens when light passes from one medium to another at an angle?

How are diopters measured?

Answer 12

The light bends based on the refractive index and angle of the beam. Perpendicular light should pass right through without bending.

A diopter is 1/(focal length of the lens in meters)

Question 13

Define Focal Point

Define Focal Length

Answer 13

1. The point through which all parallel rays of light will pass after passing through each part of the lens.
2. The distance from the center of the lens to the focal point.

More diopters means an increased focal length

Question 14

Describe a real image and how it forms

Answer 14

• Requires a convex lens converging onto a focal point, and the object needs to OUTSIDE that focal point.
• Real image is inverted, can be projected onto a screen, and can be magnified
• Greatest magnification occurs when focal length is very small, and the object is as close to focal point as possible.

Question 15

Describe a virtual image and how it is formed

Answer 15

• Object must be placed INSIDE the focal point
• Upright (not inverted), CANNOT be projected onto a screen, but CAN be magnified
• No point exists when the rays radiating off of the object are brought into focus

Question 16

Define Resolution

How would one increase resolution?

Answer 16

• The ability for a microscope to distinguish two small points as separate points
• This requires the diameter of diffraction lines around the points to be reduced
• Increased resolution = Higher Refractive Index, shorter wavelength

Question 17

What is the formula for finding resolution? Know what each symbol represents

Answer 17

Resolution d = (0.61 * Lambda)/(n * sin(alpha))
0.61 = Constant measuring human capability of the eye i think
Lambda = Wavelength of Light (3800)
n = Refractive index of medium surrounding the object (Air is 1.0)
sin(alpha) - Angle of the cone of light entering the Objective Lens (max angle = 180°)
nsin(alpha) = Numerical Aperture. N has a maximum of 1.4 (oil), which means highest possible NA is 1.4

Question 18

What is the N.A. of 4x magnification?
100x
What are methods to decrease the wavelength (lambda)?

Answer 18

At 4x magnification, NA = about 0.10
At 100x, magnification = about 1.2
Instead of solar light, U.V. Light only has about 2500 A
Electrons even smaller at 0.05A

Question 19

What is the highest possible resolution in a light microscope?

How about in comparison to a transmission electron microscope?

Answer 19

For light microscopy, the strongest resolution possible is “about” 5 um.

For transmission electron microscopes, the theoretical resolution is about 0.01 angstroms, but we’ve only ever gotten it down to about 5-10 angstroms (1 angstrom = 0.1 nm)

Question 20

What are the components of a Compound Light Microscope (Also called a Bright Field Microscope)

Answer 20

• Light Source
• Condenser
• Stage - Holds stuff. Usually 6-10 um
• Objective Lens - Also called magnification lens - close to object
• Ocular Lens
• (Short version): From the light source, object passes through objective lens and produces an real image in front of the ocular lens. That image is viewed by the eye as an inverted virtual image.

Question 21

What are the pros and cons to a compound light microscope?

Answer 21

+ Ability to magnify
+ Ability to resolve structural detail
- Specimen must be thin
- Relatively little contrast in the unstained specimen

Question 22

How does a phase contrast microscope work. List it’s advantages.

Answer 22

• This weird one converts invisible phase shifts in light passing through a transparent specimen into changes in brightness.
• These can be used to examine unstained tissues, including live cells.
• Different structures of the cell have different n’s, which defracts the light into out-of-phase.
• Non-slowed light waves are compared to retarded light-waves.

Question 23

Briefly say what flourescence microscopes do (oddly not in the objectives)

Answer 23

• Can detect specific wavelengths that molecules radiate (autoflourescent), which can be seen under a U.V. Light source.
• Usually used to detect antibodies and antigens in immunochemical staining.
• Flourescent “tracer” molecules can be injected into animals to be detected later

Question 24

Define how a confocal scanning microscope works

Answer 24

• Invented in the 50s by Minsky, this weird one adds a pinhole to the confocal plane of the lens to eliminate out-of-focus light, which helps construct 3D images
• Combines the technology (components) of a light microscope, fluorescence equipment, and laser scanning.
• If I’m understanding this right, the laser moves across the specimen using a rotating mirror, and a photomultiplier tube absorbs the light it produces to be analyzed
• Eventually, a 3D shape of the specimen is produced across a monitor

Question 25

List the advantages of a Confocal Microscope

Answer 25

• Very thin optical images of the specimen are created (typically 1 μm thick).
• Out-of-focus images are subtracted from the image by the computer program.
• Computer can make 3-D reconstructions of the specimen by stacking individual images.

Question 26

Describe how a Transmission Electron Microscope Works

Answer 26

• TEMs utilize electrons rather than light. A specific voltage is set between an anode and a cathode; potential difference usually ranges between 20k and 200k Volts
• The electrons are derived from a heated tungsten filament
• Electromagnets act as the condenser, objective, and projection lens
• The specimen needs a holder to hold in place, and a viewing screen and photographic film for viewing

Question 27

Not sure if this is worth it, but…

In 12 steps, describe the path of an electron in a TEM

Answer 27

sigh Alright so…… 1. Electron is shot out of an Electron Gun, 2. Passes through a vaccum manifold. 3. The electron enters a beam where it is 4. positioned correctly. 5. The diameter of the beam gets reduced in the 6. Condenser and passes through the aperture 7. Changer and 8. Diaphragm where it is converted to… something else. 9. Passing through the focusing lens onto the 10. Stage, the image is projected onto the 11. Visual Transmission as it enters the 12. Vaccum Chamber.