Histology & Its Method of Study: Light Microscopy

Question 1

Bright-field microscopy

Answer 1

• most common method used by students and pathologists
• uses ordinary light
• colors imparted by tissue staining

Question 2

What are the optical components of a bright-field microscope?

Answer 2

• condenser: focuses light on object
• objective: lens enlarging & projecting image of object toward observer
• eyepiece: (ocular lens) further magnifies image and projects unto viewer’s retina or a charge coupled device (CCD) highly sensitive to low light levels with a camera and monitor (DOES NOT IMPROVE RESOLUTION)

Question 3

How do you obtain total magnification?

Answer 3

objective X eyepiece/ocular

Question 4

define resolving power

Answer 4

the smallest distance between two structures at which they can be seen as separate objects
- critical factor in obtaining crisp, detailed image with a light microscope
- determines image quality, clarity

Question 5

Which optical component does resolving power mainly depend on?

Answer 5

the objective lens and its quality

Question 6

Virtual microscopy

Answer 6

• permits tissue study sans-stained slide or microscope
• cost efficient, easy, tech savvy

Question 7

Fluorescence microscopy

Answer 7

• uses UV light to for molecules to be visible
• allows localization of fluorescent probes (much more specific than routine stains)
• acridine orange acid binds DNA & RNA
• DAPI & Hoechst blue bind DNA
• important in immunohistochemistry

Question 8

Phase-contrast microscopy

Answer 8

• uses differences in refractive index of various natural cell & tissue components to produce an image without staining or fixation
• allows observation of living cells
• uses lens system that produces visible images from transparent objects

Question 9

What principle is phase-contrast microscopy based on?

Answer 9

“Light changes its speed when passes through cellular and extracellular structures with different refractive indices.”

Question 10

What is a modification of phase-contrast microscopy?

Answer 10

differential interference microscopy with Nomarski optics
- produces image of living cells with 3D aspect

Question 11

Confocal microscopy

Answer 11

• involves scanning the specimen at successive focal planes with a focused light beam, often from a laser
• produces a 3D reconstruction from the images
• high resolution, precision
• digital 3D
• sharp, small point high-intensity, laser

Question 12

Polarizing microscopy

Answer 12

allows recognition of stained or unstained of highly organized and complex subunits
- light passes through polarizing filter and exits vibrating only one way
- produces repetitive, periodic macromolecular structures

Question 13

What is birefringence in polarizing microscopy?

Answer 13

the ability to rotate the direction of vibration of polarized light

Question 14

What are the two microscopes used in Electron Microscopy?

Answer 14

• Transmission electron microscope (TEM)
• Scanning electron microscope (SEM)

Question 15

How are contrast and resolution improved in TEM?

Answer 15

heavy metal ions are added to the fixative to bind to macromolecules and increase their electron density and visibility

Question 16

What techniques allow TEM to study cells without fixation or embedding?

Answer 16

cyrofracture and freeze etching use frozen specimens in liquid nitrogen with vaporized metal atoms like platinum and split the lipid bilayer to expose the protein’s size, shape, and distribution

Question 17

What happens in SEM?

Answer 17

surface of specimen is first dried and spray-coated with a very thin layer of heavy metal like gold to reflect electrons in a beam of scanning the specimen

Question 18

what are the main differences between TEM and SEM?

Answer 18

• TEM requires thin samples (less than 100 nm thick) while SEM can accommodate bulkier samples since it only requires the surface to be exposed to the electron beam
• TEM offers higher resolution while SEM gives less
• TEM uses transmitted electrons to form image while SEM uses secondary, backscattered ones, & X-rays emitted from the surface to form an image and provide compositional information

-SEM is easier to interpret because of 3D view

Question 19

Cell & Tissue Culture

Answer 19

Cells can be grown in vitro (outside of body) and in the organism (in vivo) cells bathed in blood plasma fluid to survive and grow and can be examined in the living state by phase-contrast light microscopy.

• used to analyze infectious viruses like and chromosomal analyses, cervical cells, HeLa cells first line of cells in 1951

Question 20

primary cell structures

Answer 20

complex solutions of salts, amino acids, vitamins with added growth factors

Question 21

cell line

Answer 21

forms when cells remain in vitro for long periods and become immortalized

Question 22

transformation

Answer 22

the process in which certain changes (some related to oncogenes) promote cell immortality similar to a normal cell becoming a cancer cell