Instrumentation CH 3

Question 1

Microscopes

Answer 1

• Very important piece of equipment. All slides to be sent to a pathologist should
  be reviewed under a microscope to ensure quality.

Question 2

Types of Microscopes

Answer 2

• Light Microscopes
• Polarizing Microscopes
• Phase-Contrast Microscopes
• Darkfield Microscopes
• Flourescence Microscope
• Electron Microscopes

Question 3

Light Microscopes consist of…

Answer 3

• Simple Microscopes
• Compound Microscopes

Question 4

Light Microscopes
◦ Ocular lens
◦ Objectives

Answer 4

◦ Ocular lens
- 10X magnification power

◦ Objectives
- Scanning lens 2.5X-4X
- Intermediate lens: 10X-20X
- High power dry lens: 40X-45X
- Oil Immersion lens: 90X-100X

Question 5

Light Microscopes
◦ Total magnification power

Answer 5

Lens magnification multiplied by Objective Magnification

Question 7

Polarizing Microscope

Answer 7

• Useful for detection of crystal (talc, silica, or urate)
• Used for tissue showing birefringence: when a light polarized is split and takes
  two separate paths. Creating a
  phenomenon known as double refraction

Question 8

birefringence

Answer 8

when a light polarized is split and takes

Question 9

Polarizing Microscopes

Answer 9

• Polarizer is between light source and specimen
• Analyzer is a polarizing filter between specimen and eyepiece
• Blocking light rays, except for what shows birefringence
• Creating a dark background, with bright birefringence

Question 10

Phase-Contrast Microscope

Answer 10

• Used for unstained specimens
• Cells can be living
• Appear transparent
• Not common in most histopathology laboratories

Question 11

Darkfield Microscope

Answer 11

• Only uses scattered light
• Creates images that appear larger than reality
• Rarely used in histopathology lab

Question 12

Fluorescence Microscope

Answer 12

• Fluorescence: when a substance absorbs light, then emits it as light of longer
  wavelength
• Fluorescence Microscopy is when UV lights are emitted (short wavelength) then the substance emits it back as visible light
• Mercury or Halogen lamps are common light source
• Thioflavin T, Thioflavin S, Auramine- rhodamine, FITC, DAPI, Texas Red Instrumentation

Question 13

Autofluorescence

Answer 13

• Some naturally fluorescing components include collagen

◦ Why would this be a problem?
- Photobleaching limits time

Question 14

Electron Microscope
◦ Two types

Answer 14

1.) Scanning (SEM)
- SEM: electron gun sweeps surface creating a 3D image and is used to study the surface of many things Instrumentation

2.) Transmission (TEM)
- TEM: specimen either transmits electrons or deflects electrons. 2D black and white image
shown. Has the HIGHEST magnification power

Question 15

Electron Microscope

Answer 15

• Can identify structures 0.35nm apart
• Has magnification range of 1000-5000X
• Electron gun instead of light source

Question 16

Microtomes
- 3 types

Answer 16

1. Rotary
2. Sliding
3. Freezing

Question 17

Rotary Microtomes

Answer 17

• Block moves up and down
• Either the blade, or the block itself will advance
• Also found in cryostats
• Can be semi-auto, or fully automatic
• Most common, and used in our lab!

Question 18

Sliding Microtome

Answer 18

• Block is stationary, and blade is moved through cutting facet of block.
• Not commonly used in routine
  histopathology labs
• Used mainly for sectioning large paraffin blocks

Question 19

Freezing Microtome

Answer 19

• Replaced by cryostat in most labs
• Used for stains that require free floating sections
• More hazardous due to airborne transmission as it used carbon dioxide to freeze

Question 20

Cryostat

Answer 20

• “Refrigerated microtome” cools and maintains temperature at -20 C
• Certain tissues require warmer temps
• Slow freezing can cause ice crystal artifact, so isopentane kept in liquid nitrogen is recommended for skeletal
  muscle in particular

Question 21

Microtome Blades

Answer 21

• Glass knives: used for plastic embedded tissue mainly in Electron Microscopy
• Disposable knives: high and low profile, what we use in our lab
• Diamond Knives: also can be used for electron microscopy

Question 22

2 Types of Tissue Processors

Answer 22

1. Conventional
2. Microwave

Question 23

Conventional Tissue Processor

Answer 23

• Most commonly a Closed system, where fluid is transferred. Open systems transfer the specimen.
• Closed systems reduce vapor smells, and keep specimen from drying out
• Temperature of paraffin is important, over 4 degrees Celsius above melting point will
  leave tissue brittle and over hardened

Question 23

Stainers
- Automatic Stainer
- Linear
- Robotic

Answer 23

• Automatic Stainer: linear or robotic
• Linear: transfers from one station to next with same amount of time in each
• Robotic: can move specimens throughout at separate durations

Question 24

Microwave Tissue Processor

Answer 24

- Shorter turn around time for larger or smaller tissue - Can start at fixation step - VERY useful in preparation of biopsies

Question 25

Misc. Instruments

Answer 25

- Automatic coverslipper: can coverslip many slides at a given time - Floatation baths: used to pull sections onto slides after cutting. 5-10 degrees BELOW melting point of paraffin - Dryers and Ovens: used to dry slides prior to staining. Typically 55-60 degree Celsuis - Refrigerator and Freezers: 4 degree C for fridge, and -20 for freezers. -70 to -80 freezers used for long term storage

Question 26

Misc. Instrumentation

Answer 26

- Embedding Center: where you embed the tissue into molds for cutting. Typically 2-4 degree C ABOVE melting point of paraffin - Pipettes: used to measure small amounts ranging from 1mL- to 2uL. - Balances and Scales: Used to weigh reagents - CALIBRATION IS ESSENTIAL FOR ALL EQUIPMENT IN THE LABORATORY!!!