Dr. Mhawi 1 Microscopy and Histological Techniques

Question 1

Why do electrons have a higher resolution then a light microscope?

Answer 1

electrons have a very short wavelength

therefore, electron microscope has higher resolution than light microscope

Question 2

What are the resolution powers of SEM, TEM, and light microscope?

Answer 2

• Light microscope (LM) – 0.2 µm
• Scanning Electron Microscope (SEM) – 2.5 nm
• Transmission Electron Microscope (TEM) – 0.5 nm

Question 3

What are the 5 types of microscopes?

Answer 3

•Conventional Light Microscope

•Phase-Contrast Microscope

•Fluorescent Microscope

•Confocal Microscope

•Polarizing Microscope

Question 4

_____ is used •Used to examine fixed and stained specimen

Answer 4

Conventional Light Microscope

Question 5

______ is used for immunocytochemistry techniques and uses UV light to excite fluorescent molecules

Answer 5

Fluroescent

Question 6

•The ______ method uses one type of fluorescent molecules-labelled antibodies directed against an antigen of interest within cells and tissues

–Not widely used nowadays because of the suboptimal sensitivity

•The ______ uses two different types of antibodies

Answer 6

direct

indirect

• •Primary antibodies directed against the antigen of interest and fluorescent molecule-labelled secondary antibodies directed against the primary antibodies

Question 7

What is this image?

Answer 7

Cultured cell treated with green fluorescent phalloidin and then stained with blue fluorescent DAPI (Di Amid Phenyl Indole). Phalloidin binds to actin filaments (appear green). DAPI has affinity to DNA of the nucleus (appears blue). Note the dark background.

Question 8

______ uses laser as source of illumination to excite fluorescent molecules and provides a sharper and more focused image than the conventional fluorescent microscopy

Answer 8

Confocal Microscope

Question 9

Electron microscopes use beam of electrons as a source of “illumination” rather than beam of light

_______

•In this type electrons transmit through the sample

–Resolve the internal structure

_______

•Electrons do not pass through the sample

–i.e., deflected and create 3D image of the outer surface

Answer 9

Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

Question 10

Comparison of LM vs TEM

Answer 10

8.5.6

Question 11

What are the basic steps used for preparing tissue sections for LM? (8 steps)

Answer 11

ØSampling: obtaining sample from biopsy or autopsy

ØFixation: prevent structural changes to the cell or tissue with 4% Formaldehyde

ØDehydration: taking water out of the sample by alcohol

Lipids usually extracted during dehydration [unless it is postfixed with osmium tetroxide (OsO4)]

ØClearing: taking alcohol out of the sample with xylol

ØInfiltration: impregnation of the sample with wax (paraffin) or resin (plastic)

ØSectioning: obtaining thin (4-10 µ) paraffin or plastic sections

ØStaining: stain the sections with the desired dye (usually H&E)

ØObservation: under the light microscope

Question 12

H&E is most common stain for Lm

______ is a basic dye, blue in color, stains acidic part of the cell, the nucleus appears blue

_____ is acidic dye pink in color, stains basic part of the cell, stains cytoplasm pink

Answer 12

Hematoxylin

Eosin

8.5.6

Question 13

______ is required for immediate evaluation of biopsy obtained form surgery

Answer 13

Forzen sections, uses CO2 (dry ice)

Question 14

______ is used to satin elastic tissues dark blue and black

_____ is used to stain collagen blue and nucleus red

____ is used to stain reticular fibres black

Answer 14

1. VAN GIESON’S STAIN (resorcinol)
2. TRICHROME
3. SILVER STAIN

Question 15

What is used in this image?

Answer 15

Cross section of a muscular artery stained with Van Gieson’s stain. The elastic tissue, appears as dark blue structure, is found in the inner and outer sides of the artery wall.

Question 16

What is used in this image?

Answer 16

Kidney section stained with trichrome stain. Collagen fibers stained blue and nuclei red.

Question 17

What is used in this image?

Answer 17

Reticular fibers (red arrows) in lymph node. Silver stain.

Question 18

______ is used for specific carbohydrates

______ is used to stain blood cells on air-dried smears

____ stains mylein sheath in myelinated nerve fibres

8.5.6

Answer 18

4. PAS-paeriodic acidc Schiff
5. GIEMSA
6. Myelin

Question 19

What is this image used for?

Answer 19

PAS stain for mucus in fundic stomach. Carbohydrates ,which are abundant in the mucus, appeared magenta in color when stained with PAS.

Question 20

What is this image used for?

8.5.6

Answer 20

WBCs stained with Giemsa.

Question 21

What is this image used for?

Answer 21

Myelinated axons in a peripheral nerve. Myelin, which is visible as circles around each axon, stained black.

Question 22

What are some artifacts that can occur that cause poor fixation?

8.5.6

Answer 22

Ø The main cause of artifacts is poor fixation

Ø Autolysis

Ø Shrinkage

Ø Folds

Ø Stain precipitation and dust

Ø Defects in the knife