1.13 - light and fluorescent microscopy - OB

Question 1

What does magnification mean in microscopy?

Answer 1

degree to which an object appears larger than its actual size

Question 2

What are the types of magnification in light microscopy?

Answer 2

Objective Magnification: Magnification provided by the objective lens.

Eyepiece Magnification: Magnification provided by the ocular lens (eyepiece).

Question 3

What is resolution in microscopy?

Answer 3

ability of a microscope to distinguish between two closely spaced objects as separate entities.

Question 4

What is numerical aperture (NA) and why is it important?

Answer 4

Numerical aperture is a dimensionless number that characterizes the light-gathering ability of a microscope objective.

Higher NA values indicate better resolution due to improved light collection and clarity of the image.

Question 5

What factors affect the resolution of a light microscope?

Answer 5

Wavelength of Light: Shorter wavelengths provide better resolution.

Numerical Aperture (NA): A higher NA increases the resolution by allowing more light to enter the lens.

Question 6

resolution formula

Answer 6

pic from powerpoint OB mT1

Question 7

what can light microscope show

Answer 7

bacteria
details within nucleated cells - nuclei / mitochondria / storage granules

Question 8

What are the main steps in preparing a tissue for light microscopy?

Answer 8

Fixation

Dehydration and Embedding

Sectioning

Staining

Question 9

What is fixation in tissue preparation?

Answer 9

using chemicals (such as formaldehyde) to preserve tissue structure by cross-linking proteins, preventing degradation, and maintaining cellular morphology.

Question 10

What is dehydration and embedding in tissue preparation?

Answer 10

specimen is dehydrated using solvents like ethanol, followed by xylene

tissue is then embedded in a medium (commonly paraffin wax) to provide support for sectioning

Question 11

What is sectioning in tissue preparation?

Answer 11

involves cutting the embedded tissue into thin slices (5-10 micrometers thick) using a microtome, allowing light to penetrate for better visualization.

Question 12

What are acidophilic stains?

Answer 12

have an affinity for acidic structures, typically staining proteins and cytoplasmic components.

usually appear pink or red under the microscope.

Question 13

What is eosin, and what does it stain?

Answer 13

cidophilic stain that specifically stains cytoplasmic proteins, red blood cells, and extracellular matrix components pink

commonly used in histological preparations.

Question 14

What are basophilic stains?

Answer 14

have an affinity for basic (alkaline) structures, primarily nucleic acids and certain proteins.

typically appear blue or purple under the microscope.

Question 15

What is hematoxylin, and what does it stain?

Answer 15

basophilic stain that primarily stains nuclei and other structures rich in nucleic acids blue or purple, making it essential for identifying cellular morphology

Question 16

What is the general histological appearance of an H&E stained section?

Answer 16

In an H&E stained section:

Nuclei and structures rich in nucleic acids stain purple (due to hematoxylin).

Most proteins stain pink (due to eosin), especially in the cytoplasm of muscle, red blood cells, and epithelial cells.

Question 17

Why is H&E staining important in histology?

Answer 17

provides contrast between different tissue components

Question 18

resolution of a light microscope

Answer 18

200nm

half the wavlength of visible light

Question 19

What is immunocytochemistry?

Answer 19

a technique used to visualize the localisation of specific proteins or antigens in cells using antibodies tagged with a detectable marker (e.g., fluorescent dye or enzyme).

Question 20

What is the purpose of immunocytochemistry?

Answer 20

determine the spatial distribution and expression levels of specific proteins in cellular contexts, which helps in understanding cellular functions and disease states.

Question 21

Can you give an example of immunochemistry in action?

Answer 21

An example is the labeling of different cell types in the pancreatic islets of Langerhans, where unique protein markers are used to differentiate between various cell populations (e.g., alpha cells and beta cells).

Question 22

What is the mechanism by which immunohistochemistry works?

Answer 22

A primary antibody is introduced that specifically binds to a target antigen in the tissue or cell sample, forming an antigen-antibody complex.

A secondary antibody, designed to bind to the primary antibody, is applied = typically conjugated to a marker (e.g., fluorescent dye or enzyme).

binding of the secondary antibody amplifies the signal, as multiple secondary antibodies can attach to each primary antibody, increasing detection sensitivity.

The sample is then visualized using appropriate techniques

Question 23

fluorescence microscopy