Microscopy and Cell Visualization

Question 1

Describe the unity behind the variety of organisms

Answer 1

they are all composed of the same classes of biomolecules, have same essential elements, principal architecture.

Question 2

What kind of technology was the initial step towards understanding tissues and cells, also essential to cell biology?

Answer 2

use of microscopes (light, electron, fluorescence), slicing open a cell and viewing its parts

Question 3

what are the uses of light microscopy (bright-field microscopy)?

Answer 3

use beam of light to illuminate specimens, and create magnified image. composed of magnifying lenses and light source

Question 4

what are the limitations of light microscopy?

Answer 4

poor contrast, and lack of light ; cannot resolve structures smaller than 0.2 um or 200 nm (1/2 wavelength of visible light), low resolution, cannot see internal structures (appear colorless, transparent )

Question 5

Discuss the invention of the light microscope

Answer 5

discovery made in 17th century. Robert Hook first used lens with cork in 1675 (noticed it was made of minute chambers ) and first introduced term cell; Antonio Leeuwenhoek was the first to observe unicellular organisms, including bacteria in 1673

Question 6

What is light microscopy also known as? What are the requirements for light microscopy?

Answer 6

Bright field microscopy- still routinely used to observe cultured cells; you need light, cells/tissues must be fixed (preserved, embedded in resin, cut) and stained

Question 7

What are internal structures that can be seen under light microscope?

Answer 7

chromatids (condensed chromosomes after duplication) may be visible due to high density.

Question 8

Describe the cell theory and the scientists behind it

Answer 8

Cell theory states that all living cells come from cells that undergo growth and division of preexisting cells. This theory was postulated by Schleiden and Schwann

Question 9

What kind of microscopy has the highest magnification and best resolution?

Answer 9

Electron Microscopy

Question 10

Which form of microscopy is most widely used today?

Answer 10

Fluorescence Microscopy

Question 11

what enhances contrast and reduces glare?

Answer 11

Polarized light (polarized sun glasses)

Question 12

What is the difference between polarized and unpolarized light?

Answer 12

Their wave behavior differs. Polarized light waves vibrate in one plane (overlap perfect). unpolarized light wave vibrates in any direction/plane ( reflect light in multiple directions, random overlap)which reduces clarity/sharpness of image.

Question 13

Compare and contrast the two kinds of interference of polarized light.

Answer 13

Constructive interference- when 2 polarized, coherent waves are in phase, causing light to be brighter; Destructive interference- when 2 polarized, coherent waves are in opposite phases and cause light to be dimmer or even disappear completely.

Question 14

What kind of technique is used to increase contrast between intracellular structures of similar transparency?

Answer 14

Interference of polarized light (DIC, Phase contrast)

Question 15

What is phase contrast?

Answer 15

form of light microscopy modification in which changes in phase shifts convert into changes in brightness. important: utilizes the differences in DENSITY of intracellular elements.

Question 16

what is Differential Interference Contrast (DIC)? what is another name for this technique?

Answer 16

DIC is a light microscopy modification that produces pseudo 3D images (shadow) based on diffrence in THICKNESS. DIC is also known as Nomarski and good to use for unstained samples.

Question 17

Explain what Nomarski methods reveals in terms of cells.

Answer 17

Normarksi shows that all human cells have similar principle organization. for instance, viewing a fibroblast, you can see the plasma membrane, nucleus (genetic info stored/expressed), nucleolus, membrane vesicles (sort, transport, digest materials) , and cytoskeletal fibrils (support cell structure, intracellular transport)

Question 18

What is Biological Staining? What are two examples of the specific staining procedures?

Answer 18

method developed in the 1800s to help identify cell structures of complex tissues that are colorless. initially discovered artificially made alanine dyes Gram staining which helps distinguish between 2 forms of bacteria (gram negative and gram positive). Golgi staining uses silver nitrate to stain neutron (dendrites and axon).
many of stains affected cell viability by denaturation (affect chemical structure of biomolecule)

Question 19

What are vital stains?

Answer 19

The few stains that did not affect viability of cells.

Question 20

What kind of method is widely used in live/dead (viability) assay?

Answer 20

Trypan Blue staining- that penetrates through dead, non-viable cells without killing viable cells.

Question 21

What is the most common staining technique used in histopathology?

Answer 21

H & E stain (Hematoxylin/Eosin). This method is commonly used in blood smears, complete blood count, and helps visualize and Identify types of WBC’s

Question 22

Compare and contrast the staining in Hematoxylin and Eosin staining method.

Answer 22

Hematoxylin (positively charged, basic) stains nucleic acids (negatively charged due to phosphate backbone) in the nuclei with strong deep blue color; Eosin is the dye that has bromine and stains all proteins with pink color, to highlight cell bodies.

Question 23

what characterizes the different types of WBC’s? Provide examples of the WBC’s and the stain that it includes.

Answer 23

WBC’s characterized by shape of nuclei and cytoplasmic intrusions (granules). Basophils contain hematoxylin in their nuclei and granules. Their granules contain heparin (blood thinner, or anticoagulant) that is negatively charged like neg-charged DNA that binds Hematoxylin. Eosinophils have a large number of eosin positive granules that are filled with proteolytic enzymes cells release to kill infectious agents.
Hemocytometer used to count WBC’s and detect pathogenic forms like leukemia

Question 24

Explain what Immunohistochemistry is. Provide an example.

Answer 24

Staining method that uses antibodies which are linked to a enzyme or fluorescent dye (immunoglobulins) to detect the locations of specific proteins or antigens in tissue sections. EX: conjugated enzyme HRP (horseradish-peroxidase) linked rabbit antibody will produce large stain molecules to highlight antigen of interest (human antigen)

Question 25

What is required for immunohistochemistry staining to occur?

Answer 25

the cells must be chemically FIXED and SLICED. common fixatives or preservatives are formaldehyde, glutaraldehyde, ethanol . Fixation will preserve shape, while the membrane is treated with DETERGENTS (create holes) to allow protein detecting antibodies to go inside cell. after fixation, tissues are embedded in harder material (ice wax, resin) and will be cut with MICROTOMES.

Question 26

What is the relationship between frequency, wavelength and energy of electromagnetic waves ?

Answer 26

The shorter the wavelength, the higher the energy of em waves, higher frequency; longer the wavelength, the lower the energy of EM waves; gamma/violet has shortest wavelength and radio/red has longest wavelength

Question 27

Compare and contrast Electron microscopy and light microscopy.

Answer 27

Light microscopy- uses beam of light to illuminate, work on dead or live cells, needs to be fixed, longer wavelength than EM.
Electron microscopy- uses beam of electrons, only works on dead cells, expensive tool, shorter wavelength, higher resolution and magnification

Question 28

Describe the two types of Electron Microscopy

Answer 28

TEM (transmission electron microscopy)- a beam of electrons is transmitted (passed through) (1 direction) a specimen to form an image. Uses electron gun and allows visualization of internal cell structures on tissue cross-sections. Must be thin tissues.
SEM (Scanning electron microscopy)- scan the surface with a beam of electrons to detect the secondary e- reflected off the region (different directions). used to study Surface and shape of multicellular structures.

Question 29

Compare and contrast TEM AND SEM

Answer 29

TEM- only have 2D images, internal structures, highest resolution (0.2 nm)
SEM- 10 times lower resolution (0.2nm ) than TEM, 3D images, see surface of solid objects
must be preserved, dried without shrinkage (critical point drying) and and be coated with gold or platinum.

Question 30

How do you prepare a sample for Electron microscopy?

Answer 30

Tissue sections must be very THIN (50 nm or 500 Angstrom). materials must be embedded in very hard (epoxy) RESINS, and cut with ultramicrotomes with diamond knives. Use microscopic COPPER grids to hold the samples and stain them with URANIUM and LEAD SALTS for enough contrast.

Question 31

Why does EM have higher resolution than Light microscopy?

Answer 31

Electrons are both negatively charged particles and waves, hence they have a shorter wavelength and higher resolution.

Question 32

Explain the immunogold technique, and kinds of tech involved.

Answer 32

method of visualizing SPECIFIC molecules using TEM and immunostaining with GOLD BEAD-CONJUGATED ANTIBODIES.
process- thin sample cut, stained with antibody that will bind molecule of interest, then secondary antibody attached with gold beads attach to primary antibody, and e- dense gold particle seen under microscope as black dot, indirectly labeling molecule of interest.

Question 33

What is a freeze- fracture technique?

Answer 33

Method of rapidly freezing a frozen bio sample in and making it solid, stuck in one place, in order to break apart (fracture) the sample and view the plane or surface, details of structure as it creates replica. It is very useful to image fluid structures like CELLULAR MEMBRANES,

Question 34

Explain the process of Fluorescent microscopy.

Answer 34

method of using fluorescence to visualize internal structures of cells. highly specific, sensitive and reliable. process of absorbing light at excitation spectrum (e- to high energy state) and emitting light at lower wavelength, to lower energy state.

Question 35

What is a fluorescent chromophore?

Answer 35

The part of the molecule that is responsible for its color and defines their fluorescent properties. They are all derived from proteins found in MARINE ORGANISMs(polyps, corals). The first chromophore was green fluorescent. specific color and technical characteristics.

Question 36

Describe the Discovery of GFP and how it is used.

Answer 36

GFP (Green Fluorescent Protein)- intrinsic fluorescent protein from Jelly fish that has greatly revolutionized biomed research. GFP absorbs blue light and emits green fluorescent light. GFP used for whole body imaging, highlight tumors, cancers, lipid. GFP has amino acids serine, tyrosine and glycine form chromophore.
GFP co-discovered with fluorescent protein aequorin (emits light in presence of Ca+)

Question 37

Explain the importance of immunofluorescence.

Answer 37

Immunofluorescence is method of antibodies conjugated to fluorophore to make a single protein fluoresce. normally requires FIXATION and MEMBRANE PERMEABILIZATION. label cells with precision.

Question 38

What is Super-resolution fluorescent microscopy?

Answer 38

allows proportional imaging of objects as small as 20 nm. Uses STED (Stimulated-emission Depletion) phenomenon- when excited fluorophore reaches photon that is closer in energy to normal photon emitted, it will prevent fluorophore from emitting original photon (emit lower energy photon that is eliminated in visual field). STED uses two layers to simultaneously excite an object (one beam inside another). enhances resolution (sharper image than confocal)

Question 39

what are 2 advantages fluorescent microscopy has over bright-field microscopy?

Answer 39

1. fluorescence not limited by light wavelength, since the light of molecule is not reflected. Object is source of light.
2. Higher contrast and sensitivity because the only fluorescence light is detected (not light from background surface)

Question 40

What is FRET? Explain what it is used for.

Answer 40

FRET (Fluorescence or Foster Resonance Emission Technology)- used to detect protein-protein interactions in vivo with molecular precision when protein interacts with fluorescence fused protein, instead of emitting and being fluorescent itself, it will excite another fluorescent protein in close proximity (so it will fluoresce without direct excitation of additional light)

Question 41

What are Fusion proteins used for?

Answer 41

Fusion proteins used to visualize proteins in live cells. Fluorescence proteins may be light emitting indicators (sensors) to rapidly measure changes in intracellular ion concentration, pH and membrane potential.

Question 42

What is FRET? Explain what it is used for.

Answer 42

FRET (Fluorescence or Foster Resonance Emission Technology)- used to detect protein-protein interactions in vivo with molecular precision when protein interacts with fluorescence fused protein, instead of emitting and being fluorescent itself, it will excite another fluorescent protein in close proximity (so it will fluoresce without direct excitation of additional light) Excite violet light and emit blue light- no interaction. If excite violet light and emit yellow light- there is an interaction .

Question 43

Describe two-multi photon microscopy.

Answer 43

method to achieve images of living things that are high in thickness. Simultaneously excites 2 photons with longer wavelength than emission and are lower in energy to penetrate deep tissues also creating less tissue damage and imaging farther in samples reduces out of focus emission, since only objects in focus have enough red light intensity to be emitted.
2 photons of low energy are coherent (same frequency) have same excitation of fluorophore that is achieve with one photon

Question 44

What makes red fluorescence proteins unique?

Answer 44

Red, longer wavelengths are able to penetrate tissues, because wavelength that are equal or larger can bypass the object. However, Em waves of shorter wavelength (high energy) reflect better off objects. 
 Near Infrared (far red) light can penetrate deeper in tissues in living organisms non-destructive form of red light can detect near infrared FP's that measure size of tumor and its location without harming organism.

Question 45

What are the resolution ranges that can be seen with different forms of microscopy?

Answer 45

Human naked eye- 0.2 mm or 200 um, Light microscopy- no smaller than 0.2 um or 200 nm. Super resolution microscopy- 20 nm. EM microscopy- 0.2 nm.

Question 46

What are the resolution ranges that can be seen with different forms of microscopy?

Answer 46

Human naked eye- 0.2 mm or 200 um (cells), Light microscopy- no smaller than 0.2 um or 200 nm; cells and organelles. Super resolution microscopy- 20 nm; cells-molecules. EM microscopy- 0.2 nm, cells- atoms.

Question 47

What is stoke’s shift?

Answer 47

Difference (shift) between excitation and emission and wavelengths
maximum of absorption wave and max of fluorescence wave. Defines fluorescent emission occurs at longer wavelength than absorbed light.

Question 48

What is hyperemia?

Answer 48

Swelling and redness caused that occurs when histamine dilates (widen) blood vessels. Histamine is found in Basophilic granules and is a mediator of allergic and inflammatory responses.

Question 49

Neutrophils

Answer 49

most common WBC. neutral staining (neither hemtoxylin, nor eosin). They are part of innate immune system, nuclei are hematoxylin positve, and release cytokines at site of inflammation.