fluorescence 1

Question 1

What is fluorescence?

Answer 1

Certain molecules (fluorochromes) absorb energy from high-energy light & then release a portion of the energy also in the form of light but with a longer wavelength

Question 2

State the first law of thermodynamics

Answer 2

Energy cannot be created or destroyed.
Energy can be transferred from one system to another in many forms

Question 3

Range of visible light

Answer 3

400 - 700 nm

Question 4

Range of UV

Answer 4

short wave: 200-280nm
middle wave: 280-315nm
long wave: 315-400nm

Question 5

What is 1nm in m?

Answer 5

10−9 m

Question 6

What are some features of fluorescence?

Answer 6

• Outside Energy is Required
• Absorption of Light in 10 -15 sec
• Emission of Light in 10 -12 sec
• Peak of Wavelength: Ex < Em

Question 7

What happens during ‘photobleaching’?

Answer 7

the fluorophore is exposed to intense light for too long
–> excited electrons interact with ROS or undergo photochemical reactions
–> photobleaching

Question 8

Suggests applications of photo-bleaching in fluorescence.

Answer 8

1. study the fluidity of membranes and the Membrane Mosaic Model
2. photodynamic therapy (PDT)

Question 9

Explain the principle of photodynamic therapy

Answer 9

Cancer cells loaded with photo-sensitizer (fluorochromes) –> High Energy Laser –> Photo-bleaching –> Tissue O2 (e- Acceptor) –> ROS –>Toxic Effects –> Cancer Killing

Question 10

What is used for skin care involving photodynamic therapy?

Answer 10

ALA (5-AminoLevulinic Acid)

–> blue light laser

Question 11

What are the 3 categories of fluorophore?

Answer 11

1. from conjugate with DNA
2. conjugate with Ca2+ –> FURA-2
3. does not need to form conjugate to exhibit fluorescence

Question 12

Name fluorophores that form conjugate with DNA

Answer 12

Ethidium bromide (EB)
Propidium iodide (PI)

Question 13

Name fluorophores that do not need to form conjugate to exhibit fluorescence

Answer 13

Fluorescein (FITC)
Rhodamine (Rh)

Question 14

What are the steps involved in Immunofluorescence (IF) Assay to reveal cellular structures?

Answer 14

1. Living Human Cell in Culture Dish
2. Fixation
3. membrane permeabilization
4. first Ab, from mouse (against tubulin)
5. 2nd Ab which is against mouse (from rabbit),
   conjugated with FITC
6. DAPI stains nuclear DNA
7. flurescent microscope
8. indirect immuno-fluorescence assay

Question 15

What is used for cross-linking and coagulation during immunoflourescence assays>

Answer 15

• Formaldehyde (Cross-linking)
• Ethanol (Coagulation)

Question 16

What is the purpose of cross linking in immmunofluorescence assay ?

Answer 16

Fixatives like formaldehyde or glutaraldehyde react with functional groups of proteins (e.g., amines, sulfhydryls) to form covalent bonds

Question 17

What is the purpose of coagulation in immmunofluorescence assay ?

Answer 17

Coagulation involves the precipitation or aggregation of proteins, effectively denaturing them to immobilize cellular components.

Question 18

What is used for membrane permeabilization in immunofluorescence (IF) assays?

Answer 18

Triton-X 100 or Saponin

Question 19

Name a fluorescent dye used to stain DNA in immunofluorescence assays

Answer 19

DAPI (4′,6-diamidino-2-phenylindole)

Question 20

What are some methods to overcome photobleaching?

Answer 20

• Reducing light intensity exposure to fluorophores.
• Reduce sample exposure
• Use Antifade agent (reactive oxygen species scavengers
  like Vitamin C)
• Use different dyes, Some dyes are more susceptible to
  bleaching than others

Question 21

What are the advantages of indirect immunofluorescence assays over direct ones?

Answer 21

1. Amplified signal because multiple secondary antibodies can bind to one primary antibody.
2. Cheaper because unconjugated primary antibodies are more affordable.
3. More flexible; the same secondary antibody can be used with different fluorophores.
4. Preferred for detecting low-abundance proteins or enhancing signal in detailed imaging.
5. One secondary antibody can be used with multiple primary antibodies from the same species, reducing the need for multiple reagents.

Question 22

What are the advantages of direct immunofluorescence assays over indirect ones?

Answer 22

Simpler and faster, requiring only one antibody step.

Question 23

A Super-Resolution Imaging Technique was developed in 2018 using (1) Imidazole, (2) a His-tag
ligand conjugated with a fluorophore and (3) a recombinant His-tag protein target (Sci Rep,
2018, 8:5507). How does this technique improve the image quality? Explain your answers

Answer 23

Imadazole compete for non-specific binding which leads to higher signal to noise ratio

Question 24

In immunofluorescence assay, what is the controll set up?

Answer 24

• Control 1: No 1st & 2nd Abs Added –> Test the Sample Autofluorescence (from Vitamin B, FAD, FMN & Pigments)
• Control 2: Test the Non-Specific Binding from 2nd Ab without 1st Ab Addition

Question 25

How to avoid bacterial infection when restoring Ab for furthur use?

Answer 25

Add Sodium Azide

Question 26

Ethidium bromide is a carcinogen. Name an alternative safe DNA-binding dye.

Answer 26

SYBR Green

Question 27

What is the purpose of immunofluorescence assay?

Answer 27

to Reveal Cellular Structures

Question 28

State the design of a spectrophotometer

Answer 28

Light source --> Collimator (lens) --> mochochromator --> wavelength selector (slit) --> sample solution --> detector

Question 29

What is the conc of Ca2+ in cell cytoplasm during resting and activated phase?

Answer 29

Resting: 100 - 150 nM Activated: 1000 - 1500 nM

Question 30

What is structure of Fura-2 (penta potassium salt) base on?

Answer 30

Use EGTA as the Backbone to bind Ca2+ ions Add Ring Structures --> Fluorescence

Question 31

Ex and Em of Fura-Ca2+

Answer 31

Ex at 340nm Em at 510nm

Question 32

Why is it not possible to prepare standard solutions with known conc directly by checmical weighting and serial dilutions?

Answer 32

Ca2+ in conc of 1mM or less is easily affected by environment

Question 33

What is used as a buffer system to generate stable low Ca2+ solution?

Answer 33

EGTA, which is a pH buffering agent to control solution pH

Question 34

What is a dichroc mirror?

Answer 34

A Glass Lens with special coating: Light lamda > xx nm Pass-through; Light lamda < xx nm Reflected.

Question 35

What is the isosbestic point?

Answer 35

At 360 nm, fluorescence is Insensitive to the change in [Ca2+] --> Isosbestic Point.

Question 36

Describe what happens at 340nm, 360nm and 380nm for ex during fura-Ca2+

Answer 36

At 360 nm --> Isosbestic Point. At 340 nm for Ex, fluorescence intensity increase as [Ca2+] increase At 380 nm for Ex, fluorescence intensity decrease as [Ca2+] increase

Question 37

What if there is no convergent point at 360nm?

Answer 37

Pipetting Errors

Question 38

How is ratiomertric measurement carried out?

Answer 38

lamda1(380nm)/lamda3(340nm) The bigger the ratio, the more sensitive the measuremtn

Question 39

What are the advantages of ratiometric measurement?

Answer 39

* Eliminate Photo-bleaching Effect * More Sensitive Measurement * Removal of Artifacts from Uneven Cell Thickness * Internal Normalization --> Fair Comparison

Question 40

What happens during membrane depolarization?

Answer 40

Voltage-gated Ca2+ Channel Opening --> increase in Ca2+ gradient --> Ca2+ influx

Question 41

How to Load Fura-2 into Cells for Cell-based Assay?

Answer 41

- Microinjection or - Fura-2 / AcetoxyMethyl Ester (for permeability) - diffusion - shaking & incubation --> washing - esterase cleave AM ester group

Question 42

What happens to Fura-2 / AcetoxyMethyl Ester when it is diffused into cell?

Answer 42

esterase cleave Fura2 / AM ester group into Fura 2 and acetic acid

Question 43

Fura 2 is polar while Fura/AM ester is nonpolar. True or false

Answer 43

True

Question 44

What are some parameters that have to be considered during microinjection of Fura-2 into cells?

Answer 44

Needle shape Volume: Nano Liter Pressure Angle: 45 degrees Duration: < 5s

Question 45

Advantages of Fura-2/AM:

Answer 45

1. Ease of Use: -No need for specialized equipment like microinjection needles. - Allows simultaneous loading of multiple cells in a population. 2. Non-Invasive: -Passive diffusion avoids physical damage to cells. 3. Efficient Loading: -Quick and effective for large-scale experiments

Question 46

Disadvantages of Fura-2/AM:

Answer 46

1. Dependence on Esterase Activity 2. Background Signal --> Excess Fura-2/AM may remain outside the cell, causing non-specific fluorescence. 3. Potential Toxicity by High concentrations of Fura-2/AM 4. Limited Control --> not possible to control the exact amount of dye loaded into individual cells.