fluorescence 2

Question 1

What is a longpass filter?

Answer 1

Reflects light shorter than a certain wavelength
Transmits light wavelength that are longer than that

Question 2

What is a bandpass filter?

Answer 2

Transmit light centered around a value, within a range specified

Question 3

What is a shortpass filter?

Answer 3

Reflects light wavelength longer than a certain than a certain value
Transmit light equal to or shorter than the wavelength

Question 4

What is a dichroic mirror also called?

Answer 4

Beam Splitter

Question 5

How is dicrhoic mirror placed and what is its purpose?

Answer 5

45 angle to the incident (Ex or Em) light
–> can be shortpass or longpass
–>Part of the light is reflected at 90 degree to the incident light, and part of the light is transmitted

Question 6

How does the <Fluorescence>
Improve Your Fluorescence Experiments?</Fluorescence>

Answer 6

1. visualize the excitation & emission profiles of different fluorochromes
2. Identify areas of spectral spillover with the use of >1 fluorochromes
3. select laser excitation lines, filter configurations & fluorochromes
4. optimize the use of emission filters to pick useful signals
5. avoid experimental failure

Question 7

What is the purpose of a pinhole being placed in front of a detector in fluorescence plate reader?

Answer 7

to Reduce Light Contaminations

Question 8

Name a biological technique that utilizes fluorescence plate reader

Answer 8

Real-Time - Polymerase Chain Reaction

Question 9

What do the terms ‘bottom-up’ and ‘top-down’ imply in fluorescence plate readers?

Answer 9

bottom up: light directed at sample from bottom

top up: light directed at sample from top

Question 10

What are the different well formats and what are their characterisitcs?

Answer 10

Flat- (higher sample vol, higher light transmission),

U- (Washing Mixing & Shaking),

V-Bottom ( lower Sample Vol required)

Question 11

Plates with how many wells are commonly used as microplates for plate reader? What is their volume?

Answer 11

96 (100-300 μL)

384 (30-100 μL)

1,536 (1-5 μL)

Question 12

What could be used for surface coating of wells in microplate for fluorescence? What purpose does it serve?

Answer 12

Poly-D-Lysine (+ve Charge, Protease-Resistant) for Adherent Cells

Question 13

Which fluorophore is used for RT PCT?

Answer 13

SYBR Green (SyberGreen)

Question 14

What are the characteristics of SYBR green that make it suitable for RT PCR?

Answer 14

1. binds to minor groove of dsDNA –> not a carcinogen
2. does not bind to ssDNA
3. stable over a range of temperatures
4. wide dynamic range of up to 4 orders of magnitude
   –> accurate DNA quantification

Question 15

What are the 3 steps of PCR?

Answer 15

1) Denaturation (95C)
2) Annealing (55C)
3) Extension (72C)

Question 16

What happens during denaturation in PCR?

Answer 16

dsDNA template –> 2 ssDNAs

Question 17

What happens during annealing in PCR?

Answer 17

Primers anneals to each original strand for new DNA strand synthesis

Question 18

What happens during extension in PCR?

Answer 18

New DNA strands from the primers, DNA Elongation from 5’- to -3’-End

Question 19

What kind of ‘replication’ in PCR?

Answer 19

Semi-Conservative Replication

Question 20

Around how many cycles of amplification are there in PCR?

Answer 20

40 cycles

Question 21

What enzyme is used in PCR?

Answer 21

Taq polymerase

Question 22

At what step do we measure SYBR Green-I fluorescence in PCR?

Answer 22

extention (at 72C)

–> The extension step is when the maximum amount of dsDNA is present in the reaction.

Question 23

Define ‘Ct’ in PCR

Answer 23

Ct (Threshold Cycle Value) :

Cycle Number at which fluorescence generated in a PCR reaction crosses a pre-set fluorescence threshold

Question 24

What are fluorescence polarization assays commonly used for?

Answer 24

To Identify Enzyme Inhibitors

Question 25

What is light?

Answer 25

interaction of Electric ( Moves in One Direction) & Magnetic ( in a Perpendicular Direction) fields traveling through space

Question 26

Does orientation of light change during propagation?

Answer 26

does NOT change during Propagation.

Question 27

What is the difference between measuring fluorescence and fluorescence polarization?

Answer 27

FP is the measurement of an output of Em in Polarization Planes

Question 28

How does FP differ in small molecules and large molecules?

Answer 28

Larger molecules: Rotate more slowly within the fluorescence lifetime. Emit light that retains polarization, resulting in high FP. Smaller molecules: Rotate faster (e.g. free fluorophores) Emit depolarized light

Question 29

What is meant by F lifetime?

Answer 29

time it takes for a fluorochrome to return to its ground state after Ex

Question 30

What is the typical range of F time?

Answer 30

Pico (10-12) - Nano (10-9) sec

Question 31

Explain how FP could be used to study enzyme inhibitors

Answer 31

1.enzyme substrate attached with fluorochrome 2. add enzyme and potential inhibitor 3. inhibitor block enzyme activity --> less substrate cleaved --> higher polarization

Question 32

What are the advantages of FP?

Answer 32

* Non-Radioactive Assay * Only one binding partner has to be fluorescent * Simple, Fast, Convenient (No Need to Wash) --> Sample-to-Answer Approach * Fast Screening of Inhibitors against a given Enzyme * Automation possible --> High-throughput Screening by IC50 Comparison

Question 33

What is the limitation of FP?

Answer 33

Limited by the Change in Molecular Size after Interactions, Dye’s Lifetime

Question 34

How does fluorescence microscope differ from conventional microscope?

Answer 34

Fluorescence Microscope : Use the Same Objective Lens for 1) Focusing Ex Light & 2) Collecting Em Light from the specimen. * Epi: On the Same Side

Question 35

Why is immersion oil used in inverted microscope with 60x and 100x lens?

Answer 35

60X, 100X with Short Focal Length: Coverslip Facing the Objective Lens --> less work distance Thus immersion oil reduce signal loss

Question 36

In inverted microscopy, where is the immersion oil use when 60x or 100x lens are used?

Answer 36

between the objective lens and the coverslip

Question 37

What is the purpose of using immersion oil in inverted microscopy?

Answer 37

because refraction index of immersion oil is similar to that of glass (1.52) --> reduce signal loss

Question 38

What are some limitations of epi-fluorescence microscopy??

Answer 38

1. converts 3D image to 2D image -->Loss of depth information 2. Lack of Optical Sectioning --> overlap of signals from different planes thus blurred image 3. Fixed Light Source Lifetime 4. Photobleaching and Phototoxicity

Question 39

What is the major difference between epi-fluorescence microscope and confocal microscope in terms of their design?

Answer 39

Epi: Both n-focus and out-of-focus emitted light are collected by the detector --> blurry image Confocal: USE PINHOLE -->Only in-focus emitted light from the focal plane pass thru pinhole and reaches the detector --> sharper image

Question 40

Does confocal microscopy require a sensitive em detector?

Answer 40

Yes

Question 41

What is the purpose of scanning mirrors in confocal microscopy?

Answer 41

Moves the laser beam across the sample in the XY plane.

Question 42

What does the scanning rate in confocal microscopy depend on?

Answer 42

Mirror rotation speed on scanning mirrors higher speed = faster scanning rate

Question 43

What is the standard video rate in confocal microscopy?

Answer 43

30 frames/sec

Question 44

What are the 2 main applications of confocal microscopy?

Answer 44

1. structural analysis 2. temporal analysis (kinetic study)

Question 45

Briefly explain 'temporal analysis' which is done using confocal microscopy

Answer 45

Confocal microscopy can capture the same optical section repeatedly over time (step size = 0) to monitor dynamic processes.

Question 46

What are the advantages of using confocal microscopy?

Answer 46

1.Spatial & temporal studies in 3D or 4D (x, y, z, time) way 2.Single cell assay, Excellent sub-cellular resolution 3.Live or fixed cell analysis possible 4.Multiplex, depends on the use of the fluorescent dyes 5.High resolution

Question 47

What are the disadvantages of using confocal microscopy?

Answer 47

1. Small sample size 2. Time-consuming for analysis 3. High degree of skill required 4. Low throughput 5. Semi-quantitation