L7: Fluorescence Spectroscopy

Question 1

What is fluorescence spectroscopy?

Answer 1

A technique that studies how light interacts with matter, focusing on the excitation and emission of light by fluorophores, which are molecules that fluoresce

Question 2

How does fluorescence spectroscopy compare in sensitivity to UV spectroscopy?

Answer 2

Fluorescence spectroscopy is approximately 1,000 times more sensitive than UV spectroscopy

Question 3

What is the Stokes shift in fluorescence spectroscopy?

Answer 3

• Assesses the fluorophores efficiency
• Measures the energy difference between the absorption and emission wavelengths, indicating how much energy is lost before emission occurs /fluorescence quality

Question 4

Why is the detector placed at a 90-degree angle in fluorescence spectroscopy?

Answer 4

To minimize signal-to-noise interference and improve measurement accuracy

Question 5

What is the function of fluorophores?

Answer 5

Molecules that absorb light at one wavelength and emit it at another, allowing visualization and tracking in biological samples

Question 6

Define intrinsic and extrinsic fluorophores

Answer 6

Intrinsic fluorophores are naturally fluorescent, while extrinsic fluorophores require an external dye or stain to fluoresce

Question 7

What factors affect fluorescence intensity?

Answer 7

Factors include excitation wavelength, emission wavelength, solvent polarity, viscosity, and pH of the environment

Question 8

Give an example of an intrinsic fluorophore

Answer 8

Tryptophan, an amino acid often found in proteins, naturally fluoresces and is used in biological studies

Question 9

How can fluorescence spectroscopy be used in live cell imaging?

Answer 9

It can monitor real-time cellular processes like protein folding, cell viability, and calcium influx

Question 10

What is GFP, and why is it significant in fluorescence studies?

Answer 10

• GFP (Green Fluorescent Protein) is a protein from jellyfish that fluoresces green, widely used as a marker in cell and molecular biology
• Allows for the tracking and visualisation of cellular processes without the need for additional staining

Question 11

Describe how GFP (Green Fluorescent Protein) is modified for different studies

Answer 11

Through genetic mutation, GFP can be engineered to emit various colors, allowing for detailed multicolor imaging of cellular components

Question 12

How does solvent viscosity impact fluorescence?

Answer 12

Higher viscosity restricts molecular movement, enhancing fluorescence intensity by reducing vibrational energy loss

Question 13

What is the relationship between fluorescence emission intensity and excitation intensity?

Answer 13

Emission intensity is directly proportional to the excitation intensity

Question 14

Why is pH stability important in fluorescence spectroscopy?

Answer 14

• Variations in pH can change the emission and absorption properties of fluorophores
• Changes in pH can alter the fluorescence spectra, making pH buffers crucial for consistent results

Question 15

Name an application of fluorescence spectroscopy in cancer research

Answer 15

It can assess tumor stages by measuring emission intensity variations in response to different concentrations of tryptophan in tumor tissues

Question 16

What is flow cytometry, and how does it use fluorescence?

Answer 16

• A technique that sorts and counts cells based on fluorescence, and for biomarker identification
• Cells are tagged with fluorophores, and are sorted by the flow cytometry based on fluorescence
• Allows for detailed analysis of cell populations, such as immune cell markers

Question 17

What is meant by “excitation wavelength” in fluorescence spectroscopy?

Answer 17

The specific wavelength of light absorbed by a fluorophore, causing it to emit light at a different, longer wavelength

Question 18

What is the primary purpose of a monochromator in fluorescence spectroscopy?

Answer 18

To separate light into different wavelengths and select the optimal wavelength for excitation

Question 19

Explain the significance of the emission spectrum in fluorescence spectroscopy

Answer 19

The emission spectrum represents the range of wavelengths emitted by a sample after excitation, indicating the sample’s fluorescent properties

Question 20

Why are fluorophores used in biological imaging?

Answer 20

They enable visualisation of biological structures and processes that are otherwise invisible, enhancing details through fluorescence

Question 21

How does fluorescence spectroscopy help monitor protein folding?

Answer 21

It detects changes in protein structure by tracking the emission spectrum, showing folding and unfolding events in real-time

Question 22

Describe an application of fluorescence in cancer diagnosis

Answer 22

By measuring fluorescence intensity from tryptophan in tumor cells, it helps determine cancer stages, with higher intensity indicating more advanced stages

Question 23

What role does viscosity play in fluorescence intensity?

Answer 23

Higher viscosity limits molecular rotations and vibrations, enhancing fluorescence by conserving energy for emission

Question 24

What is a key difference between UV and fluorescence spectroscopy?

Answer 24

Fluorescence spectroscopy measures both excitation and emission, whereas UV spectroscopy only measures absorbance

Question 25

How does solvent polarity affect fluorescence?

Answer 25

Polar solvents can shift the emission wavelength, altering the fluorescence intensity and spectrum observed

Question 26

What is an extrinsic fluorophore, and when is it used?

Answer 26

An externally added dye or stain used to induce fluorescence in samples that lack intrinsic fluorescence

Question 27

What does the term “signal-to-noise ratio” mean in fluorescence spectroscopy?

Answer 27

The ratio of useful fluorescence signal to background noise, with higher ratios indicating clearer, more accurate spectra

Question 28

How is fluorescence spectroscopy used in drug testing?

Answer 28

By detecting metabolite levels in urine, it reveals how the body processes drugs, useful in both medical diagnostics and anti-doping tests

Question 29

Why is fluorescence spectroscopy useful in live cell analysis?

Answer 29

Its sensitivity allows for non-invasive, real-time monitoring of cellular functions, like metabolic activity and protein interactions

Question 30

What is the relationship between energy and wavelength in fluorescence?

Answer 30

Higher energy corresponds to shorter wavelengths, while lower energy corresponds to longer wavelengths

Question 31

In what way does fluorescence spectroscopy assist in calcium ion studies?

Answer 31

By using fluorophores sensitive to calcium, it monitors calcium influx in cells, critical for understanding cellular signaling and muscle contraction

Question 32

What kind of fluorophores are typically used for cancer imaging?

Answer 32

Intrinsic fluorophores like tryptophan and extrinsic dyes are used to differentiate cancer stages based on fluorescence intensity

Question 33

Why is fluorescence spectroscopy preferred for studying metabolic processes?

Answer 33

Its high sensitivity allows for detection of low metabolite concentrations, essential for tracking metabolic changes and diagnosing disorders

Question 34

How does fluorescence help in studying cardiac tissue?

Answer 34

By tagging tissue with voltage- and calcium-sensitive fluorophores, fluorescence spectroscopy can monitor heart contractions and action potentials

Question 35

What advantage does fluorescence spectroscopy offer over traditional staining in microscopy?

Answer 35

It enables dynamic, high-sensitivity observation of cells with minimal sample quantities, allowing real-time analysis without excessive manipulation