L7: Fluorescence Spectroscopy Flashcards
What is fluorescence spectroscopy?
A technique that studies how light interacts with matter, focusing on the excitation and emission of light by fluorophores, which are molecules that fluoresce
How does fluorescence spectroscopy compare in sensitivity to UV spectroscopy?
Fluorescence spectroscopy is approximately 1,000 times more sensitive than UV spectroscopy
What is the Stokes shift in fluorescence spectroscopy?
- 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
Why is the detector placed at a 90-degree angle in fluorescence spectroscopy?
To minimize signal-to-noise interference and improve measurement accuracy
What is the function of fluorophores?
Molecules that absorb light at one wavelength and emit it at another, allowing visualization and tracking in biological samples
Define intrinsic and extrinsic fluorophores
Intrinsic fluorophores are naturally fluorescent, while extrinsic fluorophores require an external dye or stain to fluoresce
What factors affect fluorescence intensity?
Factors include excitation wavelength, emission wavelength, solvent polarity, viscosity, and pH of the environment
Give an example of an intrinsic fluorophore
Tryptophan, an amino acid often found in proteins, naturally fluoresces and is used in biological studies
How can fluorescence spectroscopy be used in live cell imaging?
It can monitor real-time cellular processes like protein folding, cell viability, and calcium influx
What is GFP, and why is it significant in fluorescence studies?
- 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
Describe how GFP (Green Fluorescent Protein) is modified for different studies
Through genetic mutation, GFP can be engineered to emit various colors, allowing for detailed multicolor imaging of cellular components
How does solvent viscosity impact fluorescence?
Higher viscosity restricts molecular movement, enhancing fluorescence intensity by reducing vibrational energy loss
What is the relationship between fluorescence emission intensity and excitation intensity?
Emission intensity is directly proportional to the excitation intensity
Why is pH stability important in fluorescence spectroscopy?
- 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
Name an application of fluorescence spectroscopy in cancer research
It can assess tumor stages by measuring emission intensity variations in response to different concentrations of tryptophan in tumor tissues
What is flow cytometry, and how does it use fluorescence?
- 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
What is meant by “excitation wavelength” in fluorescence spectroscopy?
The specific wavelength of light absorbed by a fluorophore, causing it to emit light at a different, longer wavelength
What is the primary purpose of a monochromator in fluorescence spectroscopy?
To separate light into different wavelengths and select the optimal wavelength for excitation
Explain the significance of the emission spectrum in fluorescence spectroscopy
The emission spectrum represents the range of wavelengths emitted by a sample after excitation, indicating the sample’s fluorescent properties
Why are fluorophores used in biological imaging?
They enable visualisation of biological structures and processes that are otherwise invisible, enhancing details through fluorescence
How does fluorescence spectroscopy help monitor protein folding?
It detects changes in protein structure by tracking the emission spectrum, showing folding and unfolding events in real-time
Describe an application of fluorescence in cancer diagnosis
By measuring fluorescence intensity from tryptophan in tumor cells, it helps determine cancer stages, with higher intensity indicating more advanced stages
What role does viscosity play in fluorescence intensity?
Higher viscosity limits molecular rotations and vibrations, enhancing fluorescence by conserving energy for emission
What is a key difference between UV and fluorescence spectroscopy?
Fluorescence spectroscopy measures both excitation and emission, whereas UV spectroscopy only measures absorbance
How does solvent polarity affect fluorescence?
Polar solvents can shift the emission wavelength, altering the fluorescence intensity and spectrum observed
What is an extrinsic fluorophore, and when is it used?
An externally added dye or stain used to induce fluorescence in samples that lack intrinsic fluorescence
What does the term “signal-to-noise ratio” mean in fluorescence spectroscopy?
The ratio of useful fluorescence signal to background noise, with higher ratios indicating clearer, more accurate spectra
How is fluorescence spectroscopy used in drug testing?
By detecting metabolite levels in urine, it reveals how the body processes drugs, useful in both medical diagnostics and anti-doping tests
Why is fluorescence spectroscopy useful in live cell analysis?
Its sensitivity allows for non-invasive, real-time monitoring of cellular functions, like metabolic activity and protein interactions
What is the relationship between energy and wavelength in fluorescence?
Higher energy corresponds to shorter wavelengths, while lower energy corresponds to longer wavelengths
In what way does fluorescence spectroscopy assist in calcium ion studies?
By using fluorophores sensitive to calcium, it monitors calcium influx in cells, critical for understanding cellular signaling and muscle contraction
What kind of fluorophores are typically used for cancer imaging?
Intrinsic fluorophores like tryptophan and extrinsic dyes are used to differentiate cancer stages based on fluorescence intensity
Why is fluorescence spectroscopy preferred for studying metabolic processes?
Its high sensitivity allows for detection of low metabolite concentrations, essential for tracking metabolic changes and diagnosing disorders
How does fluorescence help in studying cardiac tissue?
By tagging tissue with voltage- and calcium-sensitive fluorophores, fluorescence spectroscopy can monitor heart contractions and action potentials
What advantage does fluorescence spectroscopy offer over traditional staining in microscopy?
It enables dynamic, high-sensitivity observation of cells with minimal sample quantities, allowing real-time analysis without excessive manipulation
