Ch 9: Fluorescence Flashcards
Why is a fluorescent dye superior to colorimetric dyes?
Fluorescent dyes can be excited at specific wavelengths, enabling selective detection and higher sensitivity
What is photobleaching? How can it be minimized?
Photobleaching is the loss of fluorescence due to prolonged exposure to excitation light. It can be minimized by using lower light intensity, anti-fade reagents, or minimizing exposure time Loss of fluorescence due to light exposure; minimized with anti-fade agents and reduced light intensity.
Describe the fluorescence of NAD+ vs. NADH.
NAD+ does not fluoresce, whereas NADH emits fluorescence at ~460 nm upon excitation
What is fluorescence?
The emission of light by a substance that has absorbed light or electromagnetic radiation, typically at a longer wavelength.
What are fluorophores?
Molecules that absorb light at a specific wavelength and re-emit it at a longer wavelength.
Define Stokes shift.
The difference between the excitation wavelength and the emission wavelength of a fluorophore.
What are common uses of fluorescence in biosensors?
DNA detection, protein analysis, and cellular imaging.
How is autofluorescence different from fluorescence?
Autofluorescence is the natural emission of light by biological substances without the addition of fluorophores.
Why is fluorescence more sensitive than colorimetric detection?
Fluorescence can be detected at lower concentrations due to its higher signal-to-noise ratio.
What is photobleaching?
The irreversible loss of fluorescence due to prolonged light exposure.
How can photobleaching be minimized?
Using anti-fade reagents, reducing light intensity, or limiting exposure time.
What are GFP and SYBR Green?
GFP (Green Fluorescent Protein) is a natural protein-based fluorophore; SYBR Green is a synthetic dye used for nucleic acid staining.
What is a DNA intercalating dye?
A dye that inserts itself between DNA base pairs and fluoresces upon binding.
How do optical filters enhance fluorescence detection?
They isolate specific wavelengths for excitation and emission, reducing background noise.
What is the role of NADH in fluorescence?
NADH fluoresces under UV light, useful in metabolic studies.
What is the difference between 180° and 90° fluorescence detection?
180° detects fluorescence in the same direction as excitation light, while 90° detects it orthogonally to minimize scattering.
Why is fluorescence used in urine analysis?
Urine contains autofluorescent compounds that can indicate health conditions.
How does chlorophyll exhibit fluorescence?
It absorbs light in the blue and red regions, emitting fluorescence in the red region.
What are quantum dots?
Semiconductor nanocrystals that fluoresce with high brightness and stability, used in imaging and diagnostics.
What is the principle of a fluorescence spectrometer?
It measures emitted light intensity across wavelengths after a sample is excited at a specific wavelength.
Why is fluorophore stability important in biosensors?
Stability ensures consistent and reliable fluorescence signals over time.
What is FRET (Förster Resonance Energy Transfer)?
A technique where energy transfer between two fluorophores is used to study molecular interactions.
How are fluorescent dyes sorted by excitation/emission wavelength?
Based on their chemical structure, which determines the wavelengths they absorb and emit.
Q: Why is a fluorescent dye superior to colorimetric dyes?
A: Fluorescent dyes allow selective excitation and higher sensitivity.
Q: How do you sort fluorescent dyes by excitation/emission wavelength?
A: Examine their chemical structures to determine absorption/emission properties.
Q: What is a DNA intercalating dye?
A: A dye that inserts between DNA bases and fluoresces upon binding.
Q: What is photobleaching? How can it be minimized?
A: Loss of fluorescence due to light exposure; minimized with anti-fade agents and reduced light intensity.
Q: How does NAD+ fluorescence differ from NADH?
A: NADH fluoresces, while NAD+ does not.
Q: Which urine component causes autofluorescence?
A: Urobilinogen; excitation: blue, emission: green.
Q: What colors does chlorophyll absorb and emit?
A: Absorbs red/blue, emits red fluorescence.
Q: How can fluorescence emission be isolated?
A: Using optical filters, angled detection, or optical fibers.
Q: How does fluorescein intensity differ above/below the air-water interface?
A: Reflectance varies due to refractive index changes.
