Questions for Introduction for Fluorescence!

1. What is the application of fluorescence? This the technique that is used to analyze a variety of
multiple parameters of cells. There are different applications that ranges from immune phenotyping to
ploidy analysis to cell counting and GFP expression analysis.

2. How do stains emit different colors? The emitted light has lower energy, and therefore has longer
wavelength, than the absorbed light. This implies that the color of the light that is emitted is different
from the color of the light that has been absorbed. Emission of light returns the fluorophore to its
ground state. This is due to excitation and emission processes.

3. What is excitation? The process of “excitation” is when light from an external source hit a
fluorophore molecule, the molecule can absorb this light energy. If the energy that absorbed is sufficient
for the molecule, the molecule reaches a higher-energy state, which in turn is called an excited state.
The electrons of a fluorochrome can be excited by a range of wavelengths of light.

4. What is emission? Then, the fluorophore rearranges from the semi-stable excited state back to the
ground state, and the excess energy is released and emitted in a form of light. In other words, emission
is the process that creates a photon and takes the molecule in an excited state back to the ground state.
The emitted light has lower energy, and therefore has longer wavelength, than the absorbed light. This
implies that the color of the light that is emitted is different from the color of the light that has been
absorbed. Emission of light returns the fluorophore to its ground state. The fluorophore can absorb light
energy again and go through the entire process repeatedly.

5. What is fluorescence? The process or an ability to give off of the radiation usually as visible light when
exposed to radiation from another source such as ultraviolet light for example.

6. What is fluorophore? It is a molecule with fluorescence properties, it can fluoresce, the molecule that
absorbs the light and then emit it.

7. What is the excited lifetime? There are multiple excited states or energy levels that the fluorophore
can attain, depending on the wavelength and energy of the external light source. Since the fluorophore
is unstable at high-energy configurations, it eventually adopts the lowest-energy excited state, which is
semi-stable. The length of time that the fluorophore is in excited states is called the excited lifetime, and
it lasts for a very short time, ranging from 10-15 to 10-9 seconds.

8. Why does emitted light has a longer wavelength? Because the emitted light has lower energy than the
absorbed light.

9. What is the cycling of fluorescence? The cycling of fluorescence refers to the process by which a
fluorescent molecule absorbs light, becomes excited, emits light at a longer wavelength, and returns to
its ground state, potentially repeating the process multiple times. This process is the basis for many
fluorescence-based applications in science and technology.

10. What is the photobleaching? Photobleaching is a process that occurs when a fluorescent molecule is
exposed to intense or prolonged light, causing irreversible damage to the molecule and resulting in a
loss of fluorescence. Molecule now is unable to fluoresce, typically due to the breaking of covalent
bonds within the molecule.
11. What is the visible spectrum of the light is composed of? The visible spectrum of light is composed of
the colors of the rainbow: red, orange, yellow, green, blue, indigo, and violet. These colors have
different wavelengths, with red having the longest wavelength and violet having the shortest
wavelength. When these colors are combined, they can create a range of different colors and shades.
The visible spectrum is just a small part of the electromagnetic spectrum, which includes other types of
electromagnetic radiation such as X-rays, ultraviolet light, and radio waves.

12. What is the excitation range? The excitation range refers to the range of wavelengths of light that
can be absorbed by a fluorescent molecule to bring it to an excited state. This range is specific to each
molecule and is determined by its chemical structure and electronic properties. Excitation usually occurs
at shorter wavelengths (in the ultraviolet or visible range) than the emission wavelength, which is the
range of wavelengths of light that the molecule emits as it returns to its ground state.

13. What is the fluorescence emission spectrum? The fluorescence emission spectrum is the range of
wavelengths of light that a fluorescent molecule emits as it returns to its ground state from an excited
state. This spectrum is specific to each fluorophore and is usually at longer wavelengths than the
excitation range.

Question on Anatomy of Fluorescence Spectra

1. What is the excitation maximum? The excitation maximum is the specific wavelength of light at which
a fluorescent molecule has the highest probability of being excited from its ground state to an excited
state. This wavelength is determined by the molecular structure and electronic properties of the
fluorophore and is often listed in the molecule's specifications.

2. What is the emission maximum? The emission maximum is the specific wavelength of light at which a
fluorescent molecule emits the most light as it returns from an excited state to its ground state. This
wavelength is determined by the molecular structure and electronic properties of the fluorophore and is
often listed in the molecule's specifications.

3. Can the illumination of the fluorophore at the excitation or emission maximum produce emission?
Yes, illuminating a fluorophore at its excitation maximum can produce emission as the molecule
becomes excited and then returns to its ground state, emitting fluorescence in the process. Similarly,
illuminating a fluorophore at its emission maximum can also produce emission as the molecule is
already in an excited state and can continue to emit fluorescence upon excitation by the light source.
However, the intensity of the emitted fluorescence may be weaker when the molecule is excited at its
emission maximum, as a portion of the emitted light may be re-absorbed by the molecule before it has a
chance to escape and be detected.

4. What is the Stocks shift? Stokes shift is the difference in wavelength between the excitation maximum
and the emission maximum of a fluorescent molecule. The Stokes shift is caused by the relaxation of the
excited state of the molecule, which typically releases energy in the form of heat, vibration or emission
of a photon at a longer wavelength than the excitation wavelength.

5. Please find the excitation and emission maximum/spectrum of the following fluorescent dyes using
Fluorescent spectra viewer (web address is listed in Lecture, slide5):

Fluorescent dye Excitation max/Spectrum Emission max/Spectrum

 Alexa Fluor 350 346 (violet) 442 (blue)
Alexa Fluor 488 495 (blue) 519 (green)
Alexa Fluor 550 555 (yellowish green) 565 (greenish yellow)
Alexa Fluor 647 650 (red) 655 (red)

Overview of Filters and Light Sources

1. List the major excitation sources: Mercury and Xenon arc lamps, Laser diodes, Light emitting diodes
(LEDs), Tungsten filament lamps, Pulsed LEDs or lasers, Synchrotron radiation.

2. What is the best source in relation to the illumination spectrum? It is probably laser, it is
advantageous when the dye's peak excitation wavelength is near the wavelength of the laser.

3. What is the purpose of using optical filters (excitation and emission)? To selectively allow light of
certain wavelengths to be transmitted while not allowing undesirable wavelengths to be passed.

4. What is the band path filters? Bandpass filters are optical filters that transmit a narrow range of
wavelengths, while blocking others.

5. What is the long path filters. Long pass filters are optical filters that transmit longer wavelengths of
light while blocking shorter wavelengths,

Find the most suitable dye for each filter using Spectral viewer

1. Alexa Fluor 350

2. Alexa Fluor 488
3. Alexa Fluor 568
4. Alexa Fluor 660

Filters:
1.Excitation wavelengths range - 335 – 379 nm
Filter: 357 - 44
1. Emission wavelengths range - 387 – 507 nm
Filter: 447 - 120
2. Excitation wavelengths range - 459 – 481 nm
Filter: 470 - 22
2. Emission wavelengths range - 489 – 531 nm
Filter: 510 - 42
3. Excitation wavelengths range - 511 – 551 nm
Filter: 531 - 40
3. Emission wavelengths range - 573 – 613 nm
Filter: 593 - 40
4. Excitation wavelengths range - 608 – 648 nm
Filter: 628 - 40
4. Emission wavelengths range - 672 – 712 nm
Filter: 692 - 40