18. Which color of light excites a natural GFP to emit green fluorescence?

(A) Blue

(B) Green

(C) Infrared

(D) Red

Which Color of Light Excites Natural GFP to Emit Green Fluorescence?

Correct Answer

Correct Option: (A) Blue

Natural green fluorescent protein (GFP) is excited by shorter-wavelength light, particularly blue light, and subsequently emits light at a longer wavelength in the green region of the visible spectrum. Therefore, when GFP absorbs blue light, its chromophore enters an excited electronic state. As the chromophore returns to its ground state, part of the absorbed energy is released as green fluorescence.

Thus, the correct sequence is:

Blue light → GFP excitation → Green fluorescence

Therefore, Option (A) Blue is the correct answer.

What Is Green Fluorescent Protein?

GFP Is a Naturally Fluorescent Protein

Green fluorescent protein, commonly abbreviated as GFP, is a naturally occurring fluorescent protein originally discovered in the jellyfish Aequorea victoria. GFP has become one of the most important tools in cell biology, molecular biology, developmental biology and fluorescence microscopy because it can be used as a visible marker of gene expression and protein localization.

The remarkable feature of GFP is its ability to produce green fluorescence without requiring an externally supplied fluorescent cofactor. The protein forms its own fluorescent structure, called a chromophore, through chemical reactions involving amino acids within the GFP polypeptide chain.

When the GFP chromophore absorbs light of an appropriate wavelength, its electrons move to a higher-energy state. The excited state is temporary. When the electrons return to a lower-energy state, energy is released in the form of visible green light.

This fundamental phenomenon is known as fluorescence.

Why Blue Light Excites GFP to Emit Green Fluorescence

Blue Light Provides the Energy Required for GFP Excitation

The color of light absorbed by a fluorescent molecule is not necessarily the same as the color it emits. GFP absorbs relatively high-energy, shorter-wavelength light and emits lower-energy, longer-wavelength light.

Blue light has a shorter wavelength and therefore higher energy than green light. When blue photons strike the GFP chromophore, their energy can be absorbed by the chromophore. This absorbed energy promotes electrons from the ground electronic state to an excited electronic state.

The excited GFP molecule does not remain in this high-energy condition for long. Some of the absorbed energy is lost through non-radiative processes, including molecular vibrations and interactions with the surrounding environment. The remaining energy is then emitted as a photon.

Because some energy has already been lost before photon emission, the emitted photon has less energy and a longer wavelength than the absorbed photon. Consequently, GFP can absorb blue light and emit green light.

Therefore:

Blue light is absorbed → GFP becomes excited → Energy is partially lost → Green light is emitted

This is why blue light excites GFP to emit green fluorescence.

Understanding the Excitation and Emission Wavelengths of Natural GFP

Natural GFP Has Characteristic Excitation Peaks

Natural or wild-type GFP has characteristic absorption and excitation properties. Wild-type GFP is known for a major excitation peak near the ultraviolet region and another excitation peak in the blue region. The blue excitation band allows GFP fluorescence to be produced using blue light.

The emitted fluorescence is centered in the green region, approximately around 509 nm. Because this emitted wavelength falls within the visible green region, the protein appears green when it fluoresces.

For the purpose of this question, the essential concept is that blue light excites GFP and GFP emits green fluorescence.

It is also important to understand that different engineered GFP variants can have somewhat different excitation maxima. However, among the colors given in the options, blue is the correct excitation color for producing green fluorescence from GFP.

How Does GFP Fluorescence Work?

Step 1: GFP Absorbs Excitation Light

The process begins when the GFP chromophore absorbs a photon of suitable energy. Blue light contains photons energetic enough to excite the chromophore.

The energy of light is related to its wavelength by the equation:

E = hc/λ

where E is the energy of the photon, h is Planck’s constant, c is the speed of light and λ is the wavelength.

According to this relationship, shorter wavelengths possess higher energy. Therefore, blue light has more energy per photon than green, red or infrared light.

Step 2: The Chromophore Enters an Excited State

After absorbing the blue photon, an electron in the GFP chromophore moves from a lower-energy electronic state to a higher-energy state.

This excited state exists only for a very short period. During this time, some of the absorbed energy is dissipated.

Step 3: Green Fluorescence Is Emitted

When the excited electron returns toward its ground state, the GFP chromophore releases a photon.

Because some energy was lost before emission, the emitted photon has lower energy than the absorbed photon. Lower-energy light has a longer wavelength, which is why the emitted light shifts from the blue excitation region to the green emission region.

The result is the characteristic green fluorescence of GFP.

What Is the Stokes Shift in GFP Fluorescence?

Emission Occurs at a Longer Wavelength Than Excitation

The difference between the wavelength of maximum excitation and the wavelength of maximum fluorescence emission is associated with the Stokes shift.

In simple terms, a fluorescent molecule absorbs light at a shorter wavelength and emits light at a longer wavelength. Since shorter-wavelength light has higher energy, the excitation photon carries more energy than the emitted fluorescence photon.

For GFP, the basic relationship can be understood as:

Higher-energy blue light → absorption and excitation

Lower-energy green light → fluorescence emission

This separation between excitation and emission wavelengths is extremely useful in fluorescence microscopy because optical filters can separate the incoming excitation light from the outgoing fluorescence.

Detailed Explanation of Every Option

Option (A): Blue

Option (A) is correct. Blue light can excite the GFP chromophore, causing it to enter an excited electronic state. When the chromophore returns to its lower-energy state, it emits fluorescence in the green region of the visible spectrum.

The important relationship is:

Blue excitation → Green emission

Blue light has a shorter wavelength and higher energy than green light. This makes it suitable for exciting GFP so that the protein subsequently emits lower-energy green fluorescence.

Therefore, Option (A) Blue is the correct answer.

Option (B): Green

Option (B) is incorrect. Green is primarily the color of fluorescence emitted by GFP rather than the expected excitation color in this question.

A fluorescent molecule generally absorbs higher-energy light and emits lower-energy light. Since green light has lower energy and a longer wavelength than blue light, it represents the emitted fluorescence rather than the primary excitation color among the given options.

Therefore, green should not be confused with the light used to excite GFP.

Option (C): Infrared

Option (C) is incorrect. Infrared radiation has a much longer wavelength and lower photon energy than visible blue light. It does not provide the appropriate photon energy for conventional excitation of natural GFP.

Standard GFP fluorescence imaging does not use infrared light for direct one-photon excitation of the GFP chromophore. Therefore, infrared light is not the correct answer.

Option (D): Red

Option (D) is incorrect. Red light has a longer wavelength and lower energy than blue light. Its photons do not match the conventional excitation requirements of natural GFP.

Red light is more closely associated with the excitation or emission properties of other fluorescent proteins and fluorophores designed to operate at longer wavelengths. It is not the appropriate choice for exciting natural GFP to produce green fluorescence.

Therefore, Option (D) is incorrect.

Difference Between Excitation Light and Emitted Fluorescence

Excitation Light Supplies Energy

Excitation light is the incoming light absorbed by the fluorophore. Its function is to provide enough energy to move the chromophore into an excited electronic state.

In the case of this question, the excitation light is blue.

Emitted Light Is the Observed Fluorescence

Emission occurs when the excited chromophore returns to a lower-energy state and releases a photon. The emitted photon has less energy and a longer wavelength than the excitation photon.

In GFP, the observed fluorescence is green.

Therefore, the two colors must not be confused:

Why GFP Is Important in Biological Research

GFP Can Be Used as a Reporter of Gene Expression

Researchers can attach the gene encoding GFP to a promoter or regulatory sequence. When the gene is expressed, GFP is produced, and the cells can be visualized by fluorescence.

This allows scientists to investigate where and when a gene is active.

GFP Can Reveal Protein Localization

The GFP gene can also be fused to a gene encoding another protein. The resulting fusion protein carries a fluorescent tag, allowing researchers to observe where the protein is located inside a living cell.

For example, GFP-tagged proteins can be tracked in the nucleus, cytoplasm, mitochondria, cell membrane or other cellular compartments.

GFP Can Be Observed in Living Cells

One of the major advantages of GFP is that fluorescence can often be observed in living cells without destroying the sample. This has made GFP extremely valuable for studying dynamic biological processes in real time.

Final Answer

Natural GFP is excited by higher-energy, shorter-wavelength light and subsequently emits lower-energy green fluorescence. Among the given options, blue light provides the appropriate excitation energy.

The correct relationship is:

Blue light → Excitation of GFP → Green fluorescence

Therefore:

Correct Option: (A) Blue