Fluorescence Microscopy Principle and Uses/Applications

Fluorescence Microscopy Principle and Uses/Applications

By: Dr. Akalesh Kumar Verma, Asst. Professor, Cotton University

(Educational materials)

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. Fluorescence is a phenomenon that involves absorbance and emission of a small range of light wavelengths by a fluorescent molecule known as a fluorophore. Fluorescence microscopy is accomplished in conjunction with the basic light microscope by the addition of a powerful light source, specialized filters, and a means of fluorescently labeling a sample.

• A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances.
• Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation while phosphorescence is a specific type of photoluminescence related to fluorescence.
• Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs.
• The fluorescence microscope was devised in the early part of the twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann, among others.

Fig 1: Fluorescence Microscope, Image credit: Google.

1. Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes.
2. Fluorescent dyes, also known as fluorophores or fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds.
3. The emission light can then be filtered from the excitation light to reveal the location of the fluorophores.

• Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength.
• The image produced is based on the second light source or the emission wavelength of the fluorescent species rather than from the light originally used to illuminate, and excite, the sample.

WORKING PRINCIPLE

The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter (see figure below). The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused on the detector by the objective. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light.

Fig .2: Principle of Fluorescence microscope. Image credit: Google.

Forms (Types)

The “fluorescence microscope” refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image.

Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective).

Typical components / requirements of a fluorescence microscope are:

Fluorescent dyes (Fluorophore)

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds. Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules that are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains like DAPI and Hoechst, phalloidin which is used to stain actin fibers in mammalian cells. 

A light source

Four main types of light sources are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, and high- power LEDs.

Lasers are mostly used for complex fluorescence microscopy techniques, while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used for wide-field epifluorescence microscopes.

The excitation filter

The exciter is typically a bandpass filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing the excitation of other sources of fluorescence and blocking excitation light in the fluorescence emission band. 

The dichroic mirror

A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.

The emission filter.

The emitter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.

By blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence, optical filters ensure the darkest background. 

APPLICATIONS

• To identify structures in fixed and live biological samples. 
• Fluorescence microscopy is a common tool for today’s life science research because it allows the use of multicolor staining, labeling of structures within cells, and the measurement of the physiological state of a cell.
• Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live-cell imaging.
• This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material.
• The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.
• Different molecules can now be stained with different colors, allowing multiple types of the molecule to be tracked simultaneously.
• These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.
• Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence.
• Cells are susceptible to phototoxicity, particularly with short-wavelength light. Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.
• Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence. 

Related Terms

The Differences between Fluorescence and Phosphorescence

Despite them being similar in nature, there are differences. The key mechanistic difference is that fluorescence emits light immediately after photon adsorption, whereas there is a delay with phosphorescence.

Additionally, once the excitation source is removed from a fluorescing molecule, the light emission stops. In comparison, the emission stays for a while once the excitation source is removed in phosphorescence. Their visible appearance is also slightly different. Fluorescence gives an immediate flash, whereas phosphorescence is a lower ‘glow in the dark’ appearance.

The Mechanism of Fluorescence

Molecules which can fluoresce contain fluorophores, which are regions of electronic structure that exhibit fluoresence. At room temperature these molecules are usually in their lowest energy, ground-state. There are many different vibrational levels within the ground state, and molecules in their un-excited form adopt the lowest vibrational level.

When exposed to UV/Vis light the flurophore will absorb photons. Upon absorption, photons make the molecule's electrons adopt a higher molecular and vibrational state. This is usually the first or second excited singlet state, termed S₁ and S₂, respectively.

The molecule now has additional energy to lose, some of which can be lost as kinetic energy. The extra energy causes the molecules to collide with the other molecules in a sample. These collisions cause the molecules to lose their vibrational energy - these energy losses are known as non-radiative transitions. Once some of the energy is lost, the molecule returns to the lowest vibrational level of the excited state. Once this point is reached no more energy can be lost kineticly and, to return to the ground state, the molecule must relax via fluoresence.

The S₁ or S₂ electron returns to the ground state by ejecting the extra energy as a photon. As some energy has been lost   the molecule’s energy levels return to the ground state (to any of the ground state vibrational levels). The return to the ground state causes a photon to be ejected from the molecule. Due to the earlier, non-transitive energy losses, this photon is of a different wavelength as the photon that excited the molecule. This is characteristic of fluorescence.

Fig: 3. A diagram illustrating the mechanism of fluorescence - photon absorption causes an excitation, which is then followed by non-radiative (kinetic) and radiative (fluorescence) relaxations. Image Credits: Google.  

Because fluorescence always occurs as a transition from the lowest energy excited state to the ground state, the shape of a fluorescence emission spectrum is always the same, regardless of the wavelength of light used to excite the molecule. Additionally, there is no perturbation of the molecular shape under excitation, so the distribution of vibrational levels in the excited and ground states are very similar. The spin direction of the electrons is also preserved in fluorescence.

It is not only molecules which can fluoresce. Atoms exhibit a phenomenon known as resonance fluorescence. There are no vibrational levels in atoms, therefore they can’t exhibit the same type of fluorescence that a molecule can. Additionally, the wavelength emitted by atoms is the same as the incident wavelength. It is a phenomenon often seen in quantum dots.

The Mechanism of Phosphoresence

Phosphorescence follows the same initial excitation mechanism. That is, a monochromatic beam of light is fired towards a molecule causing it to go from the ground state to either the S₁ or S₂ excited singlet states. It is in the return to the ground state that it is very different.

Unlike in fluorescence, the spin of the excited (and promoted) electron can be reversed. This creates a scenario where two electrons are no longer paired, and the molecule has two electrons of the same spin in different electronic orbitals. Following quantum mechanical laws, these electrons can exist in 3 states. This gives rise to an excited triple triplet state if the spin is reversed, and a singlet state if the spin is not reversed.

Upon decay, phosphorescence doesn’t return immediately to the ground state. Instead it transitions through a metastable state, known as T₁. This transition is known as an intersystem crossing, the metastable state occurs because the decay of an excited triplet state is forbidden in molecules with even numbers.

The decay via the T₁ state is much slower, as it is technically forbidden, and the molecule is only considered to be back at the ground state when all the energy has been released. Phosphorescence is most commonly seen with heavier molecules and many are crystalline in nature, as this confers enough stability for them to cope with the forbidden transition.

Fig 4: Mechanism of fluorescence and phosphorescence emission. Image credit: Google.

Stokes shift 

Stokes shift is the difference (in energy, wavenumber or frequency units) between positions of the band maxima of the absorption and emission spectra (fluorescence and Raman being two examples) of the same electronic transition. It is named after Irish physicist George Gabriel Stokes.

Fig: 5 Stokes Shift pattern. Image credit: Google.