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.

Light Microscope Principle and Applications or Uses

Light Microscope Principle & Uses

(By: Dr. Akalesh K Verma, Asst. Prof., Cotton University)

Fig 1: Components of Light Microscope.

  Parts of Light Microscope

a.      Illuminator: the light source in the base of the microscope;

b.      Abbe Condensor: a two lens system that collects and concentrates light from the illuminator and directs it to the iris diaphragm;

c.      Iris diaphragm: regulates the amount of light entering the lens system;

d.      Mechanical stage: a platform used to place the slide on which has a hole in the center to let light from the illuminator pass through. Often contains stage clips to hold the slide in place;

e.      Body tube: houses the lens system that magnifies the specimens;

f.       Upper end of body tube—oculars/eye pieces: what you view through;

g.      Lower end of body tube—nose-piece: revolves and contains the objectives.

h.      Essentially, a light microscope magnifies small objects and makes them visible. The science of microscopy is based on the following concepts and principles:

i.        Magnification is simply the enlargement of the specimen. In a compound lens system, each lens sequentially enlarges or magnifies the specimen;

j.        The objective lens magnifies the specimen, producing a real image that is then magnified by the ocular lens resulting in the final image;

k.      The total magnification can be calculated by multiplying the objective lens value by the ocular lens value.

KEY TERMS IN MICROSCOPY

Magnification

In most light microscopes, the objective lens, which is closest to the specimen, magnifies objects 100× (times), and the ocular lens, which is nearest the eye, magnifies 10×. Using these two lenses in combination, organisms in the specimen are magnified 1000× their actual size when viewed through the ocular lens. Objective lenses of lower magnification are available so that those of 10×, 20×, and 40× magnification power can provide total magnifications of 100×, 200×, and 400×, respectively. Magnification of 1000× allows for the visualization of fungi, most parasites, and most bacteria, but it is not sufficient for observing viruses, which require magnification of 100,000× or more (Electron Microscopy needed).

Total magnification:

The total magnification obtained in a compound microscope is the product of objective magnification and ocular magnification.

M_t = M_ob X M_oc

Where,

M_t = Total magnification,

M_ob = Objective magnification and

M_oc = Ocular magnification

Resolution

In microscopy, the term 'resolution' is used to describe the ability of a microscope to distinguish in details. In other words, this is the minimum distance at which two distinct points of a specimen can still be seen - either by the observer or the microscope camera - as separate entities.

                                  Fig 2: Difference between high and low resolution image of an object.

To optimize visualization, other factors besides magnification must be considered. Resolution, defined as the extent to which detail in the magnified object is maintained, is also essential. Without it everything would be magnified as an indistinguishable blur. Therefore, resolving power, which is the closest distance between two objects that when magnified still allows the two objects to be distinguished from each other, is extremely important. The resolving power of most light microscopes allows bacterial cells to be distinguished from one another but usually does not allow bacterial structures, internal or external, to be detected.

To achieve the level of resolution desired with 1000× magnification, oil immersion must be used in conjunction with light microscopy. Immersion oil has specific optical and viscosity characteristics designed for use in microscopy. Immersion oil is used to fill the space between the objective lens and the glass slide onto which the specimen has been affixed. When light passes from a material of one refractive index to a material with a different refractive index, as from glass to air, the light bends. Light of different wavelengths bend at different angles creating a less distinct distorted image. Placing immersion oil with the same refractive index as glass between the objective lens and the coverslip or slide decreases the number of refractive surfaces the light must pass through during microscopy. The oil enhances resolution by preventing light rays from dispersing and changing wavelength after passing through the specimen. A specific objective lens, the oil immersion lens, is designed for use with oil; this lens provides 100× magnification on most light microscopes.

Lower magnifications (i.e., 100× or 400×) may be used to locate specimen samples in certain areas on a microscope slide or to observe microorganisms such as some fungi and parasites. The 1000× magnification provided by the combination of ocular and oil immersion lenses usually is required for optimal detection and characterization of bacteria.

Contrast

The another key component to light microscopy is contrast, which is needed to make objects stand out from the background. Because microorganisms are essentially transparent, owing to their microscopic dimensions and high water content, they cannot be easily detected among the background materials and debris in patient specimens. Lack of contrast is also a problem for the microscopic examination of microorganisms grown in culture. Contrast is most commonly achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris. In the absence of staining, the simplest way to improve contrast is to reduce the diameter of the microscope aperture diaphragm increasing contrast at the expense of the resolution. Setting the controls for bright field microscopy requires a procedure referred to as setting the Kohler illumination.

Fig 3: High and low contrast images.

The resolving power

The resolving power is the inverse of the distance between two objects that can be just resolved. On the other hand it is the ability of an optical instrument or type of film to separate or distinguish small or closely adjacent images.

It is given by Abbe's criterion

Resolving power =  ​ 1/delta 'd' = 2a / 'λ'

 where a is the numerical aperture

d is distance

λ is the wavelength of the objective lens used

(a) When wavelength of incident light is decreased, the resolving power increases.

(b) When aperture of the objective lens is decreased, the resolving power decreases.

Numerical Aperture

The numerical aperture (NA) of an optical system (e.g. an imaging system) is a measure for its angular acceptance for incoming light. It is defined based on geometrical considerations and is thus a theoretical parameter which is calculated from the optical design. It cannot be directly measured, except in limiting cases with rather large apertures and negligible diffraction effects.

Numerical Aperture of an Optical System

Numerical aperture is a numerical value concerned with the diameter of the objective lens in relation to its focal length. Thus, it is related to the size of the lower aperture of the objective, through which light enters into it. In a microscope, light is focused on the object as a narrow pencil of light, from where it enters into the objective as a diverging pencil.

Numerical aperture (n.a.) = n sin θ

Where,

n = Refractive index of the medium between the object and the objective and

θ = Half aperture angle

NA of a Lens

A simple case is that of a collimating lens:

Fig 4: A collimating lens can theoretically accept light from a cone, the opening angle of which is limited by its size.

PRINCIPLES

The light microscope is an important tool in the study of microorganisms, particularly for identification purposes. The light microscope is an instrument for visualizing fine detail of an object. It does this by creating a magnified image through the use of a series of glass lenses, which first focus a beam of light onto or through an object, and convex objective lenses to enlarge the image formed. In the majority of light microscopes, the image is viewed directly through binocular eyepieces that act as a secondary lens in the form of a magnifying glass to observe the projected image. Such instruments are termed ‘compound microscopes,’ and the total magnification is the sum of the objective magnification and the eyepiece magnification. The magnification range extends from ×10 to ×1000, with a resolving power of the order of 0.2 μm, depending on the type and numerical aperture (area available for passage of light) of the objective lenses.

                                                Fig 5: Path of light in Light microscope.

Fig 6: Principle of Light Microscope

APPLICATIONS OF LIGHT MICROSCOPE

1.      For details study of cells or tissues section under high magnification and contrast.

2.      For histopathological study to understand toxic effect of drugs/chemicals.

3.      For studying anatomical structures of bones, tissues (Liver, kidney, spleen etc).

4.      For counting WBC, RBC and platelets Counts in patients sample.

5.      Rapid final identification of certain organisms by direct visualization in patient specimens

6.      Detection of different organisms or contaminations present in the specimen.

7.      Detection of organisms not easily cultivated in the laboratory.

8.      Evaluation of patient specimens for the presence of cells indicative of inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells).

9.      Determination of an organism’s clinical significance; bacterial contaminants usually are not present in patient specimens at sufficiently high numbers (×10⁵ cells/mL) to be seen by light microscopy.

10.  Provide preculture information about which organisms might be expected to grow so that appropriate cultivation techniques are used.

11.  Determine which tests and methods should be used for identification and characterization of cultivated organisms.

12.  Provide a method for investigating unusual or unexpected laboratory test results.