Fluorescence Microscopy- Definition, Principle, Parts, Uses

Fluorescence microscopy is a powerful and versatile technique that allows researchers to visualize biological structures and processes with high specificity, sensitivity, and contrast. Fluorescence microscopy uses fluorescent molecules, called fluorophores or fluorochromes, to label the target molecules or structures of interest in a biological sample. When these fluorophores are exposed to light of a specific wavelength, they absorb the light and emit light of a longer wavelength. This emitted light can be detected and separated from the excitation light by using optical filters, resulting in a bright image of the fluorescently labeled sample against a dark background.

Fluorescence microscopy has many advantages over conventional light microscopy, such as:

• It can reveal the spatial distribution and temporal dynamics of specific molecules or structures within cells or tissues, which are otherwise invisible or indistinguishable under normal light.
• It can enable multiplexing, which means simultaneous detection of multiple targets using different fluorophores with distinct emission spectra.
• It can provide quantitative information about the concentration, localization, interaction, and activity of the fluorescently labeled molecules or structures.

Fluorescence microscopy has a wide range of applications in various fields of biology and medicine, such as:

• Cell biology: to study the structure and function of cellular organelles, cytoskeleton, membrane proteins, signaling pathways, gene expression, cell cycle, apoptosis, etc.
• Developmental biology: to track the fate and differentiation of stem cells, embryonic tissues, organogenesis, morphogenesis, etc.
• Neuroscience: to investigate the anatomy and physiology of neurons, synapses, neural circuits, neurotransmission, neurogenesis, etc.
• Immunology: to examine the immune system components, such as antibodies, antigens, cytokines, lymphocytes, macrophages, etc.
• Microbiology: to identify and characterize microorganisms, such as bacteria, viruses, fungi, parasites, etc.
• Pathology: to diagnose and monitor diseases, such as cancer, inflammation, infection, etc.

Fluorescence microscopy is not without limitations, however. Some of the challenges and drawbacks of fluorescence microscopy include:

• Photobleaching: the loss of fluorescence due to irreversible chemical damage of the fluorophores by the excitation light.
• Phototoxicity: the harmful effect of the excitation light on the biological sample, causing damage or death of cells or tissues.
• Autofluorescence: the unwanted fluorescence from non-target molecules or structures in the biological sample or the microscope components.
• Spectral overlap: the interference between different fluorophores with similar emission spectra or between the excitation and emission spectra of the same fluorophore.

To overcome these limitations and enhance the performance of fluorescence microscopy, various modifications and innovations have been developed over time. These include:

• New types of fluorophores with improved brightness, stability, specificity, and diversity.
• New types of light sources with higher intensity, tunability, coherence, and pulse duration.
• New types of optical filters with better transmission, reflection, and discrimination properties.
• New types of detectors with higher sensitivity, resolution, speed, and dynamic range.
• New types of microscopes with different configurations and modes of operation.

In this article, we will introduce the basic principles and components of fluorescence microscopy. We will also describe some of the common forms and applications of fluorescence microscopy. Finally, we will discuss some of the advantages and limitations of fluorescence microscopy.

Fluorescence microscopy is a type of optical microscopy that uses fluorescence and phosphorescence to visualize biological specimens. Fluorescence is the phenomenon of emitting light of a longer wavelength after absorbing light of a shorter wavelength. Phosphorescence is a similar phenomenon, but with a longer delay between absorption and emission. Both fluorescence and phosphorescence are forms of luminescence, which is the emission of light by a substance that is not due to heat.

The principle of fluorescence microscopy is based on the use of fluorescent dyes, also known as fluorophores or fluorochromes, that can bind to specific molecules or structures in the specimen and emit light when excited by a light source. The emitted light can be detected by a detector, such as a camera or an eyepiece, after passing through an emission filter that blocks the excitation light. The image produced by fluorescence microscopy is thus based on the location and intensity of the fluorophores in the specimen, rather than on the reflection or absorption of the excitation light.

Fluorescence microscopy can reveal information about the structure, function, and dynamics of biological specimens that are not visible by other microscopy techniques. For example, fluorescence microscopy can be used to label and track different proteins, nucleic acids, organelles, or cells in living or fixed samples. Fluorescence microscopy can also be used to measure physiological parameters, such as pH, calcium concentration, membrane potential, or oxygen levels, by using fluorophores that change their fluorescence properties in response to these factors.

Fluorescence microscopy has many advantages over other optical microscopy techniques, such as high sensitivity, specificity, contrast, resolution, and versatility. However, fluorescence microscopy also has some limitations, such as photobleaching, phototoxicity, autofluorescence, and background noise. These factors can affect the quality and accuracy of the fluorescence images and require careful optimization of the experimental conditions.

Fluorescence microscopy works by exploiting the phenomenon of fluorescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emits 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.

The basic steps involved in fluorescence microscopy are:

• Sample preparation: The sample is prepared by staining it with fluorescent dyes, also known as fluorophores or fluorochromes, that bind to specific molecules or structures of interest. Alternatively, the sample can be genetically modified to express fluorescent proteins, such as green fluorescent protein (GFP), that can be used as markers for cellular processes or localization.
• Illumination: The sample is illuminated with a light source that emits light of a specific wavelength range, usually in the ultraviolet (UV) or blue region of the spectrum. This light is filtered by an excitation 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.
• Detection: The fluorescence emitted by the sample is collected by an objective lens and focused on a detector, such as a camera or a photomultiplier tube (PMT). Since most of the excitation light is transmitted through the sample, only reflected excitatory light reaches the objective together with the emitted light. To separate the two, a dichroic mirror or beam splitter is used, which reflects the excitation light and transmits the emission light. The emission light is further filtered by an emission 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 and highest contrast for the fluorescence image.
• Image formation: The detector converts the fluorescence signal into an electrical signal that can be processed and displayed on a monitor or stored on a computer. Depending on the type of fluorescence microscope, different methods of image formation can be used, such as wide-field imaging, confocal imaging, multiphoton imaging, super-resolution imaging, etc.

There are different forms of fluorescence microscopes that vary in their design, complexity, and applications. Some of the most common forms are:

• Epifluorescence microscope: This is the simplest and most widely used form of fluorescence microscope. It uses a single light path for both excitation and emission of fluorescence. The light source passes through an excitation filter, a dichroic mirror, and the objective lens to illuminate the specimen. The fluorescence emitted by the specimen is collected by the same objective lens, passes through the dichroic mirror, and an emission filter, and reaches the detector (such as an eyepiece, a camera, or a photomultiplier tube). The dichroic mirror reflects the excitation light and transmits the emission light, thus separating them. The emission filter blocks any residual excitation light and allows only the desired fluorescence wavelength to reach the detector. Epifluorescence microscopes can be used for both fixed and live samples, and can be combined with other techniques such as phase contrast or differential interference contrast (DIC).

• Confocal microscope: This is a more advanced form of fluorescence microscope that uses a pinhole aperture to eliminate out-of-focus light and improve the resolution and contrast of the image. The light source (usually a laser) passes through an excitation filter and a scanning device (such as a mirror or a galvanometer) to scan the specimen point by point. The fluorescence emitted by the specimen is collected by an objective lens, passes through a dichroic mirror and an emission filter, and reaches a pinhole aperture that is conjugated with the focal plane of the objective lens. Only the light from the focal plane can pass through the pinhole, while the light from above or below the focal plane is blocked. The light that passes through the pinhole reaches a detector (such as a photomultiplier tube or a camera). By scanning the specimen in different planes, a three-dimensional image can be reconstructed from the collected data. Confocal microscopes can provide higher resolution, better contrast, and optical sectioning than epifluorescence microscopes, but they require more complex equipment and longer acquisition time.

• Two-photon microscope: This is a special form of confocal microscope that uses two photons of lower energy (usually infrared) to excite a fluorophore instead of one photon of higher energy (usually visible or ultraviolet). The two photons are absorbed simultaneously by the fluorophore in a nonlinear process that occurs only at the focal point of the objective lens, where the photon density is high enough. The fluorescence emitted by the fluorophore is collected by the same objective lens, passes through a dichroic mirror and an emission filter, and reaches a detector (such as a photomultiplier tube or a camera). Two-photon microscopy has several advantages over conventional confocal microscopy, such as deeper penetration into thick tissues, reduced photobleaching and phototoxicity, and enhanced contrast due to background suppression.

• Super-resolution microscope: This is a general term for various forms of fluorescence microscopes that can overcome the diffraction limit of light and achieve resolution beyond that of conventional optical microscopes. The diffraction limit is determined by the wavelength of light and the numerical aperture of the objective lens, and it limits the smallest distance between two points that can be resolved by an optical microscope. Super-resolution microscopy techniques use different strategies to break this limit, such as manipulating the emission states of fluorophores (e.g., stimulated emission depletion microscopy or STED), localizing single molecules with high precision (e.g., photoactivated localization microscopy or PALM), or exploiting structured illumination patterns (e.g., structured illumination microscopy or SIM). Super-resolution microscopy can reveal subcellular structures and molecular interactions that are otherwise invisible to conventional optical microscopy.

These are some of the main forms of fluorescence microscopes that are used in biological research. Each form has its own advantages and limitations, and they can be combined with other techniques or modalities to achieve different goals. Fluorescence microscopy is a powerful tool for studying biological phenomena at different scales and dimensions.

A fluorescence microscope is composed of several components that work together to produce an image based on the fluorescence emission of the specimen. The main parts of a fluorescence microscope are:

• Fluorescent dyes: These are chemical compounds that can absorb light at a specific wavelength and emit light at a longer wavelength. Fluorescent dyes are also known as fluorophores or fluorochromes. They are used to label biological molecules or structures of interest in the specimen. For example, nucleic acid stains like DAPI and Hoechst can bind to DNA and emit blue fluorescence, while phalloidin can bind to actin filaments and emit green or red fluorescence .

• Light source: This is the device that provides the illumination for excitation of the fluorescent dyes. The light source can be a xenon arc lamp, a mercury-vapor lamp, a high-power LED, or a laser, depending on the type and complexity of the fluorescence microscope. The light source should have a high intensity and a broad spectrum to cover the excitation wavelengths of different fluorophores .

• Excitation filter: This is a bandpass filter that selects the appropriate wavelength of light from the light source to excite the fluorophore. The excitation filter should match the absorption spectrum of the fluorophore and minimize the excitation of other fluorescent sources. The excitation filter also blocks the light in the emission wavelength range to prevent interference with the detection .

• Dichroic mirror: This is a special type of mirror that reflects light at certain wavelengths and transmits light at other wavelengths. The dichroic mirror is also called a dichroic beamsplitter or a dichroic filter. It is placed at an angle between the light source and the objective lens. The dichroic mirror reflects the excitation light from the light source towards the specimen, and transmits the emission light from the specimen towards the detector .

• Emission filter: This is another bandpass filter that selects the appropriate wavelength of light from the emission spectrum of the fluorophore. The emission filter should match the emission spectrum of the fluorophore and block all undesired light outside this range, especially the excitation light. The emission filter ensures that only the fluorescence signal reaches the detector and improves the contrast and specificity of the image .

• Detector: This is the device that captures and converts the fluorescence signal into an electrical signal or an image. The detector can be an eye, a camera, a photomultiplier tube, or a photodiode, depending on the type and resolution of the fluorescence microscope. The detector should have a high sensitivity and a low noise level to detect weak fluorescence signals .

The diagram below shows how these parts are arranged in a typical fluorescence microscope:

Fluorescence microscopy has a wide range of applications in various fields of science and technology. Some of the main applications are:

• Cell biology: Fluorescence microscopy is used to study the structure and function of cells and their components, such as organelles, membranes, cytoskeleton, and molecules. Fluorescent dyes and probes can be used to label specific cellular structures or molecules, such as DNA, RNA, proteins, lipids, carbohydrates, and metabolites. Fluorescence microscopy can also be used to monitor the dynamic processes that occur within and between cells, such as cell division, differentiation, migration, signaling, transport, and apoptosis. Fluorescence microscopy can also be combined with other techniques, such as immunofluorescence, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to enhance the specificity and sensitivity of detection and measurement.

• Microbiology: Fluorescence microscopy is used to identify and characterize microorganisms, such as bacteria, fungi, viruses, and parasites. Fluorescent dyes and probes can be used to stain specific structures or molecules of microorganisms, such as cell walls, membranes, nucleic acids, proteins, and antigens. Fluorescence microscopy can also be used to track the interactions and infections of microorganisms with host cells or tissues. Fluorescence microscopy can also be combined with other techniques, such as fluorescence-activated cell sorting (FACS), fluorescence lifetime imaging microscopy (FLIM), and total internal reflection fluorescence microscopy (TIRFM), to isolate and analyze individual microorganisms or populations.

• Molecular biology: Fluorescence microscopy is used to study the structure and function of biomolecules, such as nucleic acids, proteins, lipids, and carbohydrates. Fluorescent dyes and probes can be used to label specific regions or domains of biomolecules, such as bases, amino acids, fatty acids, and sugars. Fluorescence microscopy can also be used to monitor the interactions and reactions of biomolecules with each other or with other molecules or substrates. Fluorescence microscopy can also be combined with other techniques, such as single-molecule fluorescence microscopy (SMFM), fluorescence cross-correlation spectroscopy (FCCS), and fluorescence polarization microscopy (FPM), to measure the properties and kinetics of single molecules or complexes.

• Biomedical engineering: Fluorescence microscopy is used to develop and evaluate novel materials and devices for biomedical applications, such as drug delivery, tissue engineering, biosensors, bioimaging, and biotherapy. Fluorescent dyes and probes can be used to label specific components or functions of materials and devices, such as polymers, nanoparticles, micelles, vesicles, scaffolds, electrodes, transducers, and actuators. Fluorescence microscopy can also be used to monitor the performance and biocompatibility of materials and devices in vitro or in vivo. Fluorescence microscopy can also be combined with other techniques, such as multiphoton microscopy (MPM), confocal laser scanning microscopy (CLSM), super-resolution microscopy (SRM), and optical coherence tomography (OCT), to improve the resolution and depth of imaging.

• Nanotechnology: Fluorescence microscopy is used to synthesize and characterize nanomaterials and nanostructures for various applications in science and technology. Fluorescent dyes and probes can be used to label specific features or properties of nanomaterials and nanostructures, such as size, shape, surface charge, composition, and functionality. Fluorescence microscopy can also be used to monitor the assembly and manipulation of nanomaterials and nanostructures by physical or chemical means. Fluorescence microscopy can also be combined with other techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning tunneling microscopy (STM), to reveal the morphology and structure of nanomaterials and nanostructures at different scales.

These are some of the major applications of fluorescence microscopy in various fields of science and technology. Fluorescence microscopy is a powerful and versatile tool that enables the visualization and quantification of biological and non-biological phenomena at different levels of complexity and organization.

Fluorescence microscopy is a powerful technique that allows researchers to visualize specific structures or molecules within biological samples with high sensitivity, specificity, and contrast. Some of the advantages of using fluorescence microscopy are:

• Live-cell imaging: Fluorescence microscopy can be used to study the dynamic behavior of living cells and tissues, such as cell division, migration, differentiation, signaling, gene expression, and protein interactions. Fluorescent probes can be introduced into cells by various methods, such as microinjection, electroporation, transfection, viral infection, or genetic engineering. Fluorescent proteins, such as GFP and its variants, can be fused to target proteins of interest and expressed in living cells, allowing the tracking of their localization and movement over time. Fluorescence microscopy can also be used to measure physiological parameters of living cells, such as pH, calcium, membrane potential, oxygen, and reactive oxygen species.

• Multicolor staining: Fluorescence microscopy can be used to label multiple structures or molecules within the same sample with different colors, allowing the simultaneous visualization and analysis of their spatial distribution and interactions. For example, different organelles, such as the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and peroxisomes, can be stained with different fluorescent dyes or proteins and observed in the same cell. Similarly, different cellular components, such as DNA, RNA, proteins, lipids, carbohydrates, and metabolites, can be labeled with different fluorophores and detected in the same sample. The use of multicolor staining requires careful selection of fluorophores that have distinct excitation and emission spectra and minimal overlap or cross-talk.

• Three-dimensional imaging: Fluorescence microscopy can be used to obtain three-dimensional images of biological samples by collecting a series of optical sections at different focal planes. This can be achieved by using confocal microscopy or multiphoton microscopy, which use a pinhole or a nonlinear excitation process to eliminate out-of-focus light and improve the axial resolution. Three-dimensional imaging can provide more information about the morphology and organization of biological structures than conventional two-dimensional imaging. It can also enable the reconstruction of three-dimensional models and the quantification of three-dimensional parameters, such as volume, surface area, shape, and orientation.

• Super-resolution imaging: Fluorescence microscopy can be used to overcome the diffraction limit of light and achieve nanometer-scale resolution by using various super-resolution techniques. These techniques rely on manipulating the fluorescence emission of individual molecules or exploiting their stochastic switching behavior to localize them with high precision. Some examples of super-resolution techniques are stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM) , stochastic optical reconstruction microscopy (STORM) , structured illumination microscopy (SIM) , and single-molecule localization microscopy (SMLM) . Super-resolution imaging can reveal subcellular details and molecular interactions that are otherwise invisible by conventional fluorescence microscopy.

Fluorescence microscopy is a powerful and versatile technique, but it also has some limitations that need to be considered. Some of the main limitations are:

• Photobleaching: Fluorophores lose their ability to fluoresce as they are illuminated by the excitation light, resulting in a gradual fading of the signal . Photobleaching can limit the observation time and the number of images that can be acquired from a sample. Photobleaching can also affect the quantification of fluorescence intensity and the comparison of different samples or conditions. Photobleaching can be reduced by using lower light intensity, shorter exposure time, anti-fading agents, or more stable fluorophores.
• Phototoxicity: The exposure of living cells or tissues to high-intensity light can cause damage to cellular structures and functions, leading to cell death or altered behavior . Phototoxicity can affect the viability and physiology of the cells under study, and introduce artifacts or biases in the results. Phototoxicity can be minimized by using lower light intensity, shorter exposure time, longer wavelength, or less invasive fluorophores.
• Fluorophore specificity: Fluorescence microscopy relies on the specific binding or interaction of fluorophores with the target molecules or structures of interest. However, some fluorophores may have non-specific binding or cross-reactivity with other molecules or structures, resulting in false-positive signals or background noise . Fluorophore specificity can be improved by using appropriate controls, optimizing the concentration and incubation time of the fluorophores, blocking non-specific binding sites, or using more selective fluorophores.
• Fluorophore interference: The simultaneous use of multiple fluorophores or the presence of autofluorescent molecules in the sample can cause interference or quenching of the fluorescence signals, reducing the contrast and resolution of the images . Fluorophore interference can be avoided by choosing fluorophores with compatible excitation and emission spectra, using appropriate filters and detectors, removing autofluorescent molecules from the sample, or using spectral unmixing techniques.

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