Name: sharper cell images with fluorescence microscopy
Uploaded: 2023-04-30T12:00:45.000Z
Duration: 1 min 37 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

Super-resolution Fluorescence Microscopy

Conventional fluorescence microscopy can only resolve structures farther apart than 200 nm. This is because under a microscope, the diffraction of light makes the fluorescent molecules appear as blurry spots instead of sharp points. As a result, when objects are very close to each other, their images overlap.
Therefore, scientists have developed super-resolution techniques that provide better resolution to image even small subcellular structures. These techniques overcome the diffraction limits by using specific patterns of light to excite the fluorophores in the sample.
For instance, structured illumination microscopy uses a striped pattern which is then rotated to capture multiple images. The images are combined, creating an interference pattern that is computationally processed to make the final image.
Stimulated emission depletion microscopy creates a pattern using two lasers. A primary laser excites the sample while a donut-shaped beam suppresses the fluorescence around it, making the fluorescent point appear smaller.
Other techniques avoid overlapping signals by using fluorescent probes that can be switched on and off. Only some probes are activated for each image, minimizing signal overlap and allowing for the localization of a single fluorescent molecule.&#160;
Many of these images can be combined into a single high-resolution composite image.

super-resolution fluorescence microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
Photoactivated Localization Microscopy
Photoactivated localization microscopy (PALM) is a type of fluorescence microscopy that captures high-resolution images using a single-molecule detection and localization approach. For example, two fluorescent spots 75 nm apart may appear to be a single spot during imaging due to interference of their PSFs. In such cases, especially for live-cell imaging, PALM is a suitable technique to resolve the interference and provide a better resolution.
In PALM, a variant of green fluorescent protein (GFP) with a different excitation wavelength and high fluorescence is employed. In the first step, a few GFPs are activated and imaged with very high precision. In the next step, another set of GFPs is activated and imaged. Step by step, all the GFPs across the specimen are thus recorded. Finally, the data is processed to generate a high-resolution image.
Stochastic Optical Reconstruction Microscopy
In stochastic optical reconstruction microscopy (STORM), unique photo-switchable probes are used for specimen imaging. The emission from these probes can be switched on and off using lights of different wavelengths. Thus, only a few fluorophores can be activated at a point in time such that the number of activated fluorophores is significantly lesser than the number of deactivated fluorophores. This selective activation of probes helps determine their precise position in the specimen. Following this, the center of each fluorescent probe is identified and marked. The process is then repeated to record all the fluorescent probes in the specimen. Finally, a high-resolution composite image is constructed by superimposing these multiple images.

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread ...

Education

JoVE Core

Cell Biology

Unit 1

Visualizing Cells, Tissues, and Molecules

KEY TERMS AND CONCEPTS

sharper cell images with fluorescence microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear.

Several super-resolution methods have been developed to improve image detail. Photoactivated localization microscopy, or PALM, uses single-molecule detection and localization to build a high-resolution image. It is useful when two fluorescent spots are about 75 nm apart and may look like one spot during imaging. This can be especially helpful in live-cell imaging.

PALM uses a variant of green fluorescent protein, or GFP, with a different excitation wavelength and high fluorescence. First, only a few GFP molecules are activated and imaged with very high precision. Then another set is activated and imaged. This step-by-step process continues until all GFPs in the specimen are recorded, and the data is processed into a high-resolution image.

Stochastic optical reconstruction microscopy, or STORM, uses photo-switchable probes instead of GFP. These probes can be switched on and off with lights of different wavelengths. Only a few fluorophores are activated at a time, while many remain off. This makes it easier to find the precise position of each probe in the specimen.

In STORM, the center of each fluorescent probe is identified and marked. The process is repeated until all probes have been recorded across the specimen. The separate images are then superimposed to form a high-resolution composite image.

Sharper Cell Images with Fluorescence Microscopy

sharper-cell-images-with-fluorescence-microscopy

imaging biological samples with optical microscopy

imaging-biological-samples-with-optical-microscopy

How Light Microscopes Bring Samples Into View

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

phase contrast and differential interference contrast microscopy

phase-contrast-and-differential-interference-contrast-microscopy

Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

fixation and sectioning

Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

protein dynamics in living cells

Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

total-internal-reflection-fluorescence-microscopy

Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

immunogold electron microscopy

Immunogold Labeling in Electron Microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

electron-microscope-tomography-and-single-particle-reconstruction

3D Imaging with Electron Microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

key terms and concepts

Learn how super-resolution fluorescence microscopy sharpens cell images using PALM and STORM to locate fluorophores with far greater precision.

Sharper Cell Images with Fluorescence Microscopy | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

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Describe the scientific theory of cells (cell theory) and relate the history of its discovery to the process of science.

SC.912.L.14.1

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The Nucleus

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Endoplasmic ReticulumThe endoplasmic reticulum (ER) is an extensive network of membranous sacs and tubules in eukaryotic cells, continuous with the outer ...

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Microtubules

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Plant Cell Wall

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Plasmodesmata

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A key characteristic of life is the ability to separate the external environment from the internal space. To do this, cells have evolved semi-permeable ...

Membrane Fluidity

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Fluid Mosaic Model

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SC.912.L.14.2

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Background
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Compare and contrast the general structures of plant and animal cells. Compare and contrast the general structures of prokaryotic and eukaryotic cells.

SC.912.L.14.3

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Introduction to Light Microscopy

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Overview of Electron Microscopy

The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can ...

Phase Contrast and Differential Interference Contrast Microscopy

Phase-Contrast Microscopes
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Confocal Fluorescence Microscopy

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Introduction to Fluorescence Microscopy

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SC.912.L.14.5

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SC.912.L.14.6

A. Cells have characteristic structures and functions that make them distinctive.

B. Processes in a cell can be classified broadly as growth, maintenance, reproduction, and homeostasis.

C. Life can be organized in a functional and structural hierarchy ranging from cells to the biosphere.

D. Most multicellular organisms are composed of organ systems whose structures reflect their particular function.

Sharper Cell Images with Fluorescence Microscopy

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