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

La microscopie à fluorescence conventionnelle ne peut résoudre que des structures plus éloignées que 200 nm. En effet, sous un microscope, la diffraction de la lumière fait apparaître les molécules fluorescentes comme des taches floues au lieu de points acérés. Par conséquent, lorsque les objets sont très proches les uns des autres, leurs images se chevauchent.
Par conséquent, les scientifiques ont mis au point des techniques de super-résolution qui offrent une meilleure résolution pour imager même de petites structures subcellulaires. Ces techniques surmontent les limites de diffraction en utilisant des motifs spécifiques de lumière pour exciter les fluorophores dans l’échantillon.
Par exemple, la microscopie à éclairage structuré utilise un motif rayé qui est ensuite tourné pour capturer plusieurs images. Les images sont combinées, créant un motif d’interférence qui est traité par calcul pour créer l’image finale.
La microscopie à déplétion par émission stimulée crée un motif à l’aide de deux lasers. Un laser primaire excite l’échantillon tandis qu’un faisceau en forme de beignet supprime la fluorescence autour de lui, ce qui fait paraître le point fluorescent plus petit.
D’autres techniques évitent le chevauchement des signaux en utilisant des sondes fluorescentes qui peuvent être activées et désactivées. Seules quelques sondes sont activées pour chaque image, ce qui minimise le chevauchement des signaux et permet la localisation d’une seule molécule fluorescente. 
Un grand nombre de ces images peuvent être combinées en une seule image composite haute résolution.

<ol>
	<li>Baddeley, David et Joerg Bewersdorf. « Aperçu biologique de la microscopie à super-résolution : ce que nous pouvons apprendre des images basées sur la localisation. » Revue annuelle de biochimie 87 (2018) : 965-989.</li>
	<li>Chen, Xi, Yu Wang, Xuewei Zhang et Changsheng Liu. « Avancées en microscopie à fluorescence à super-résolution pour l’étude des interactions nano-cellules. » Biomaterials Science 9, n° 16 (2021) : 5484-5496.</li>
</ol>

microscopie à fluorescence super-résolution

La microscopie à fluorescence à super-résolution (SRFM) offre une meilleure résolution que la microscopie à fluorescence conventionnelle en réduisant la fonction d'étalement des points (PSF). PSF est la distribution de l'intensité lumineuse à partir d'un point qui la fait apparaître floue. En raison du PSF, chaque point fluorescent apparaît plus grand que sa taille réelle, et c'est l'interférence PSF des fluorophores proches qui provoque l'image floue. Diverses approches pour obtenir une résolution plus élevée grâce au SRFM ont récemment été développées.

Microscopie de Localisation Photoactivée

La microscopie de localisation photoactivée (PALM) est un type de microscopie à fluorescence qui capture des images haute résolution à l'aide d'une approche de détection et de localisation d'une seule molécule. Par exemple, deux points fluorescents distants de 75 nm peuvent apparaître comme un seul point lors de l'imagerie en raison de l'interférence de leurs PSF. Dans de tels cas, notamment pour l’imagerie de cellules vivantes, PALM est une technique appropriée pour résoudre les interférences et fournir une meilleure résolution.

Dans PALM, une variante de protéine fluorescente verte (GFP) avec une longueur d’onde d’excitation différente et une fluorescence élevée est utilisée. Dans un premier temps, quelques GFP sont activées et imagées avec une très haute précision. À l’étape suivante, un autre ensemble de GFP est activé et imagé. Étape par étape, toutes les GFP de l’échantillon sont ainsi enregistrées. Enfin, les données sont traitées pour générer une image haute résolution.

Microscopie de Reconstruction Optique Stochastique

En microscopie de reconstruction optique stochastique (STORM), des sondes photo-commutables uniques sont utilisées pour l'imagerie des échantillons. L'émission de ces sondes peut être activée et désactivée à l'aide de lumières de différentes longueurs d'onde. Ainsi, seuls quelques fluorophores peuvent être activés à un instant donné de telle sorte que le nombre de fluorophores activés soit nettement inférieur au nombre de fluorophores désactivés. Cette activation sélective des sondes permet de déterminer leur position précise dans l'échantillon. Suite à cela, le centre de chaque sonde fluorescente est identifié et marqué. Le processus est ensuite répété pour enregistrer toutes les sondes fluorescentes dans l’échantillon. Enfin, une image composite haute résolution est construite en superposant ces multiples images.

Microscopie à Fluorescence Super-Résolution

La microscopie à fluorescence à super-résolution (SRFM) offre une meilleure résolution que la microscopie à fluorescence conventionnelle en réduisant l’étalement du point ...

Education

Enseignement

JoVE Core

Cell Biology

Biologie cellulaire

Unit 1

Unité 1

Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

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

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 Biologie cellulaire illustre les processus cellulaires complexes dans des leçons vidéos complètes mais faciles à comprendre. Cette collection se concentre sur les composants et fonctions essentiels de la cellule ; cela inclut les organelles et les membranes ainsi que le transport et le métabolisme énergétique cellulaire. Elle est complétée par nos vidéos de scientifiques à l'action qui démontrent l'application de la biologie cellulaire dans des expériences concrètes.

What are Cells?

Cells are the smallest and basic units of life, whether it is a single cell that forms the entire organism, e.g., in a bacterium or trillions of them, ...

Characteristics of Life

Biology is a natural science that studies life and living organisms, including their structure, function, development, interactions, evolution, ...

https://cloudfront.jove.com/files/media/science-education/science-education-thumbs/10647.jpg

Animal and Plant Cell Structure

Animal and plant cells not only differ in their structure, function, and mode of nutrition but also in how they reproduce, specialize, and organize into ...

Eukaryotic Evolution

The endosymbiont theory is the most widely accepted theory of eukaryotic evolution; however, its progression is still somewhat debated. According to the ...

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The concept of a cell started with microscopic observations of dead cork tissue by Robert Hooke in 1665. Hooke coined the term &#34;cell&#34; based on the ...

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The cell is chemically composed of water, organic molecules and inorganic ions.
Water
The polarity of the water molecule and its resulting hydrogen ...

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

Cell Size

Cell sizes vary widely among and within organisms. Bacterial cells range between 1-10 micrometers (&#x3BC;m)and are considerably smaller than most ...

Eukaryotic Compartmentalization

One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and mitochondria, that carry ...

Prokaryotic Cells

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Cytoplasm

The cytoplasm consists of organelles and a framework of protein scaffolds called the cytoskeleton suspended in an aqueous solution, the cytosol. The ...

The Nucleus

The nucleus is a membrane-bound organelle that acts as a control center in a eukaryotic cell. It contains chromosomal DNA, which controls gene expression ...

Endoplasmic Reticulum

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

Ribosomes

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Golgi Apparatus

As they leave the Endoplasmic Reticulum (ER), properly folded and assembled proteins are selectively packaged into vesicles. These vesicles are ...

Microtubules

There are three types of cytoskeletal structures in eukaryotic cells&#x2014;microfilaments, intermediate filaments, and microtubules. With a diameter of ...

Peroxisomes

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Gap Junctions

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The Extracellular Matrix

In order to maintain tissue organization, many animal cells are surrounded by structural molecules that make up the extracellular matrix (ECM). Together, ...

Tissues

Cells with similar structure and function are grouped into tissues. A group of tissues with a specialized function is called an organ. There are four main ...

Plant Cell Wall

The plant cell wall gives plant cells shape, support, and protection. As a cell matures, its cell wall specializes according to the cell type. For ...

Plasmodesmata

The organs in a multicellular organism&#x2019;s body are made up of tissues formed by cells. To work together cohesively, cells must communicate. One way ...

What are Membranes?

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

Cell membranes are composed of phospholipids, proteins, and carbohydrates loosely attached to one another through chemical interactions. Molecules are ...

Fluid Mosaic Model

The fluid mosaic model was first proposed as a visual representation of research observations. The model comprises the composition and dynamics of ...

What is an Electrochemical Gradient?

Adenosine triphosphate, or ATP, is considered the primary energy source in cells. However, energy can also be stored in the electrochemical gradient of an ...

Diffusion

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Osmosis

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Tonicity in Animals

The tonicity of a solution determines if a cell gains or loses water in that solution. The tonicity depends on the permeability of the cell membrane for ...

Tonicity in Plants

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Introduction to Membrane Proteins

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Facilitated Transport

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Primary Active Transport

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Secondary Active Transport

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Receptor-mediated Endocytosis

Overview

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Pinocytosis

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Phagocytosis

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Exocytosis

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While endocytosis takes particles ...

Relate structure to function for the components of plant and animal cells. Explain the role of cell membranes as a highly selective barrier (passive and active transport).

SC.912.L.14.2

Cell Structure

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

Overview of Microscopy Techniques

The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern ...

Introduction to Light Microscopy

The light microscope is an instrument used by researchers in many different fields to magnify specimens to as much as a thousand times their original ...

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
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular ...

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability ...

Introduction to Fluorescence Microscopy

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. ...

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

Compare and contrast structure and function of various types of microscopes.

comparing-mitochondrial-chloroplast-and-prokaryotic-genomes

Comparing Mitochondrial, Chloroplast, and Prokaryotic Genomes

The present-day mitochondrial and chloroplast genomes have retained some of the characteristics of their ancestral prokaryotes and also have acquired new ...

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The Tree of Life - Bacteria, Archaea, Eukaryotes

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The Anatomy of Chloroplasts

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Explain the evidence supporting the scientific theory of the origin of eukaryotic cells (endosymbiosis).

SC.912.L.14.5

An Overview of Genetics and Disease

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Factors Affecting Illness

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Gene-Environment Interactions

Gene expression is influenced by environmental factors such as radiation, temperature, behavior, nutrition, and stress.
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Explain the significance of genetic factors, environmental factors, and pathogenic agents to health from the perspectives of both individual and public health.

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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