Name: confocal microscopy for sharp cell images
Uploaded: 2023-04-30T12:00:13.000Z
Duration: 1 min 16 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

Confocal Fluorescence Microscopy

Un microscope confocal à balayage laser ou CLSM utilise un laser et une série de sténopés pour sectionner un échantillon en fines tranches optiques. Ces tranches peuvent être compilées pour reconstruire une image nette et détaillée.
Contrairement aux microscopes à fluorescence traditionnels où tout le champ est éclairé, le CLSM utilise un faisceau laser pour limiter l’excitation des fluorophores à un seul point à un moment donné dans une section mince de l’échantillon. Il en résulte une perte de fluorescence plus lente par photoblanchiment.
Un sténopé d’éclairage focalise la lumière, et un sténopé d’émission permet à la lumière uniquement du plan focal focalisé de passer jusqu’au détecteur, réduisant ainsi la fluorescence de fond et le flou des images.
Le laser balaie l’ensemble de l’échantillon à plusieurs reprises sur différents plans focaux le long de l’axe Z, capturant les images pour générer une série de sections optiques.
Ces coupes optiques peuvent être utilisées comme une image bidimensionnelle ou compilées ensemble comme une pile Z à l’aide d’un logiciel de traitement d’image pour reconstruire une image tridimensionnelle haute résolution.

<ol>
	<li>Elliott, A.D., 2020. Microscopie confocale : principes et pratiques modernes. Protocoles actuels en cytométrie, 92(1), p.e68.</li>
	<li>Schneider, S.L., Kohli, I., Hamzavi, I.H., Conseil, M.L., Rossi, A.M. et Ozog, D.M., 2019. Technologies d’imagerie émergentes en dermatologie : Partie I : Principes de base. Journal de l’Académie américaine de dermatologie, 80(4), pp.1114-1120.</li>
</ol>

microscopie confocale à fluorescence

La microscopie confocale est une technique microscopique avancée. Le principal avantage du microscope confocal par rapport aux autres techniques de microscopie est sa capacité à bloquer la lumière floue des échantillons éclairés à l’aide de trous d’épingle. Il est largement utilisé avec l’optique de fluorescence pour obtenir des images haute résolution et à contraste net. Contrairement aux microscopes optiques, les microscopes confocaux utilisent un faisceau laser focalisé pour balayer toute la surface de l'échantillon sur différents plans z. Ces microscopes sont donc très utiles pour examiner des spécimens épais tels que des biofilms, qui peuvent être examinés vivants et non fixés.

Les microscopes confocaux utilisent deux sténopés : l'éclairage et l'épingle d'émission, pour moduler le faisceau laser afin d'obtenir des images claires et nettes. Le laser passe à travers le sténopé d’éclairage et est réfléchi par le miroir dichroïque pour scanner la surface de l’échantillon. Un sténopé d'émission confocal au plan d'éclairage ; concentre la lumière émise atteignant le détecteur. Il élimine la lumière des plans z non focalisés atteignant le détecteur pour obtenir des images bidimensionnelles à contraste élevé appelées sections optiques. Un logiciel informatique fusionne ensuite les sections optiques de différents plans focaux pour reconstruire une image tridimensionnelle.

Deux types de microscopes confocaux sont largement utilisés en fonction de leur méthode de numérisation des échantillons ; microscopie à balayage laser (LSCM) et laser à disque tournant (SDLM). Dans le LSCM, un laser ponctuel balaye chaque plan focal de l'échantillon et collecte la fluorescence émise à travers un sténopé dans les détecteurs. Ces images bidimensionnelles, appelées sections optiques, peuvent être empilées pour reconstruire l’image tridimensionnelle. En revanche, SDLM se compose de deux disques rotatifs liés avec des centaines de trous d'épingle. Il permet un balayage rapide de la surface de l'échantillon sur différents plans et une capture d'image plus rapide.

Limites de la microscopie confocale

En microscopie confocale, les longueurs d’onde limitées de la lumière dans les lasers constituent un inconvénient. Les microscopes à fluorescence traditionnels offrent une large gamme de longueurs d'onde d'éclairage, utilisant des lampes à arc au mercure ou au xénon comme sources d'éclairage. La haute intensité du laser dans les premiers microscopes confocaux était dommageable pour les cellules, problème qui a été largement surmonté dans les systèmes de microscopes multiphotoniques.

Microscopie Confocale à Fluorescence

La microscopie confocale est une technique microscopique avancée. Le principal avantage du microscope confocal par rapport aux autres techniques de microscopie est sa capacité ...

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

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 unfixed.

Confocal microscopes use two pinholes: an illumination pinhole and an emission pinhole. The laser passes through the illumination pinhole and is reflected by a dichroic mirror to scan the sample surface. The emission pinhole stays aligned with the illumination plane and lets only focused emitted light reach the detector. Light from non-focused z-planes is removed, which creates high-contrast two-dimensional optical sections.

Computer software can then combine optical sections from different focal planes to build a three-dimensional image. Two common confocal systems are laser scanning confocal microscopy, or LSCM, and spinning disc laser microscopy, or SDLM. In LSCM, a point laser scans each focal plane across the sample, and the emitted fluorescence is collected through a pinhole in the detector. In SDLM, two linked spinning disks with hundreds of pinholes allow rapid scanning and faster image capture.

Confocal microscopy also has limits. Laser systems provide only a limited range of wavelengths, unlike traditional fluorescence microscopes that use mercury or xenon arc lamps. Early confocal microscopes also used high-intensity lasers that could damage cells. This problem has been reduced greatly in multiphoton microscope systems.

Confocal Microscopy for Sharp Cell Images

confocal-microscopy-for-sharp-cell-images

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 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

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...

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 confocal microscopy uses lasers and pinholes to remove blur, scan thick samples, and build sharp 2D and 3D fluorescence images.

Confocal Microscopy for Sharp Cell Images | 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.

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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

Cell Size

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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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Golgi Apparatus

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Microtubules

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The Extracellular Matrix

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Membrane Fluidity

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Diffusion

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Osmosis

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Tonicity in Animals

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Tonicity in Plants

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Receptor-mediated Endocytosis

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Pinocytosis

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Phagocytosis

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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

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

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

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Super-resolution Fluorescence Microscopy

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

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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.

Confocal Microscopy for Sharp Cell Images

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