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 microscopio a scansione laser confocale o CLSM utilizza un laser e una serie di fori stenopeici per sezionare un campione in sottili fette ottiche. Queste sezioni possono essere compilate per ricostruire un'immagine nitida e dettagliata.
A differenza dei microscopi a fluorescenza tradizionali, in cui l'intero campo è illuminato, il CLSM utilizza un raggio laser per limitare l'eccitazione dei fluorofori a un singolo punto in un dato momento in una sezione sottile del campione. Ciò si traduce in una perdita più lenta di fluorescenza dovuta al fotosbiancamento.
Un foro stenopeico di illuminazione focalizza la luce e un foro stenopeico di emissione consente alla luce solo dal piano focale focalizzato di passare attraverso il rivelatore, riducendo la fluorescenza e la sfocatura dello sfondo nelle immagini.
Il laser scansiona ripetutamente l'intero campione su diversi piani focali lungo l'asse Z, catturando le immagini per generare una serie di sezioni ottiche.
Queste sezioni ottiche possono essere utilizzate come immagini bidimensionali o compilate insieme come Z-stack utilizzando un software di elaborazione delle immagini per la ricostruzione di un'immagine tridimensionale ad alta risoluzione.

<ol>
	<li>Elliott, A.D., 2020. Microscopia confocale: principi e pratiche moderne. Protocolli attuali in citometria, 92(1), p.e68.</li>
	<li>Schneider, S.L., Kohli, I., Hamzavi, I.H., Consiglio, M.L., Rossi, A.M. e Ozog, D.M., 2019. Tecnologie emergenti di imaging in dermatologia: Parte I: Principi di base. Giornale dell'Accademia Americana di Dermatologia, 80(4), pp.1114-1120.</li>
</ol>

microscopia confocale a fluorescenza

La microscopia confocale è una tecnica microscopica avanzata. Il vantaggio principale del microscopio confocale rispetto ad altre tecniche di microscopia è la sua capacità di bloccare la luce fuori fuoco proveniente dai campioni illuminati utilizzando fori stenopeici. È ampiamente utilizzato con l'ottica a fluorescenza per ottenere immagini ad alta risoluzione e con contrasto nitido. A differenza dei microscopi ottici, i microscopi confocali utilizzano un raggio focalizzato di luce laser per scansionare l’intera superficie del campione su diversi piani z. Questi microscopi sono quindi molto utili per esaminare campioni spessi come i biofilm, che possono essere esaminati vivi e non fissati.

I microscopi confocali utilizzano due fori stenopeici: illuminazione e foro stenopeico di emissione, per modulare il raggio laser e ottenere immagini chiare e nitide. Il laser passa attraverso il foro stenopeico di illuminazione e viene riflesso dallo specchio dicroico per scansionare la superficie del campione. Un foro stenopeico di emissione confocale con il piano di illuminazione; focalizza la luce emessa che raggiunge il rilevatore. Elimina la luce dai piani z non focalizzati che raggiungono il rilevatore per ottenere immagini bidimensionali ad alto contrasto chiamate sezioni ottiche. Un programma software per computer unisce quindi le sezioni ottiche di diversi piani focali per ricostruire un'immagine tridimensionale.

Due tipi di microscopio confocale sono ampiamente utilizzati in base al metodo di scansione dei campioni; microscopia a scansione laser (LSCM) e laser a disco rotante (SDLM). In LSCM, un laser puntiforme scansiona ciascun piano focale del campione e raccoglie la fluorescenza emessa attraverso un foro stenopeico nei rilevatori. Queste immagini bidimensionali, chiamate sezioni ottiche, possono essere impilate per ricostruire l'immagine tridimensionale. Al contrario, SDLM è costituito da due dischi rotanti collegati con centinaia di fori stenopeici. Consente una rapida scansione della superficie del campione su diversi piani e un'acquisizione più rapida delle immagini.

Limitazioni della microscopia confocale

Nella microscopia confocale, le lunghezze d’onda limitate della luce nei laser rappresentano uno svantaggio. I tradizionali microscopi a fluorescenza offrono un'ampia gamma di lunghezze d'onda di illuminazione, utilizzando lampade ad arco al mercurio o allo xeno come fonti di illuminazione. L'elevata intensità del laser nei primi microscopi confocali era dannosa per le cellule, cosa che è stata in gran parte superata nei sistemi di microscopi multifotonici.

Microscopia confocale a fluorescenza

La microscopia confocale è una tecnica microscopica avanzata. Il vantaggio principale del microscopio confocale rispetto ad altre tecniche di microscopia è la sua capacità ...

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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 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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SC.912.L.14.1

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SC.912.L.14.2

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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

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Overview of Electron Microscopy

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Phase Contrast and Differential Interference Contrast Microscopy

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

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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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