Name: microscope contrast for live, unstained cells
Uploaded: 2023-04-30T11:59:41.000Z
Duration: 1 min 26 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

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

Phase Contrast and Differential Interference Contrast Microscopy

Le cellule viventi non colorate sono trasparenti e difficili da vedere al microscopio ottico. I microscopi a contrasto di fase e a contrasto di interferenza differenziale o DIC consentono la loro visualizzazione creando immagini a risoluzione più elevata e con contrasto più nitido.
Quando un campione viene illuminato a contrasto di fase, una parte della luce viene diffratta dai componenti della cella, mentre il resto passa direttamente. L'entità della diffrazione dipende dagli indici di rifrazione dei componenti cellulari.
Poiché la diffratta e la luce diretta interferiscono, parti del campione appaiono luminose se le onde sono in fase e scure se sono fuori fase. In questo modo si creano immagini ad alto contrasto di grandi organelli e sottili sezioni di tessuto.
Cambiamenti bruschi negli indici di rifrazione producono un alone attorno all'immagine.
Tali artefatti sono assenti nel DIC, che utilizza una sorgente di luce polarizzata per modulare le onde luminose che passano attraverso il campione.
Il contrasto dell'immagine dipende dalla velocità di variazione degli indici di rifrazione nel campione. I cambiamenti più intensi generano immagini ad alto contrasto con un apparente effetto tridimensionale.
La DIC aiuta a visualizzare campioni spessi come fette di cervello, uova o embrioni.

<ol>
	<li>Parker, N. et al. Microbiologia: OpenStax., 2022. Sezione 2.4</li>
	<li>Frohlich, V.C., 2008. Microscopia a contrasto di fase e contrasto interferenziale differenziale (DIC). JoVE (Giornale degli esperimenti visualizzati), (18), p.e844.</li>
	<li>Palima, D. e Glückstad, J., 2010. Contrasto di fase generalizzato: microscopia, manipolazione e altro ancora. Fisica contemporanea, 51(3), pp.249-265.</li>
	<li>Obara, B., Roberts, M.A., Armitage, J.P. e Grau, V., 2013. Identificazione di cellule batteriche in immagini di microscopia a contrasto ad interferenza differenziale. Bioinformatica BMC, 14(1), pp.1-13.</li>
</ol>

microscopia a contrasto di fase e contrasto di interferenza differenziale

Microscopi a contrasto di fase

Nei microscopi a contrasto di fase, l'interferenza tra la luce che passa direttamente attraverso una cellula e la luce rifratta dai componenti cellulari viene utilizzata per creare immagini ad alto contrasto e ad alta risoluzione senza colorazione. È il tipo di microscopio più antico e semplice che crea un'immagine alterando le lunghezze d'onda dei raggi luminosi che passano attraverso il campione. I percorsi di lunghezza d'onda alterati vengono creati utilizzando un arresto anulare nel condensatore. Lo stop anulare produce un cono cavo di luce focalizzato sul campione prima di raggiungere la lente dell'obiettivo. L'obiettivo contiene una piastra di fase con un anello di fase. Di conseguenza, la luce che viaggia direttamente dall'illuminatore passa attraverso l'anello di fase mentre la luce rifratta o riflessa dal campione passa attraverso la piastra. Ciò fa sì che le onde che viaggiano attraverso l'anello siano sfalsate di circa metà della lunghezza d'onda rispetto a quelle che passano attraverso la piastra.

Poiché le onde hanno picchi e avvallamenti, possono sommarsi (se sono in fase) o annullarsi a vicenda (se fuori fase). Quando le lunghezze d'onda sono sfasate, le depressioni dell'onda cancellano i picchi dell'onda, fenomeno chiamato interferenza distruttiva. Le strutture che rifrangono la luce appaiono quindi scure su uno sfondo luminoso di sola luce non rifratta. Più in generale, le strutture che differiscono per caratteristiche come l'indice di rifrazione differiranno nei livelli di oscurità. Poiché aumenta il contrasto senza richiedere colorazioni, la microscopia a contrasto di fase viene spesso utilizzata per osservare campioni vivi. Strutture cellulari specifiche, come gli organelli nelle cellule eucariotiche e le endospore nelle cellule procariotiche, sono ben visualizzate con la microscopia a contrasto di fase.

Microscopi a contrasto di interferenza differenziale

I microscopi a contrasto di interferenza differenziale (DIC) (noti anche come ottica Nomarski) sono simili ai microscopi a contrasto di fase in quanto utilizzano modelli di interferenza per migliorare il contrasto tra le diverse caratteristiche di un campione. In un microscopio DIC vengono creati due fasci di luce in cui la direzione del movimento delle onde (polarizzazione) è diversa. Una volta che i raggi attraversano il campione o lo spazio libero dal campione, vengono ricombinati. Gli effetti dei campioni causano differenze negli schemi di interferenza generati dalla combinazione dei raggi. Ciò si traduce in immagini ad alto contrasto di organismi viventi con un aspetto tridimensionale. Questi microscopi sono particolarmente utili per distinguere le strutture all'interno di campioni vivi e non colorati.

Microscopia a contrasto di fase e contrasto di interferenza differenziale

Microscopi a contrasto di fase
I microscopi a contrasto di fase, l'interferenza tra la luce che passa direttamente attraverso una cellula e la luce rifratta ...

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

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 ring. Light from the illuminator passes through the ring, while refracted light passes through the plate. This makes the light waves about one-half wavelength out of phase.

When waves are out of phase, their peaks and troughs can cancel each other out. This is called destructive interference. As a result, structures that bend light appear dark against a bright background. Differences in refractive index can also change how dark a structure looks. Because it does not require stains, phase-contrast microscopy is often used for live specimens. It can clearly show organelles in eukaryotic cells and endospores in prokaryotic cells.

Differential interference contrast (DIC) microscopy, also called Nomarski optics, uses a similar idea. It creates two beams of light with different polarization directions. After the beams pass through the specimen or through empty space, they are recombined. The specimen changes the interference pattern that forms when the beams come together.

DIC microscopy produces high-contrast images of living organisms with a three-dimensional look. It is especially useful for telling apart structures inside live, unstained specimens. Like phase-contrast microscopy, it helps reveal details that are hard to see with ordinary bright-field light.

Microscope Contrast for Live, Unstained Cells

microscope-contrast-for-live-unstained-cells

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 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

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 phase-contrast and DIC microscopy reveal live, unstained cells by using light interference to boost contrast and show fine structures clearly.

Microscope Contrast for Live, Unstained Cells | JoVE K12

cell biology

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

Microscope Contrast for Live, Unstained Cells

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