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

Niebarwione żywe komórki są przezroczyste i trudne do zobaczenia pod mikroskopem optycznym. Mikroskopy DIC z kontrastem fazowym i kontrastem różnicowo-interferencyjnym umożliwiają ich wizualizację poprzez tworzenie obrazów o wyższej rozdzielczości i ostrzejszym kontraście.
Gdy próbka jest oświetlona kontrastem fazowym, część światła jest dyfrakcjonowana przez składniki ogniwa, podczas gdy reszta przechodzi bezpośrednio. Zakres dyfrakcji zależy od współczynników załamania światła składników komórek.
Ponieważ dyfrakcja i bezpośrednie światło interferują, części próbki wydają się jasne, jeśli fale są w fazie i ciemne, jeśli są poza fazą. W ten sposób powstają obrazy o wysokim kontraście dużych organelli i cienkich skrawków tkanek.
Ostre zmiany współczynników załamania światła tworzą aureolę wokół obrazu.
Takie artefakty są nieobecne w DIC, który wykorzystuje spolaryzowane źródło światła do modulowania fal świetlnych przechodzących przez próbkę.
Kontrast obrazu zależy od szybkości zmian współczynników załamania światła w całej próbce. Strome zmiany generują obrazy o wysokim kontraście z pozornym efektem trójwymiarowości.
DIC pomaga wizualizować grube próbki, takie jak plastry mózgu, jaja lub zarodki.

<ol>
	<li>Parker, N. i in. Mikrobiologia: OpenStax., 2022. Sekcja 2.4</li>
	<li>Frohlich, V.C., 2008. Mikroskopia kontrastu fazowego i kontrastu różnicowego interferencyjnego (DIC). JoVE (Dziennik Eksperymentów Wizualnych), (18), s. e844.</li>
	<li>Palima, D. i Glückstad, J., 2010. Uogólniony kontrast fazowy: mikroskopia, manipulacja i inne. Fizyka współczesna, 51(3), s.249-265.</li>
	<li>Obara, B., Roberts, M.A., Armitage, J.P. i Grau, V., 2013. Identyfikacja komórek bakteryjnych w obrazach z mikroskopii kontrastowej z interferencją różnicową. Bioinformatyka BMC, 14(1), s.1-13.</li>
</ol>

mikroskopia kontrastowo-fazowa i różnicowo-interferencyjna

<h4>Mikroskopy z kontrastem fazowym</h4> 

 W mikroskopach z kontrastem fazowym interferencja pomiędzy światłem przechodzącym bezpośrednio przez komórkę a światłem załamanym przez składniki komórkowe jest wykorzystywana do tworzenia obrazów o wysokim kontraście i wysokiej rozdzielczości bez plam. Jest to najstarszy i najprostszy typ mikroskopu, który tworzy obraz poprzez zmianę długości fal promieni świetlnych przechodzących przez preparat. Zmienione ścieżki długości fal tworzone są za pomocą pierścieniowego ogranicznika w skraplaczu. Pierścieniowy ogranicznik wytwarza wydrążony stożek światła skupionego na preparacie przed dotarciem do soczewki obiektywu. Obiektyw zawiera płytkę fazową z pierścieniem fazowym. W rezultacie światło przechodzące bezpośrednio z oświetlacza przechodzi przez pierścień fazowy, podczas gdy światło załamane lub odbite przez próbkę przechodzi przez płytkę. Powoduje to, że fale przechodzące przez pierścień są przesunięte w fazie o około połowę długości fali w stosunku do fal przechodzących przez płytkę. 

 Ponieważ fale mają szczyty i doliny, mogą się sumować (jeśli są w fazie) lub znosić się nawzajem (jeśli są w przeciwfazie). Kiedy długości fal są przesunięte w fazie, doliny fal znoszą szczyty fal, co nazywa się zakłóceniami destrukcyjnymi. Struktury załamujące światło wydają się wówczas ciemne na jasnym tle składającym się wyłącznie z niezałamanego światła. Mówiąc bardziej ogólnie, struktury różniące się takimi cechami, jak współczynnik załamania światła, będą różnić się poziomem zaciemnienia. Ponieważ zwiększa kontrast bez konieczności barwienia, do obserwacji żywych okazów często stosuje się mikroskopię z kontrastem fazowym. Specyficzne struktury komórkowe, takie jak organelle w komórkach eukariotycznych i endospory w komórkach prokariotycznych, są dobrze uwidocznione za pomocą mikroskopii z kontrastem fazowym. 
 <h4>Mikroskopy różnicowo-interferencyjne kontrastowe</h4> 

 Mikroskopy z kontrastem różnicowo-interferencyjnym (DIC) (znane również jako optyka Nomarskiego) są podobne do mikroskopów z kontrastem fazowym, ponieważ wykorzystują wzorce interferencyjne w celu zwiększenia kontrastu między różnymi cechami próbki. W mikroskopie DIC powstają dwie wiązki światła, w których różni się kierunek ruchu fali (polaryzacja). Gdy wiązki przejdą przez próbkę lub przestrzeń wolną od próbek, zostają ponownie połączone. Oddziaływanie próbek powoduje różnice we wzorach interferencji generowanych przez połączenie wiązek. Dzięki temu powstają obrazy żywych organizmów o wysokim kontraście i trójwymiarowym wyglądzie. Mikroskopy te są szczególnie przydatne w rozróżnianiu struktur w żywych, niezabarwionych okazach.

Mikroskopia kontrastowo-fazowa i różnicowo-interferencyjna

Mikroskopy z kontrastem fazowym
Mikroskopy z kontrastem fazowym, interferencja między światłem przechodzącym bezpośrednio przez komórkę a światłem załamywanym przez komórkę ...

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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

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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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, ...

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

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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 ...

Cell Diversity

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Chemistry of the Cell

The cell is chemically composed of water, organic molecules and inorganic ions.
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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

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

Prokaryotes are small unicellular organisms that include the domains&#x2014;Archaea and Bacteria. Bacteria include many common organisms, such as ...

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

Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that ...

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

Peroxisomes and mitochondria are two important oxygen-utilizing organelles in eukaryotic cells. Mitochondria carry out cellular respiration&#x2014;the ...

Gap Junctions

Multicellular organisms employ a variety of ways for cells to communicate with each other. Gap junctions are specialized proteins that form pores between ...

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

Diffusion is the passive movement of substances down their concentration gradients&#x2014;requiring no expenditure of cellular energy. Substances, such as ...

Osmosis

Approximately 60% to 95% of the weight of living organisms is attributed to water. Therefore, maintaining appropriate water balance within cells is of ...

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

Tonicity describes the capacity of a cell to lose or gain water. It depends on the quantity of solute that does not penetrate the membrane. Tonicity ...

Introduction to Membrane Proteins

The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the ...

Facilitated Transport

The chemical and physical properties of plasma membranes cause them to be selectively permeable. Since plasma membranes have both hydrophobic and ...

Primary Active Transport

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Secondary Active Transport

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Receptor-mediated Endocytosis

Overview

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Pinocytosis

Cells use energy-requiring bulk transport mechanisms to transfer large particles, or large amounts of small particles, into or out of the cell. The cells ...

Phagocytosis

Cells pull particles inward and engulf them in spherical vesicles in an energy-requiring process called endocytosis. Phagocytosis (&#x201C;cellular ...

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 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

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

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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.

Microscope Contrast for Live, Unstained Cells

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