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

Boyanmamış canlı hücreler şeffaftır ve optik mikroskop altında görülmesi zordur. Faz kontrastı ve diferansiyel girişim kontrastı veya DIC mikroskopları, daha yüksek çözünürlüklü ve daha keskin kontrastlı görüntüler oluşturarak görselleştirmelerine izin verir.
Bir numune faz kontrastında aydınlatıldığında, ışığın bir kısmı hücre bileşenleri tarafından kırılırken, geri kalanı doğrudan geçer. Kırınımın derecesi, hücre bileşenlerinin kırılma indekslerine bağlıdır.
Kırınım ve doğrudan ışık karıştıkça, dalgalar faz içi ise numunenin bazı kısımları parlak, faz dışıysa karanlık görünür. Bu, büyük organellerin ve ince doku bölümlerinin yüksek kontrastlı görüntülerini oluşturur.
Kırılma indislerindeki keskin değişiklikler, görüntünün etrafında bir hale oluşturur.
Numuneden geçen ışık dalgalarını modüle etmek için polarize bir ışık kaynağı kullanan DIC'de bu tür artefaktlar yoktur.
Görüntü kontrastı, numune boyunca kırılma indislerinin değişim hızına bağlıdır. Dik değişiklikler, belirgin bir üç boyutlu efekte sahip yüksek kontrastlı görüntüler oluşturur.
DIC, beyin dilimleri, yumurtalar veya embriyolar gibi kalın örnekleri görselleştirmeye yardımcı olur.

<ol>
	<li>Parker, N. ve ark. Mikrobiyoloji: OpenStax., 2022. Bölüm 2.4</li>
	<li>Frohlich, V.C., 2008. Faz kontrastı ve diferansiyel girişim kontrastı (DIC) mikroskobu. JoVE (Görselleştirilmiş Deneyler Dergisi), (18), s.e844.</li>
	<li>Palima, D. ve Glückstad, J., 2010. Genelleştirilmiş faz kontrastı: mikroskopi, manipülasyon ve daha fazlası. Çağdaş fizik, 51(3), s.249-265.</li>
	<li>Obara, B., Roberts, MA, Armitage, J.P. ve Grau, V., 2013. Diferansiyel girişim kontrast mikroskobu görüntülerinde bakteri hücresi tanımlaması. BMC biyoinformatik, 14(1), s.1-13.</li>
</ol>

faz kontrast ve diferansiyel İnterferans kontrast mikroskobu

<h4>Faz-Kontrast Mikroskopları</h4> Eş fazlı kontrast mikroskopları, doğrudan bir hücreden geçen ışık ile hücresel bileşenler tarafından kırılan ışık arasındaki girişim, lekelenme olmadan yüksek kontrastlı, yüksek çözünürlüklü görüntüler oluşturmak için kullanılır. Numunenin içinden geçen ışık ışınlarının dalga boylarını değiştirerek görüntü oluşturan en eski ve en basit mikroskop türüdür. Değiştirilmiş dalga boyu yolları, yoğunlaştırıcıdaki halka şeklinde bir durdurma kullanılarak oluşturulur. Halka şeklindeki durdurucu, objektif merceğe ulaşmadan önce numuneye odaklanan içi boş bir ışık konisi üretir. Objektif, faz halkalı bir faz plakası içerir. Sonuç olarak, doğrudan aydınlatıcıdan gelen ışık faz halkasından geçerken numune tarafından kırılan veya yansıtılan ışık plakasının içinden geçer. Bu, halkadan geçen dalgaların, plakadan geçenlerle dalga boyunun yaklaşık yarısı kadar faz dışı olmasına neden olur. Dalgaların tepe ve dip noktaları olduğundan, bir araya gelebilirler (eğer aynı fazdalarsa) veya birbirlerini iptal edebilirler (eğer faz dışıysalar). Dalga boyları faz dışı olduğunda, dalga çukurları dalga tepe noktalarını iptal eder, buna yıkıcı girişim denir. Işığı kıran yapılar, yalnızca kırılmamış ışıktan oluşan parlak bir arka plana karşı karanlık görünür. Daha genel olarak, kırılma indisi gibi özellikler bakımından farklı olan yapıların karanlık seviyeleri de farklı olacaktır. Leke gerektirmeden kontrastı artırdığından, canlı örnekleri gözlemlemek için faz kontrast mikroskobu sıklıkla kullanılır. Ökaryotik hücrelerdeki organeller ve prokaryotik hücrelerdeki endosporlar gibi spesifik hücresel yapılar, faz kontrast mikroskobu ile iyi bir şekilde görselleştirilir. <h4>Diferansiyel İnterferans Kontrast Mikroskopları</h4> Diferansiyel interferans kontrast (DIC) mikroskopları (Nomarski optiği olarak da bilinir), bir numunenin farklı özellikleri arasındaki kontrastı artırmak için girişim desenleri kullanmaları bakımından faz kontrast mikroskoplarına benzer. Bir DIC mikroskobunda, dalga hareketinin (polarizasyon) yönü farklı olan iki ışık ışını oluşturulur. Kirişler numuneden veya numunesiz alandan geçtikten sonra yeniden birleştirilir. Numunelerin etkileri, kirişlerin birleştirilmesiyle oluşturulan girişim desenlerinde farklılıklara neden olur. Bu, canlı organizmaların üç boyutlu görünüme sahip yüksek kontrastlı görüntüleri ile sonuçlanır. Bu mikroskoplar özellikle canlı, lekesiz örneklerdeki yapıların ayırt edilmesinde kullanışlıdır.

Faz Kontrast ve Diferansiyel İnterferans Kontrast Mikroskobu

Faz-Kontrast Mikroskopları
Faz içi kontrast mikroskoplar, bir hücreden doğrudan geçen ışık ile hücresel tarafından kırılan ışık arasındaki girişim ...

Education

JoVE Core

Cell Biology

Hücre Biyolojisi

Unit 1

Ünite 1

Visualizing Cells, Tissues, and Molecules

Hücrelerin, Dokuların ve Moleküllerin Görülebilir Kılınması

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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 Hücre Biyolojisi, anlaşılması kolay ancak kapsamlı animasyonlu video derslerde karmaşık hücresel süreçleri gösterir. Bu koleksiyon, organeller ve zarlar, hücresel taşıma ve hücre enerjileri dahil olmak üzere hücrelerin temel bileşenlerine ve işlevlerine odaklanır. Gerçek dünya araştırma deneylerinde hücre biyolojisinin uygulanmasını gösteren bilim insanı eylem videoları ile tamamlanmaktadır.

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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

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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

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Phase-Contrast Microscopes
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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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