Name: how light microscopes bring samples into view
Uploaded: 2023-04-30T11:59:33.000Z
Duration: 1 min 18 s
Channel: JoVE

Optik mikroskoplar, numuneleri aydınlatmak için ışık kaynakları kullanır. Işık bir dizi mercekten geçer; Kondansatör, ışığı numuneye odaklarken, objektif ve oküler lensler büyütülmüş ve ters çevrilmiş iki boyutlu bir görüntü oluşturur.
Bir merceğin sayısal açıklığı, ışık toplama yeteneğini belirler. Toplanan ışık miktarı, ışık konisinin açısına ve ışığın içinden geçtiği ortamın, örneğin hava, su veya yağın kırılma indisine bağlıdır.
Optik mikroskopların çözünürlük sınırı 0,2 mikrometredir. Çapı sıfır nokta beş mikrometre civarında olan ökaryotik hücreler içindeki prokaryotik hücreleri veya büyük organelleri çözebilirler. 
Objektif merceğin çözünürlüğü, ışığın dalga boyu ve merceğin sayısal açıklığı ile ters orantılıdır; değer ne kadar küçükse, büyütülmüş görüntünün çözünürlüğü o kadar yüksek olur.
Bir optik mikroskop, objektif ve oküler lenslerin büyütmelerinin çarpılmasıyla hesaplanan, maksimum 1000 kat büyütmeli bir görüntü üretebilir.

biyolojik Örneklerin optik mikroskopi ile görüntülenmesi

Imaging Biological Samples with Optical Microscopy

Optik mikroskopi, numunelerin ayrıntılı görüntülerini sağlamak için optik prensipleri kullanır. Antonie van Leeuwenhoek, 17. yüzyılda kan hücrelerini, bakterileri ve maya hücrelerini görselleştirmek için ilk bileşik optik mikroskobu tasarladı. 1830'da Joseph Jackson Lister esasen modern bir ışık mikroskobu yarattı. 20. yüzyılda gelişmiş büyütme ve çözünürlüğe sahip mikroskopların gelişimi görüldü. Optik mikroskopide, görüntülenecek örnek bir cam slayt üzerine yerleştirilir ve sahneye (bir platform) kırpılır. Slayt sabitlendikten sonra slaydın üzerindeki numune, x-y mekanik kademe düğmeleri kullanılarak ışığın üzerine konumlandırılır. Bu düğmeler sahnenin yüzeyindeki sürgüyü hareket ettirir ancak sahneyi yükseltmez veya alçaltmaz. Numune ışık üzerinde ortalandıktan sonra sahne konumu, görüntüye odaklanmak için yükseltilebilir veya alçaltılabilir. Kaba odaklama düğmesi, 4⨯ ve 10⨯ objektif lenslerle büyük ölçekli hareketler için kullanılır; ince odaklama düğmesi, özellikle 40⨯ veya 100⨯ objektif lenslerle küçük ölçekli hareketler için kullanılır. Görüntüler büyütüldüğünde, görüntünün birim alanına daha az ışık düştüğü için soluklaşırlar. Mikroskoplar oldukça büyütülmüş görüntüler üretir, bu nedenle yoğun aydınlatma gerektirir. Aydınlık alan mikroskobunda, bu ışığı bir aydınlatıcı, tipik olarak sahnenin altında yüksek yoğunluklu bir ampul ile sağlar. Aydınlatıcıdan gelen ışık, aydınlatmayı en üst düzeye çıkarmak için tüm ışık ışınlarını numuneye odaklayan kondansatör mercekten (sahnenin altında bulunur) geçer. Kondansatörün konumu, takılı kondansatör odak düğmesi kullanılarak optimize edilebilir; optimum mesafe belirlendikten sonra, parlaklığı ayarlamak için kondansatör hareket ettirilmemelidir. Maksimumdan daha düşük ışık seviyelerine ihtiyaç duyulursa, numuneye çarpan ışık miktarı, kondansatör ile numune arasındaki diyaframın açılması veya kapatılmasıyla kolayca ayarlanabilir. Bazı durumlarda parlaklık, aydınlatıcının yoğunluğunu kontrol eden bir kısma anahtarı olan reosta kullanılarak da ayarlanabilir. Parlak alan mikroskobu, aydınlatıcıdan gelen ışığı numuneye yönlendirerek bir görüntü oluşturur; bu ışık farklı yapılar tarafından farklı şekilde iletilir, emilir, yansıtılır veya kırılır. Objektif mercek, numunenin gerçekten ters çevrilmiş bir görüntüsünü oluşturur. Göz merceği veya göz merceğinde görselleştirilen son görüntü, objektif tarafından oluşturulan görüntünün büyütülmüş sanal görüntüsüdür. Farklı renkler, numunenin bazı kısımlarındaki kromoforlarla (belirli ışık dalga boylarını emen ve yansıtan pigmentler) etkileşime girdikçe farklı davranabilir. Çoğunlukla kromoforlar, lekeler kullanılarak numuneye yapay olarak eklenir, kontrast ve çözünürlük artar. Genel olarak, numunedeki yapılar parlak arka plana göre çeşitli düzeylerde daha koyu görünecek ve yaklaşık 1000⨯'ye kadar büyütmelerde maksimum keskinlikte görüntüler yaratacaktır. Daha fazla büyütme daha büyük bir görüntü oluşturacaktır ancak çözünürlük artmayacaktır. Bu, yaklaşık 400°'de görülebilen, bakteri kadar küçük nesneleri görmemizi sağlar, ancak virüsler gibi daha küçük nesneleri göremez. Çok yüksek büyütmelerde, ışık numune ile mercek arasındaki az miktarda havanın içinden geçtiğinde çözünürlük bozulabilir. Bunun nedeni hava ile camın kırılma indisleri arasındaki önemli farktır; hava, ışık ışınlarını mercek onları odaklayamadan dağıtır. Bu sorunu çözmek için, numune ile immersiyon yağlarıyla kullanılmak üzere tasarlanmış özel bir mercek olan immersiyon yağı merceği arasındaki boşluğu doldurmak için bir damla yağ kullanılabilir. Yağın kırılma indisi camınkine çok benzer olduğundan, numuneden çıkan ışığın merceğe çarpabileceği maksimum açıyı arttırır. Bu, toplanan ışığı ve dolayısıyla görüntünün çözünürlüğünü artırır. Farklı ışık türleri için çeşitli yağlar kullanılabilir.

Biyolojik Örneklerin Optik Mikroskopi ile Görüntülenmesi

Optik mikroskopi, numunelerin ayrıntılı görüntülerini sağlamak için optik prensipleri kullanır. Antonie van Leeuwenhoek, ilk bileşik optik mikroskobu tasarladı ...

imaging-biological-samples-with-optical-microscopy

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

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 slide is then moved over the light with the x-y mechanical stage knobs. These knobs move the slide across the stage but do not lift it. After the specimen is centered over the light, the stage is raised or lowered to bring the image into focus.

The coarse focus knob is used for large adjustments with 4x and 10x objective lenses. The fine focus knob makes small adjustments, especially with 40x and 100x lenses. As images become more magnified, they also become dimmer because less light reaches each part of the image. For that reason, microscopes need strong lighting.

In a brightfield microscope, a high-intensity bulb below the stage acts as the illuminator. Light from the illuminator passes through the condenser lens, which focuses the light onto the specimen. The condenser can be set with the condenser focus knob. After the best distance is found, the condenser should not be moved just to change brightness. If less light is needed, the diaphragm between the condenser and the specimen can be opened or closed. In some microscopes, the rheostat, or dimmer switch, also changes the illuminator intensity.

A brightfield microscope forms an image because the specimen transmits, absorbs, reflects, or refracts light in different ways. The objective lens makes a real inverted image. The eyepiece, also called the ocular, then shows a magnified virtual image of that picture. Chromophores, which are pigments that absorb and reflect certain wavelengths of light, can affect how colors appear. Stains are often added to increase contrast and resolution. This makes structures in the sample appear darker than the bright background and allows sharp images at magnifications up to about 1000x.

Higher magnification does not keep improving resolution. It can make the image larger without making it clearer. This is why bacteria can be seen at about 400x, but smaller objects such as viruses cannot. At very high magnifications, resolution can drop because light passes through the thin air gap between the specimen and the lens. Air and glass have very different refractive indices, so air scatters light before it can be focused.

Oil immersion solves this problem. A drop of oil fills the space between the specimen and an oil immersion lens, which is designed to work with immersion oils. Because the oil has a refractive index very close to glass, it lets light leave the specimen at a wider angle and reach the lens more easily. This increases the amount of light collected and improves image resolution. Different oils can be used for different types of light.

How Light Microscopes Bring Samples Into View

how-light-microscopes-bring-samples-into-view

imaging biological samples with optical microscopy

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...

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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...

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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...

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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...

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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...

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Surface-Only Imaging with TIRF Microscopy

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Atomic Force Microscopy for Surface Mapping

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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...

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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...

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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...

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Electron Microscopy Sample Prep Methods

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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...

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electron-microscope-tomography-and-single-particle-reconstruction

3D Imaging with Electron Microscopy

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key terms and concepts

Learn how optical and brightfield microscopes use light, lenses, stains, and oil immersion to view cells, bacteria, and other tiny samples clearly.

How Light Microscopes Bring Samples Into View | 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.

How Light Microscopes Bring Samples Into View

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