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

光学顕微鏡は、光源を使用してサンプルを照らします。光は一連のレンズを通過します。コンデンサーはサンプルに光を集中させ、対物レンズと接眼レンズは拡大および反転した2次元画像を作成します。
レンズの開口数によって、光を集める能力が決まります。収集される光の量は、光円錐の角度と、光が通過する媒体(空気、水、油など)の屈折率によって異なります。
光学顕微鏡の分解能限界は0.2マイクロメートルです。彼らは、原核細胞または真核細胞内の大きな細胞小器官(直径が約0ポイント5マイクロメートル)を分解できます。 
対物レンズの解像度は、光の波長とレンズの開口数に反比例します。値が小さいほど、拡大像の解像度は高くなります。
光学顕微鏡は、対物レンズと接眼レンズの倍率を掛けて計算した最大1000倍の倍率で画像を生成できます。

光学顕微鏡による生体サンプルの画像化

Imaging Biological Samples with Optical Microscopy

光学顕微鏡では、光学原理を使用してサンプルの詳細な画像を提供します。 アントニー・ファン・レーウェンフックは、血球、細菌、酵母細胞を視覚化するために、17 世紀に最初の複合光学顕微鏡を設計しました。 1830 年、ジョセフ ジャクソン リスターは、本質的に近代的な光学顕微鏡を作成しました。 20 世紀には、倍率と解像度が向上した顕微鏡が開発されました。

光学顕微鏡では、観察する標本をスライドガラス上に置き、ステージ (プラットフォーム) にクリップで留めます。 スライドが固定されたら、X-Y メカニカル ステージのノブを使用して、スライド上の標本を照明の上に配置します。 これらのノブはステージ表面のスライドを動かしますが、ステージを上げたり下げたりするものではありません。 標本がライトの中心に配置されたら、ステージの位置を上げたり下げたりして画像の焦点を合わせることができます。 粗動フォーカスノブは、4⨯ および 10⨯ 対物レンズを使用した大規模な移動に使用されます。 微調整ノブは、特に 40⨯ または 100⨯ の対物レンズを使用する場合、小規模な移動に使用されます。

画像を拡大すると、画像の単位面積あたりの光が少なくなるため、画像が暗くなります。 顕微鏡は高倍率の画像を生成するため、強力な照明が必要です。 明視野顕微鏡では、照明装置、通常はステージの下の高輝度電球がこの光を提供します。 照明器からの光は集光レンズ (ステージの下にあります) を通過し、すべての光線が標本上に焦点を合わせて照明を最大化します。 付属のコンデンサーフォーカスノブを使用してコンデンサーの位置を最適化できます。 最適な距離が確立されたら、明るさを調整するためにコンデンサーを移動する必要はありません。 最大未満の光レベルが必要な場合は、コンデンサーと標本の間の絞りを開閉することで標本に当たる光の量を簡単に調整できます。 場合によっては、加減抵抗器（照明器の強度を調節する調光スイッチ）を使用して明るさを調整することもできます。

明視野顕微鏡は、照明装置からの光を標本に向けることによって画像を作成します。 この光は、さまざまな構造によって差動的に透過、吸収、反射、または屈折します。 対物レンズは標本の実倒立像を形成します。 接眼レンズまたは接眼レンズで視覚化される最終的な画像は、対物レンズによって形成された仮想画像を拡大したものです。

色が異なれば、標本の一部の発色団（光の特定の波長を吸収および反射する色素）と相互作用するため、異なる動作をする可能性があります。 多くの場合、染色を使用して発色団が標本に人工的に追加され、コントラストと解像度が向上します。 一般に、標本内の構造は明るい背景よりもさまざまな程度に暗く見え、最大約 1000⨯ までの倍率で最大限に鮮明な画像が得られます。 さらに拡大すると、より大きな画像が作成されますが、解像度は向上しません。 これにより、約 400⨯ 程度で見える細菌のような小さな物体を見ることができますが、ウイルスなどの小さな物体は見ることができません。

非常に高い倍率では、光が標本とレンズの間の少量の空気を通過するときに解像度が低下する可能性があります。 これは、空気とガラスの屈折率の大きな違いによるものです。 レンズが光線を集束させる前に、空気が光線を散乱させます。 この問題を解決するには、一滴の油を試料と油浸レンズ（液浸油で使用するように設計された特殊なレンズ）の間の空間を満たすのに使用できます。 オイルの屈折率はガラスの屈折率と非常に似ているため、試料から出た光がレンズに当たる最大角度が増加します。 これにより、集められる光が増加し、画像の解像度が向上します。 さまざまな種類の光にさまざまなオイルを使用できます。

このテキストは Openstax、Microbiology 2e、セクション 2.3: Instruments of Microscopy から抜粋したものです。

光学顕微鏡は、光学原理を使用してサンプルの詳細な画像を提供します。アントニー・ファン・レーウェンフックは、米国で最初の複合光学顕微鏡を設計しました...

imaging-biological-samples-with-optical-microscopy

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Visualizing Cells, Tissues, and Molecules

細胞・組織・分子の可視化

KEY TERMS AND CONCEPTS

主要用語と概念

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...

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 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 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 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

How Light Microscopes Bring Samples Into View

Description

Transcript