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

광학 현미경은 광원을 사용하여 샘플을 비춥니다. 빛은 일련의 렌즈를 통과합니다. 콘덴서는 샘플에 빛을 집중시키는 반면, 대물 렌즈와 접안 렌즈는 확대 및 반전된 2차원 이미지를 생성합니다.
렌즈의 개구수는 빛을 모으는 능력을 결정합니다. 모이는 빛의 양은 광 원뿔의 각도와 빛이 통과하는 매체의 굴절률(예: 공기, 물 또는 기름)에 따라 달라집니다.
광학 현미경의 해상도 제한은 0.2마이크로미터입니다. 그들은 진핵 세포 내에서 원핵 세포 또는 큰 세포 기관을 분해할 수 있으며, 이는 직경이 약 0점 5마이크로미터입니다. 
대물 렌즈의 해상도는 빛의 파장과 렌즈의 개구수에 반비례합니다. 값이 작을수록 확대된 이미지의 해상도가 높아집니다.
광학 현미경은 대물 렌즈와 안구 렌즈의 배율을 곱하여 계산한 최대 1000배 배율의 이미지를 생성할 수 있습니다.

광학현미경을 이용한 생물학적 샘플 이미징

Imaging Biological Samples with Optical Microscopy

광학 현미경 검사법은 광학 원리를 사용하여 샘플의 상세한 이미지를 제공합니다. 안토니 판 레이우엔훅은 혈액 세포, 박테리아 및 효모 세포를 시각화하기 위해 17세기 최초의 복합 광학 현미경을 설계했습니다. 1830년에 조셉 잭슨 리스터는 본질적으로 현대적인 광학 현미경을 만들었습니다. 20세기에는 배율과 해상도가 향상된 현미경이 개발되었습니다.

광학 현미경 검사에서는 관찰할 표본을 유리 슬라이드 위에 놓고 스테이지(플랫폼)에 고정합니다. 슬라이드가 고정되면 x-y 기계식 스테이지 손잡이를 사용하여 슬라이드의 표본이 조명 위에 배치됩니다. 이 손잡이는 스테이지 표면의 슬라이드를 움직이지만 스테이지를 올리거나 내리지는 않습니다. 표본이 조명 중앙에 위치하면 스테이지 위치를 올리거나 내려 이미지의 초점을 맞출 수 있습니다. 거친 초점 조절 손잡이는 4⨯ 및 10⨯ 대물 렌즈를 사용한 대규모 이동에 사용됩니다. 미세 초점 조절 손잡이는 특히 40⨯ 또는 100⨯ 대물 렌즈의 소규모 움직임에 사용됩니다.

이미지를 확대하면 이미지의 단위 면적당 빛의 양이 적어져 어두워집니다. 현미경은 매우 확대된 이미지를 생성하므로 강렬한 조명이 필요합니다. 명시야 현미경에서는 조명 장치(일반적으로 스테이지 아래의 고강도 전구)가 이러한 빛을 제공합니다. 조명기의 빛은 콘덴서 렌즈(스테이지 아래에 위치)를 통과하여 모든 광선을 시편에 집중시켜 조명을 최대화합니다. 부착된 콘덴서 초점 손잡이를 사용하여 콘덴서의 위치를 최적화할 수 있습니다. 최적의 거리가 설정된 후에는 밝기 조정을 위해 콘덴서를 이동해서는 안 됩니다. 최대 미만의 조명 수준이 필요한 경우 콘덴서와 표본 사이의 다이어프램을 열거 나 닫아 표본에 닿는 빛의 양을 쉽게 조정할 수 있습니다. 어떤 경우에는 조명의 강도를 제어하는 조광기 스위치인 가변저항기를 사용하여 밝기를 조정할 수도 있습니다.

명시야 현미경은 조명기에서 나온 빛을 표본에 전달하여 이미지를 생성합니다. 이 빛은 다양한 구조에 의해 차등적으로 투과, 흡수, 반사 또는 굴절됩니다. 대물렌즈는 표본의 실제 반전 이미지를 형성합니다. 접안렌즈 또는 안구에 시각화된 최종 이미지는 대물렌즈에 의해 형성된 이미지를 확대한 가상 이미지입니다.

다양한 색상은 표본의 일부에 있는 발색단(특정 파장의 빛을 흡수하고 반사하는 색소)과 상호 작용할 때 다르게 행동할 수 있습니다. 흔히 염색단을 사용하여 표본에 발색단을 인위적으로 추가하여 대비와 해상도를 높입니다. 일반적으로 표본의 구조는 밝은 배경보다 다양한 정도로 더 어둡게 나타나 최대 약 1000⨯의 배율에서 최대한 선명한 이미지를 생성합니다. 더 확대하면 더 큰 이미지가 생성되지만 해상도는 증가하지 않습니다. 이를 통해 우리는 약 400⨯ 정도에서 볼 수 있는 박테리아만큼 작은 물체를 볼 수 있지만 바이러스와 같은 더 작은 물체는 볼 수 없습니다.

매우 높은 배율에서는 빛이 표본과 렌즈 사이의 소량의 공기를 통과할 때 해상도가 저하될 수 있습니다. 이는 공기와 유리의 굴절률 사이의 상당한 차이 때문입니다. 공기는 렌즈가 광선의 초점을 맞추기 전에 광선을 산란시킵니다. 이 문제를 해결하기 위해 오일 한 방울을 사용하여 시편과 이머젼 오일과 함께 사용하도록 설계된 특수 렌즈인 오일 이머젼 렌즈 사이의 공간을 채울 수 있습니다. 오일은 유리의 굴절률과 매우 유사한 굴절률을 갖기 때문에 표본을 떠나는 빛이 렌즈에 닿을 수 있는 최대 각도가 증가합니다. 이렇게 하면 수집된 빛이 증가하여 이미지의 해상도가 높아집니다. 다양한 유형의 조명에 다양한 오일을 사용할 수 있습니다.

본 텍스트는 Openstax, Microbiology 2e, Section 2.3: Instruments of Microscopy에서 발췌한 것입니다.

광학 현미경 검사는 광학 원리를 사용하여 샘플의 상세한 이미지를 제공합니다. Antonie van Leeuwenhoek은 최초의 복합 광학 현미경을 설계했습니다.

imaging-biological-samples-with-optical-microscopy

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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 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.

How Light Microscopes Bring Samples Into View

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