Name: sharper cell images with fluorescence microscopy
Uploaded: 2023-04-30T12:00:45.000Z
Duration: 1 min 37 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

Super-resolution Fluorescence Microscopy

従来の蛍光顕微鏡では、200 nmより遠く離れた構造しか分解できません。これは、顕微鏡下では、光の回折により、蛍光分子が鋭い点ではなくぼやけた点として現れるためです。その結果、オブジェクトが互いに非常に接近している場合、それらの画像は重なります。
そのため、科学者たちは、小さな細胞内構造でもより優れた解像度で画像化できる超解像技術を開発しました。これらの技術は、特定の光パターンを使用してサンプル中の蛍光色素を励起することにより、回折限界を克服します。
例えば、構造化照明顕微鏡では、縞模様のパターンを回転させて複数の画像を撮影します。画像が結合され、干渉パターンが作成され、このパターンが計算処理されて最終的な画像が作成されます。
誘導放出量減少顕微鏡法は、2つのレーザーを使用してパターンを作成します。一次レーザーがサンプルを励起し、ドーナツ型のビームがサンプルの周囲の蛍光を抑制し、蛍光点を小さく見せます。
他の手法では、オンとオフを切り替えることができる蛍光プローブを使用することで、信号の重なりを回避します。各画像に対して一部のプローブのみが活性化されるため、シグナルのオーバーラップが最小限に抑えられ、単一の蛍光分子の局在化が可能になります。 
これらの画像の多くは、1 つの高解像度合成画像に結合できます。

<ol>
	<li>「超解像顕微鏡からの生物学的洞察:ローカリゼーションベースの画像から何を学ぶことができるか」 生化学の年次レビュー 87 (2018): 965-989.</li>
	<li>「ナノセル相互作用の研究のための超解像蛍光顕微鏡法の進歩」 Biomaterials Science 9, no. 16 (2021): 5484-5496.</li>
</ol>

超解像蛍光顕微鏡

超解像蛍光顕微鏡 (SRFM) は、点像分布関数 (PSF) を低減することにより、従来の蛍光顕微鏡よりも優れた解像度を提供します。 PSF は、ぼやけて見える点からの光の強度分布です。 PSF により、各蛍光点は実際のサイズよりも大きく見えます。ぼやけた画像の原因は近くの蛍光色素分子の PSF 干渉です。 SRFM を通じてより高い解像度を達成するためのさまざまなアプローチが最近開発されました。

光活性化局在性顕微鏡法

光活性化局在性顕微鏡法 (PALM) は、単一分子の検出および局在化アプローチを使用して高解像度画像をキャプチャする蛍光顕微鏡法の一種です。 たとえば、75 nm 離れた 2 つの蛍光スポットは、PSF の干渉により、イメージング中に単一のスポットに見える場合があります。 このような場合、特に生細胞イメージングの場合、PALM は干渉を解決し、より優れた解像度を提供するのに適した技術です。

PALM では、励起波長が異なり、高い蛍光を発する緑色蛍光タンパク質 (GFP) の変異体が使用されます。 最初のステップでは、いくつかの GFP が活性化され、非常に高い精度で画像化されます。 次のステップでは、別の GFP セットがアクティブ化され、イメージ化されます。 このようにして、段階的に、標本全体のすべての GFP が記録されます。 最後に、データが処理されて高解像度画像が生成されます。

確率的光学再構成顕微鏡法

確率的光学再構成顕微鏡法 (STORM) では、独自の光切り替え可能なプローブが標本イメージングに使用されます。 これらのプローブからの発光は、異なる波長の光を使用してオン/オフを切り替えることができます。 したがって、活性化された蛍光団の数が失活した蛍光団の数よりも大幅に少なくなるように、ある時点で活性化できる蛍光団はわずか数個だけである。 プローブのこの選択的な活性化は、標本内のプローブの正確な位置を決定するのに役立ちます。 これに続いて、各蛍光プローブの中心が特定され、マークされます。 次に、このプロセスを繰り返して、標本内のすべての蛍光プローブを記録します。 最後に、これらの複数の画像を重ね合わせて高解像度の合成画像が構築されます。

超解像蛍光顕微鏡(SRFM)は、点差を小さくすることで、従来の蛍光顕微鏡よりも優れた分解能を提供します。

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

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 microscopy, or PALM, uses single-molecule detection and localization to build a high-resolution image. It is useful when two fluorescent spots are about 75 nm apart and may look like one spot during imaging. This can be especially helpful in live-cell imaging.

PALM uses a variant of green fluorescent protein, or GFP, with a different excitation wavelength and high fluorescence. First, only a few GFP molecules are activated and imaged with very high precision. Then another set is activated and imaged. This step-by-step process continues until all GFPs in the specimen are recorded, and the data is processed into a high-resolution image.

Stochastic optical reconstruction microscopy, or STORM, uses photo-switchable probes instead of GFP. These probes can be switched on and off with lights of different wavelengths. Only a few fluorophores are activated at a time, while many remain off. This makes it easier to find the precise position of each probe in the specimen.

In STORM, the center of each fluorescent probe is identified and marked. The process is repeated until all probes have been recorded across the specimen. The separate images are then superimposed to form a high-resolution composite image.

Sharper Cell Images with Fluorescence Microscopy

sharper-cell-images-with-fluorescence-microscopy

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

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

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 super-resolution fluorescence microscopy sharpens cell images using PALM and STORM to locate fluorophores with far greater precision.

Sharper Cell Images with Fluorescence Microscopy | 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。

What are Cells?

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, ...

Characteristics of Life

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 Cell Structure

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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

The concept of a cell started with microscopic observations of dead cork tissue by Robert Hooke in 1665. Hooke coined the term &#34;cell&#34; based on the ...

Chemistry of the Cell

The cell is chemically composed of water, organic molecules and inorganic ions.
Water
The polarity of the water molecule and its resulting hydrogen ...

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

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Tonicity in Plants

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Introduction to Membrane Proteins

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Facilitated Transport

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Primary Active Transport

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Secondary Active Transport

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Receptor-mediated Endocytosis

Overview

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Pinocytosis

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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 and Differential Interference Contrast Microscopy

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 (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.

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Comparing Mitochondrial, Chloroplast, and Prokaryotic Genomes

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

SC.912.L.14.5

An Overview of Genetics and Disease

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Factors Affecting Illness

When a person&#39;s physical, emotional, intellectual, social development or spiritual functioning is compromised, this deviation from a healthy normal ...

Gene-Environment Interactions

Gene expression is influenced by environmental factors such as radiation, temperature, behavior, nutrition, and stress.
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Explain the significance of genetic factors, environmental factors, and pathogenic agents to health from the perspectives of both individual and public health.

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.

Sharper Cell Images with Fluorescence Microscopy

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