Name: confocal microscopy for sharp cell images
Uploaded: 2023-04-30T12:00:13.000Z
Duration: 1 min 16 s
Channel: JoVE

K12 Standards

Florida Department of Education

High School

Life Science

SC.912.L.14

SC.912.L.14.4

Confocal Fluorescence Microscopy

Конфокальный лазерный сканирующий микроскоп или CLSM использует лазер и ряд точечных отверстий для разрезания образца на тонкие оптические срезы. Эти срезы могут быть скомпилированы для восстановления четкого, детализированного изображения.
В отличие от традиционных флуоресцентных микроскопов, в которых освещается все поле, CLSM использует лазерный луч для ограничения возбуждения флуорофоров одной точкой в определенный момент времени на тонком участке образца. Это приводит к более медленной потере флуоресценции из-за фотообесцвечивания.
Световое отверстие фокусирует свет, а эмиссионное отверстие позволяет свету только из сфокусированной фокальной плоскости проходить к детектору, уменьшая фоновую флуоресценцию и размытость изображений.
Лазер многократно сканирует весь образец в разных фокальных плоскостях вдоль оси Z, захватывая изображения для создания серии оптических сечений.
Эти оптические сечения могут быть использованы в качестве двумерного изображения или скомпонованы в виде Z-стека с помощью программного обеспечения для обработки изображений для реконструкции трехмерного изображения с высоким разрешением.

<ol>
	<li>Эллиотт, А.Д., 2020. Конфокальная микроскопия: принципы и современная практика. Современные протоколы в цитометрии, 92(1), стр.e68.</li>
	<li>Шнайдер, С.Л., Коли, И., Хамзави, И.Х., Совет, М.Л., Росси, А.М. и Озог, Д.М., 2019. Новые технологии визуализации в дерматологии: Часть I: Основные принципы. Журнал Американской академии дерматологии, 80 (4), стр. 1114-1120.</li>
</ol>

Конфокальная флуоресцентная микроскопия

Конфокальная микроскопия – это современный микроскопический метод. Основным преимуществом конфокального микроскопа перед другими методами микроскопии является его способность блокировать расфокусированный свет от освещенных образцов с помощью точечных отверстий. Он широко используется в флуоресцентной оптике для получения резких контрастных изображений высокого разрешения. В отличие от оптических микроскопов, конфокальные микроскопы используют сфокусированный луч лазерного света для сканирования всей поверхности образца в разных z-плоскостях. Поэтому эти микроскопы очень полезны для исследования толстых образцов, таких как биопленки, которые можно исследовать живыми и нефиксированными. В конфокальных микроскопах используются два отверстия — освещение и эмиссионное — для модуляции лазерного луча, с целью получения резких и четких изображений. Лазер проходит через отверстие для освещения и отражается от дихроичного зеркала, сканируя поверхность образца. Эмиссионная обскура, конфокальная плоскости освещения; фокусирует излучаемый свет, попадающий на детектор. Он устраняет свет от несфокусированных z-плоскостей, попадающих на детектор, для получения высококонтрастных двумерных изображений, называемых оптическими секциями. Затем компьютерная программа объединяет оптические срезы из разных фокальных плоскостей для восстановления трехмерного изображения. В зависимости от метода сканирования образцов широко используются два типа конфокальных микроскопов; лазерная сканирующая (LSCM) и лазерная микроскопия с вращающимся диском (SDLM). В LSCM точечный лазер сканирует каждую фокальную плоскость образца и собирает излучаемую флуоресценцию через точечное отверстие в детекторах. Эти двухмерные изображения, называемые оптическими секциями, можно складывать друг на друга, чтобы восстановить трехмерное изображение. SDLM, напротив, состоит из двух связанных вращающихся дисков с сотнями точечных отверстий. Это позволяет быстро сканировать поверхность образца в разных плоскостях и быстрее захватывать изображения. Ограничения конфокальной микроскопии Недостатком конфокальной микроскопии является ограниченная длина волны света лазеров. Традиционные флуоресцентные микроскопы предлагают широкий диапазон длин волн освещения, используя в качестве источников освещения ртутные или ксеноновые дуговые лампы. Высокая интенсивность лазера в первых конфокальных микроскопах повреждала клетки, что было в значительной степени преодолено в системах многофотонных микроскопов.

Конфокальная микроскопия является передовым микроскопическим методом. Основным преимуществом конфокального микроскопа по сравнению с другими методами микроскопии является его способность ...

Education

JoVE Core

Cell Biology

Клеточная биология

Unit 1

Раздел 1

Visualizing Cells, Tissues, and Molecules

Визуализация клеток, тканей и молекул

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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 unfixed.

Confocal microscopes use two pinholes: an illumination pinhole and an emission pinhole. The laser passes through the illumination pinhole and is reflected by a dichroic mirror to scan the sample surface. The emission pinhole stays aligned with the illumination plane and lets only focused emitted light reach the detector. Light from non-focused z-planes is removed, which creates high-contrast two-dimensional optical sections.

Computer software can then combine optical sections from different focal planes to build a three-dimensional image. Two common confocal systems are laser scanning confocal microscopy, or LSCM, and spinning disc laser microscopy, or SDLM. In LSCM, a point laser scans each focal plane across the sample, and the emitted fluorescence is collected through a pinhole in the detector. In SDLM, two linked spinning disks with hundreds of pinholes allow rapid scanning and faster image capture.

Confocal microscopy also has limits. Laser systems provide only a limited range of wavelengths, unlike traditional fluorescence microscopes that use mercury or xenon arc lamps. Early confocal microscopes also used high-intensity lasers that could damage cells. This problem has been reduced greatly in multiphoton microscope systems.

Confocal Microscopy for Sharp Cell Images

confocal-microscopy-for-sharp-cell-images

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 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 confocal microscopy uses lasers and pinholes to remove blur, scan thick samples, and build sharp 2D and 3D fluorescence images.

Confocal Microscopy for Sharp Cell Images | 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.

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, ...

https://cloudfront.jove.com/files/media/science-education/science-education-thumbs/10647.jpg

Animal and Plant Cell Structure

Animal and plant cells not only differ in their structure, function, and mode of nutrition but also in how they reproduce, specialize, and organize into ...

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

The tonicity of a solution determines if a cell gains or loses water in that solution. The tonicity depends on the permeability of the cell membrane for ...

Tonicity in Plants

Tonicity describes the capacity of a cell to lose or gain water. It depends on the quantity of solute that does not penetrate the membrane. Tonicity ...

Introduction to Membrane Proteins

The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the ...

Facilitated Transport

The chemical and physical properties of plasma membranes cause them to be selectively permeable. Since plasma membranes have both hydrophobic and ...

Primary Active Transport

In contrast to passive transport, active transport involves a substance being moved through membranes in a direction against its concentration or ...

Secondary Active Transport

One example of how cells use the energy contained in electrochemical gradients is demonstrated by glucose transport into cells. The ion vital to this ...

Receptor-mediated Endocytosis

Overview

Receptor-mediated endocytosis is a process through which bulk amounts of specific molecules can be imported into a cell after binding to cell ...

Pinocytosis

Cells use energy-requiring bulk transport mechanisms to transfer large particles, or large amounts of small particles, into or out of the cell. The cells ...

Phagocytosis

Cells pull particles inward and engulf them in spherical vesicles in an energy-requiring process called endocytosis. Phagocytosis (&#x201C;cellular ...

Exocytosis

Exocytosis is used to release material from cells. Like other bulk transport mechanisms, exocytosis requires energy.

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
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, ...

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 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

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.

comparing-mitochondrial-chloroplast-and-prokaryotic-genomes

Comparing Mitochondrial, Chloroplast, and Prokaryotic Genomes

The present-day mitochondrial and chloroplast genomes have retained some of the characteristics of their ancestral prokaryotes and also have acquired new ...

the-tree-of-life-bacteria-archaea-eukaryotes

The Tree of Life - Bacteria, Archaea, Eukaryotes

The &#8220;tree of life&#8221; describes the evolution of life and the evolutionary relationships between organisms. The root of the tree is the common ...

The Anatomy of Chloroplasts

Green algae and plants, including green stems and unripe fruit, harbor specialized organelles called chloroplasts to carry out photosynthesis. They ...

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

Many human diseases are associated with mutations or variations in genetic sequences. Some of these genetic variants are heritable, passed down from ...

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.
The range of reaction suggests ...

Dimensions of Health and Illness

The factors influencing the health-illness continuum can be internal or external and may or may not be under conscious control. They are related to the ...

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.

Confocal Microscopy for Sharp Cell Images

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