פרוטוקול טקסט מלא לגישה זו ניסיוני זמין <a href="http://www.mrw.interscience.wiley.com/emrw/9780471143031/cp/cpcb/article/cb0401/current/abstract ">הפרוטוקולים נוכחי בביולוגיה של התא</a> .

The authors have nothing to disclose.

major components of the light microscope

מיקרוסקופ אור הוא כלי בסיסי ביולוג של התא, שהיה צריך הבנה מעמיקה של איך זה עובד, איך זה צריך להיות מתואם עבור יישומים שונים, ואיך זה צריך להישמר כפי שנדרש כדי להשיג קיבולת מקסימלית להרכיב תמונה ורזולוציה. הרכיבים של המיקרוסקופ מתוארים בפירוט כאן.

Watch this Scientific Journal Video about Major Components of the Light Microscope at JoVE.com

Major Components of the Light Microscope

הרכיבים העיקריים של מיקרוסקופ אור

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Research

JoVE Journal

Biology

22.6K Views.  University of Texas Health Science Center at San Antonio (UTHSCSA). מיקרוסקופ אור הוא כלי בסיסי ביולוג של התא, שהיה צריך הבנה מעמיקה של איך זה עובד, איך זה צריך להיות מתואם עבור יישומים שונים, ואיך זה צריך להישמר כפי שנדרש כדי להשיג קיבולת מקסימלית להרכיב תמונה ורזולוציה. הרכיבים של המיקרוסקופ מתוארים בפירוט כאן.

Video: Major Components of the Light Microscope

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

ניצוח מספר מצבי הדמיה עם מיקרוסקופ זריחה אחת

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Bioengineering

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

מדריך לבניית מאוד נוטה נסחף מיקרוסקופ אריח עבור הדמיה מולקולה בודדת שדה-of-view מורחבת

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

הגדרת מיקרוסקופ גיליון אור פשוט עבור<em&gt; בטוטו</em&gt; הדמיה של<em&gt; ג elegans</em&gt; פיתוח

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

מיקרוסקופ פלואורסצנטי הניתן להתאמה אישית להמחשת רשתות שלד ציטו-שלד

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

מחבר זרקור: קידום ידע בחומרים רחוקים משיווי משקל באמצעות מיקרוסקופ יריעות אור

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

תכנון ובנייה של מיקרוסקופ פלואורסצנטי הניתן להתאמה אישית, חד-אובייקטיבי, עבור הדמיה של רשתות שלד ציטו-שלד

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...

Imaging Biological Samples with Optical and Confocal Microscopy

Source: <a href="https://www.jove.com/author/Peiman_Shahbeigi-Roodposhti">Peiman Shahbeigi-Roodposhti</a> and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal conditions. However, optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. While the resolution of optical microscopes does not reach that of electron microscopes, they are the most valuable tools in the imaging of biological macrostructures and are a staple in any biological lab.
In conventional light microscopes, the signal produced from the imaged object is from the full thickness of the specimen, which does not allow most of it to be in focus to the observer. This causes the image to have &#34;out of focus blur&#34;. The confocal microscope, on the other hand, illuminates the sample through a pin-hole, and is therefore able to filter out the out-of-focus light from above and below the point of focus in the object.
This demonstration provides an introduction to image acquisition using optical and confocal microscopy methods. Here, a sectioned piece of mouse brain will be studied.&#160; Image acquisition and analysis, including the tools to generate topographical maps and composite images, will be covered. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to provide more information on optical and confocal microscopes to determine if these microscopy modules are the best fit for a type of biological sample.

Biological Sample Imaging with Optical and Confocal Microscopy

הדמיה דגימות ביולוגיות עם מיקרוסקופיה אופטית וקונפוקל

Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
Optical ...

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Biomedical Engineering

Cell Structure

<h4>Background</h4>
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19th century, the Cell Theory was proposed to define a cell, which states:
<ul>
	<li>Every living organism is made up of one or more cells.</li>
	<li>The cells are the functional units of all organisms.</li>
	<li>All cells arise from preexisting cells.</li>
</ul>
All cells share common features such as having a plasma membrane, a cytoplasm, DNA, and ribosomes. A plasma membrane is a phospholipid bilayer that surrounds the cell. This thin and fluid layer around the cells serves to isolate the cell&#8217;s contents from its environment and regulates the material exchange with its environment, while also facilitating interactions with other cells. Inside the plasma membrane, the cell is filled with a gel-like fluid called cytoplasm that contains organic molecules, salts, and other materials that are vital for the cell&#8217;s functions. Therefore, biochemical reactions that support life, which are known as metabolic processes, take place inside the cytoplasm. The types of metabolic processes that a cell can execute depend on its genetic information. All cells use DNA as the genetic material, which is the hereditary blueprint to construct cellular structures and products. Finally, all cells use ribosomes to synthesize their protein products.
There are two types of cells based on their genetic material location: prokaryotic, which means &#8220;before nucleus&#8221;, and eukaryotic, which means &#8220;true nucleus&#8221;. Therefore, while both types of organisms have DNA, prokaryotes like bacteria have nucleoids, or &#8220;nucleus-like&#8221; components instead of a nucleus, whereas eukaryotes possess true, membrane-bound nuclei to contain their DNA. Moreover, prokaryotes are relatively small, around 0.1&#8211;5.0 micrometers (&#181;m), in comparison to eukaryotes that can typically range from 10-100 &#181;m in size. The small size of prokaryotes allows quick and effortless distribution of materials within the cell and execution of metabolic processes, as well as swift removal of waste or other products from the cell. Hence, eukaryotic cells possess specialized structures known as organelles, such as mitochondria or Golgi apparatus, to enable the execution of vital functions.
<h4>The Eukaryotic Cell</h4>
The eukaryotic cell is a shared derived trait of all eukaryotes, meaning that it had a single origin that has since been inherited by all eukaryotes. The earliest eukaryotic cells are seen in fossils about 2.4 billion years ago, and are recognizable because they are larger than prokaryotic cells1. The origin of this cell type resulted from an endosymbiotic event in which one amoeba-like cell engulfed a micrococcal bacteria and formed a stable coexistence2. The engulfed bacteria evolved into the first energy-producing organelles, mitochondria, which are the aerobic metabolism organelles in the cell. Mitochondria have their own separate genome and are similar in size to prokaryotes. They contain two layers of membranes that enclose two distinct compartments. Some of the reactions that break down high-energy biomolecules occur in the inner compartment, whereas the outer compartment houses the reactions that capture the energy released from these compounds into adenosine triphosphate (ATP) molecules to be used as the energy currency of the cell.
Nuclei and mitochondria are not the only shared structures of eukaryotic cells. Other ubiquitous eukaryotic organelles are the smooth and rough endoplasmic reticulum (ER), Golgi apparatus, lysosomes and vacuoles. Endoplasmic reticulum simply means &#8220;network inside the plasma&#8221; and as its name indicates it is a large network of membranes inside the cell, especially around the nucleus. Parts of the rough ER extend from the nuclear membrane and are distinguished from the smooth ER by their rough appearance due to numerous ribosomes on their surface. Rough ER is the site for protein synthesis, such as the proteins that are embedded in the plasma membrane or the proteins that are secreted from the cell. In contrast, smooth ER produces lipid-based products but also contains enzymes to detoxify harmful chemicals. Hence liver cells contain abundant smooth ER. Also, muscle cells contain significant amounts of smooth ER because of the calcium-storing function of this organelle, which is essential for muscle contraction. The Golgi apparatus sorts, modifies, and packages cellular products inside vesicles, which fuse with the plasma membrane to release the products. Some of the proteins that are produced in the rough ER are intracellular digestive enzymes. These enzymes are packed in the Golgi apparatus into special vesicles called lysosomes. The major function of the lysosomes is to digest the food particles engulfed by the cell as well as old cell parts. Vacuoles are sacs of cell membrane that serve as storage units within cells. They may serve to store water to regulate the cell&#8217;s water content as well as store metabolic products or even poisonous molecules, depending on the cell type and the organism.
<h4>Kingdom-specific Organelles</h4>
Eukaryotic cells also developed distinct organelles, specific for each kingdom. For instance, kingdom Plantae and Animalia are both eukaryotic, however the organelles of plant and animal cells differ in key ways that allow them to carry out their lives as producers and consumers, respectively. Terrestrial plants need to grow tall and have rigid stems to hold leaves, which they use for photosynthesis. They also need to be able to retain water taken up by roots. Their cells reflect these specific needs. Unlike animal cells, plant cells have chloroplasts, which are used for photosynthesis and often contain the green pigment chlorophyll. Additionally, they are surrounded by cell walls, which are rigid outer layers made of cellulose to support growth and water retention. Because they need to store large amounts of water to maintain the water pressure in the cell, they have larger vacuoles than animal cells. In addition, plant cells also have another type of specialized storage organelles called plastids, which contain pigments as well as photosynthetic products such as starch. These differences are noticeable and distinguish plant cells from animal cells: plant cells generally have a regular, rectangular shape due to their rigid cell walls, while animal cells are rounded and more irregular.
<h4>Microscopy</h4>
Some cells, such as frog oocytes, are large enough to be seen with the naked eye, yet most cells cannot be seen without any visual aid. Therefore, scientists use microscopy techniques to study cell structures and distinguish cell types from one another. While microscopes are able to magnify objects that are difficult or impossible to be see with the human eye, most tissues naturally lack pigmentation. Therefore, solutions have been created that can selectively stain cells based on their molecular composition. This allows researchers to distinguish between organelles in a cell, tissue types in a plant stem, and fat layers in animals, just to name a few examples. Methylene blue dye stains nucleic acids of dead cells, binding to negatively charged DNA. Safranin solution is another biological dye that stains cell nuclei red. Cells only need to be in the staining solutions for a short period of time, and can be mounted immediately following the staining step. The commonly used mounting techniques are wet mount and oil immersion. A wet mount is created by collecting a sample and putting it on a glass slide with liquid between the slide and the coverslip. Cell samples are suspended in liquids like water or glycerol. Glycerol is better to use with live cultures, because it prevents the proliferation of bacteria3. Immersion oil can be added on top of the coverslip to improve viewing of the sample at high magnification. This is accomplished because the oil has the same refractive index as glass, which means that it allows light to pass through it as well as glass would. The glass-air interface scatters light more than oil or glass, so the clarity of the image is affected when samples are mounted &#8220;dry&#8221;, or without oil. Once the cells are stained and mounted, they are ready to be studied under the microscope.
There are various microscopy techniques from electron scanning technology that has allowed researchers to view objects on an atomic level to fluorescent live cell imaging that allows real-time monitoring of the movement of molecules within individual cells4. Bright-field microscopy is the simplest microscopy technique, requiring only a halogen source light, a condenser lens to focus the light, an ocular lens to view the image, and an objective lens to magnify the image. With any microscopy technique, it is important to understand the parts of a microscope before using one. In general, compound microscopes used for bright-field imaging have an eyepiece at the top of the scope, which is attached to the head and objectives. The eyepiece has a magnification of 10X, and objective lenses are set to a particular magnification in a range of 4X-100X. There are between three and five objectives on a standard microscope. The objectives point down to the stage, which is where a specimen is placed for viewing. The stage often has mechanical parts and stage clips to hold a slide and move it around while viewing. An aperture is a hole in the stage for light to pass through. This light is controlled by an adjustable condenser lens above an illuminator, or light source. To control the zoom of the stage for the object being viewed, microscopes feature coarse and fine focus adjustment knobs. The coarse focus knob moves on a larger scale than the fine focus, but they are on the same axis. The fine focus is useful when the object on the stage has been brought close to the objectives. It is important not to let the objective lens touch the object on the stage, as it can scratch the lens. Objects should always be first viewed on the lowest magnification objective and clearly focused before switching to higher magnification objectives.
Microscopy is an important tool for many aspects of the medical field including research, diagnosis, and treatment. This has the application of using nanotechnology in medicine, as a new treatment method in place of a more invasive surgery5. Surgeons also make use of microscopes, some of which have been modified to be mounted on a surgeon&#8217;s head and are operated with foot pedals. These are of much lower magnification than even the light microscopes used today, but they facilitate the safe execution of delicate procedures, such as optical and neurosurgery.
<h4>References</h4>
<ol class="references">
	<li>Bengtson S, Rasmussen B, Ivarsson M, Muhling J, Broman C, Marone F, Stampanoni M, Bekker A. Fungus-like mycelial fossils in 2.4-billion-year-old vesicular basalt. Nature Ecology &#38; Evolution. 2017, Vol. 1, Article number: 0141.</li>
	<li>Vellai T, Vida G. The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. Proc. R. Soc. Lond. B. 1999, Vol. 266, 1571-1577.</li>
	<li>Gouet V, Roger G, Fonty C, Andre P. Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi. Current Microbiology. 24, 1992, Vol. 4, 197-201.</li>
	<li>Cognet L, Leduc C, Lounis B. Advances in live-cell single-particle tracking and dynamic super-resolution imaging. Curr Opin Chem Biol. 2014, Jun; 20:78-85.</li>
	<li>Asiyanbola B, Soboyejo W. For the surgeon: an introduction to nanotechnology. J Surg Educ. 2008, Vol. 65, 2 (155-61).</li>
</ol>

Cell Structure: Visualizing Onion and Human Cells - Concept

מבנה התא

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

JoVE Lab Manual

Fundamentals

Introduction to Fluorescence Microscopy

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. Fluorescence is a phenomenon that involves absorbance and emission of a small range of light wavelengths by a fluorescent molecule known as a fluorophore. Fluorescence microscopy is accomplished in conjunction with the basic light microscope by the addition of a powerful light source, specialized filters, and a means of fluorescently labeling a sample. This video describes the basic principles behind fluorescence microscopy including the mechanism of fluorescence, the Stoke&#x2019;s shift, and photobleaching. It also gives examples of the numerous ways to fluorescently label a sample including the use of fluorescently tagged antibodies and proteins, nucleic acid fluorescent dyes with, and the addition of naturally fluorescent proteins to a specimen. The major components of the fluorescence microscope including a xenon or mercury light source, light filters, the dichroic mirror, and use of the shutter to illuminate the sample are all described. Finally, examples of some of the many applications for fluorescence microscopy are shown.

Fluorescence Microscopy: Concept, Instrumentation, and Applications

מבוא למיקרוסקופיה פלואורסצנטית

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. ...

Basic Biology

General Laboratory Techniques

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 size. In its simplest form, it is composed of a clear lens that magnifies the sample and a light source to illuminate it. However, most light microscopes are much more complex and house numerous fine-tuned lenses with tightly controlled dimensions all within the body of the microscope itself and in components such as the objectives and eyepieces. In this video, the major components of the light microscope are described and their uses and functions are explained in detail. The basic principles of magnification, focus, and resolution are also introduced. Basic light microscope operation begins with bringing light to the sample and ensuring that the light source is of the correct intensity, directionality, and shape in order to produce the best quality image. Next, the sample must be magnified properly and brought into focus to view the region of interest. There are many practical applications for light microscopy including the viewing of stained or unstained cells and tissues, resolving small details of specimens, and even magnifying a region of interest during surgery to assist with complex procedures on the micron scale.

Light Microscopy: Principle, Instrumentation, and Applications

מבוא למיקרוסקופיה קלה

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 ...

הרכיבים העיקריים של מיקרוסקופ אור

Summary

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Chapters in this video

ניצוח מספר מצבי הדמיה עם מיקרוסקופ זריחה אחת

מדריך לבניית מאוד נוטה נסחף מיקרוסקופ אריח עבור הדמיה מולקולה בודדת שדה-of-view מורחבת

הגדרת מיקרוסקופ גיליון אור פשוט עבור

תכנון ובנייה של מיקרוסקופ פלואורסצנטי הניתן להתאמה אישית, חד-אובייקטיבי, עבור הדמיה של רשתות שלד ציטו-שלד

תכנון ובנייה של מיקרוסקופ פלואורסצנטי הניתן להתאמה אישית, חד-אובייקטיבי, עבור הדמיה של רשתות שלד ציטו-שלד

תכנון ובנייה של מיקרוסקופ פלואורסצנטי הניתן להתאמה אישית, חד-אובייקטיבי, עבור הדמיה של רשתות שלד ציטו-שלד

הדמיה דגימות ביולוגיות עם מיקרוסקופיה אופטית וקונפוקל

מבנה התא

מבוא למיקרוסקופיה פלואורסצנטית

מבוא למיקרוסקופיה קלה