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

Mikroskopy optyczne wykorzystują źródła światła do oświetlania próbek. Światło przechodzi przez szereg soczewek; Kondensor skupia światło na próbce, podczas gdy soczewki obiektywu i okularu tworzą powiększony i odwrócony dwuwymiarowy obraz.
Apertura numeryczna obiektywu określa jego zdolność do zbierania światła. Ilość zebranego światła zależy od kąta nachylenia stożka świetlnego i współczynnika załamania światła, przez który przechodzi światło, na przykład powietrza, wody lub oleju.
Mikroskopy optyczne mają limit rozdzielczości 0,2 mikrometra. Mogą rozpuszczać komórki prokariotyczne lub duże organelle w komórkach eukariotycznych, które mają około punktu zerowego o średnicy pięciu mikrometrów. 
Rozdzielczość soczewki obiektywu jest odwrotnie proporcjonalna do długości fali światła i apertury numerycznej soczewki; im mniejsza wartość, tym wyższa rozdzielczość powiększonego obrazu.
Mikroskop optyczny może uzyskać obraz o maksymalnym powiększeniu 1000 razy, obliczonym poprzez pomnożenie powiększeń soczewek obiektywu i soczewki okularowej.

obrazowanie próbek biologicznych za pomocą mikroskopii optycznej

Imaging Biological Samples with Optical Microscopy

Mikroskopia optyczna wykorzystuje zasady optyczne, aby zapewnić szczegółowe obrazy próbek. Antonie van Leeuwenhoek zaprojektował w XVII wieku pierwszy złożony mikroskop optyczny do wizualizacji komórek krwi, bakterii i komórek drożdży. W 1830 roku Joseph Jackson Lister stworzył zasadniczo nowoczesny mikroskop świetlny. W XX wieku rozwinęły się mikroskopy o większym powiększeniu i rozdzielczości. W mikroskopii optycznej badany preparat umieszcza się na szkiełku i przypina do stolika (platformy). Po zamocowaniu szkiełka preparat na szkiełku umieszcza się nad światłem za pomocą mechanicznych pokręteł stolika x-y. Pokrętła te przesuwają suwak po powierzchni sceny, ale nie podnoszą ani nie obniżają sceny. Gdy preparat zostanie wyśrodkowany nad światłem, stolik można podnieść lub obniżyć, aby uzyskać ostrość obrazu. Pokrętło zgrubnej regulacji ostrości służy do ruchów na dużą skalę z obiektywami 4⨯ i 10⨯; pokrętło dokładnego ustawiania ostrości służy do ruchów o małej skali, szczególnie w przypadku obiektywów 40⨯ lub 100⨯. Powiększone obrazy stają się ciemniejsze, ponieważ na jednostkę powierzchni obrazu przypada mniej światła. Mikroskopy dają bardzo powiększone obrazy, dlatego wymagają intensywnego oświetlenia. W mikroskopie z jasnym polem światło to zapewnia oświetlacz, zwykle żarówka o dużej intensywności umieszczona pod sceną. Światło z oświetlacza przechodzi przez soczewkę kondensora (umieszczoną pod stolikiem), która skupia wszystkie promienie świetlne na preparacie, maksymalizując oświetlenie. Położenie kondensora można zoptymalizować za pomocą dołączonego pokrętła ostrości kondensora; po ustaleniu optymalnej odległości nie należy przesuwać kondensora w celu regulacji jasności. Jeśli potrzebny jest poziom światła niższy niż maksymalny, ilość światła padającego na próbkę można łatwo regulować, otwierając lub zamykając przesłonę pomiędzy kondensorem a próbką. W niektórych przypadkach jasność można również regulować za pomocą reostatu, czyli ściemniacza sterującego intensywnością iluminatora. Mikroskop jasnego pola tworzy obraz, kierując światło z oświetlacza na próbkę; światło to jest w różny sposób przepuszczane, pochłaniane, odbijane lub załamywane przez różne struktury. Soczewka obiektywu tworzy rzeczywisty odwrócony obraz preparatu. Ostateczny obraz wizualizowany w okularze lub okularze jest powiększonym wirtualnym obrazem obrazu utworzonego przez obiektyw. Różne kolory mogą zachowywać się inaczej, gdy wchodzą w interakcję z chromoforami (pigmentami pochłaniającymi i odbijającymi określone długości fal światła) w częściach próbki. Często chromofory są sztucznie dodawane do próbki za pomocą barwników, zwiększając kontrast i rozdzielczość. Ogólnie rzecz biorąc, struktury preparatu będą w różnym stopniu ciemniejsze niż jasne tło, tworząc maksymalnie ostre obrazy przy powiększeniach do około 1000⨯. Dalsze powiększenie spowodowałoby utworzenie większego obrazu, ale bez zwiększonej rozdzielczości. To pozwala nam widzieć obiekty tak małe jak bakterie, widoczne w odległości około 400⨯, ale nie mniejsze obiekty, takie jak wirusy. Przy bardzo dużych powiększeniach rozdzielczość może zostać pogorszona, gdy światło przechodzi przez niewielką ilość powietrza pomiędzy preparatem a soczewką. Wynika to ze znacznej różnicy między współczynnikami załamania światła powietrza i szkła; powietrze rozprasza promienie świetlne, zanim soczewka będzie w stanie je skupić. Aby rozwiązać ten problem, można użyć kropli oleju do wypełnienia przestrzeni pomiędzy próbką a soczewką immersyjną, specjalną soczewką przeznaczoną do stosowania z olejkami immersyjnymi. Ponieważ olej ma współczynnik załamania światła bardzo podobny do szkła, zwiększa on maksymalny kąt, pod jakim światło opuszczające próbkę może padać na soczewkę. Zwiększa to ilość zbieranego światła, a co za tym idzie, rozdzielczość obrazu. Do różnych rodzajów światła można stosować różne olejki. Ten tekst jest adaptacją z Openstax, Mikrobiologia 2e, Sekcja 2.3: Instrumenty mikroskopowe.

Obrazowanie próbek biologicznych za pomocą mikroskopii optycznej

Mikroskopia optyczna wykorzystuje zasady optyki do dostarczania szczegółowych obrazów próbek. Antonie van Leeuwenhoek zaprojektował pierwszy złożony mikroskop optyczny w ...

imaging-biological-samples-with-optical-microscopy

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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...

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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...

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Electron Microscopy Sample Prep Methods

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3D Imaging with Electron Microscopy

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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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