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

I microscopi ottici utilizzano sorgenti luminose per illuminare i campioni. La luce passa attraverso una serie di lenti; Il condensatore focalizza la luce sul campione, mentre l'obiettivo e le lenti oculari creano un'immagine bidimensionale ingrandita e invertita.
L'apertura numerica di un obiettivo determina la sua capacità di raccogliere la luce. La quantità di luce raccolta dipende dall'angolo del cono di luce e dall'indice di rifrazione del mezzo attraverso il quale la luce passa, ad esempio aria, acqua o olio.
I microscopi ottici hanno un limite di risoluzione di 0,2 micrometri. Possono risolvere cellule procariotiche o grandi organelli all'interno delle cellule eucariotiche, che hanno un diametro di circa zero virgola cinque micrometri. 
La risoluzione della lente dell'obiettivo è inversamente proporzionale alla lunghezza d'onda della luce e all'apertura numerica della lente; più piccolo è il valore, maggiore è la risoluzione dell'immagine ingrandita.
Un microscopio ottico può produrre un'immagine con un ingrandimento massimo di 1000 volte, calcolato moltiplicando gli ingrandimenti dell'obiettivo e delle lenti oculari.

imaging di campioni biologici con microscopia ottica

Imaging Biological Samples with Optical Microscopy

La microscopia ottica utilizza principi ottici per fornire immagini dettagliate dei campioni. Antonie van Leeuwenhoek progettò il primo microscopio ottico composto nel XVII secolo per visualizzare cellule del sangue, batteri e cellule di lievito. Nel 1830, Joseph Jackson Lister creò un microscopio ottico essenzialmente moderno. Il 20° secolo ha visto lo sviluppo di microscopi con ingrandimento e risoluzione migliorati.

Nella microscopia ottica, il campione da osservare viene posizionato su un vetrino e fissato sul tavolino (una piattaforma). Una volta fissato il vetrino, il campione sul vetrino viene posizionato sopra la luce utilizzando le manopole meccaniche xy. Queste manopole muovono la slitta sulla superficie del palco ma non alzano o abbassano il palco. Una volta centrato il campione rispetto alla luce, la posizione del tavolino può essere alzata o abbassata per mettere a fuoco l'immagine. La manopola di messa a fuoco grossolana viene utilizzata per movimenti su larga scala con obiettivi da 4⨯ e 10⨯; la manopola di messa a fuoco fine viene utilizzata per movimenti su piccola scala, soprattutto con obiettivi da 40⨯ o 100⨯.

Quando le immagini vengono ingrandite, diventano più scure perché c'è meno luce per unità di area dell'immagine. I microscopi producono immagini altamente ingrandite, pertanto richiedono un'illuminazione intensa. In un microscopio a campo chiaro, un illuminatore fornisce questa luce, in genere una lampadina ad alta intensità sotto il tavolino. La luce proveniente dall'illuminatore passa attraverso la lente del condensatore (situata sotto il tavolino), che concentra tutti i raggi luminosi sul campione per massimizzare l'illuminazione. La posizione del condensatore può essere ottimizzata utilizzando la manopola di messa a fuoco del condensatore allegata; una volta stabilita la distanza ottimale, il condensatore non deve essere spostato per regolare la luminosità. Se sono necessari livelli di luce inferiori al massimo, la quantità di luce che colpisce il campione può essere facilmente regolata aprendo o chiudendo un diaframma tra il condensatore e il campione. In alcuni casi, la luminosità può essere regolata anche utilizzando il reostato, un dimmer che controlla l'intensità dell'illuminatore.

Un microscopio a campo chiaro crea un'immagine dirigendo la luce dell'illuminatore sul campione; questa luce viene trasmessa, assorbita, riflessa o rifratta in modo differenziale da diverse strutture. La lente dell'obiettivo forma un'immagine reale invertita del campione. L'immagine finale visualizzata nell'oculare o nell'oculare è un'immagine virtuale ingrandita di quella formata dall'obiettivo.

Colori diversi possono comportarsi diversamente poiché interagiscono con i cromofori (pigmenti che assorbono e riflettono particolari lunghezze d'onda della luce) in parti del campione. Spesso i cromofori vengono aggiunti artificialmente al campione utilizzando coloranti, aumentando il contrasto e la risoluzione. In generale, le strutture del campione appariranno più scure, in varia misura, rispetto allo sfondo luminoso, creando immagini estremamente nitide con ingrandimenti fino a circa 1000⨯. Un ulteriore ingrandimento creerebbe un'immagine più grande ma senza una maggiore risoluzione. Ciò ci consente di vedere oggetti piccoli come batteri, visibili a circa 400° circa, ma non oggetti più piccoli come i virus.

A ingrandimenti molto elevati, la risoluzione può essere compromessa quando la luce passa attraverso la piccola quantità di aria tra il campione e la lente. Ciò è dovuto alla differenza significativa tra gli indici di rifrazione dell'aria e del vetro; l'aria disperde i raggi luminosi prima che l'obiettivo possa focalizzarli. Per risolvere questo problema, è possibile utilizzare una goccia d'olio per riempire lo spazio tra il campione e una lente ad immersione in olio, una lente speciale progettata per essere utilizzata con olio da immersione. Poiché l'olio ha un indice di rifrazione molto simile a quello del vetro, aumenta l'angolo massimo al quale la luce che esce dal campione può colpire la lente. Ciò aumenta la luce raccolta e, quindi, la risoluzione dell'immagine. È possibile utilizzare una varietà di oli per diversi tipi di luce.

Questo testo è adattato da Openstax, Microbiology 2e, Sezione 2.3: Strumenti di microscopia.

Imaging di campioni biologici con microscopia ottica

La microscopia ottica utilizza principi ottici per fornire immagini dettagliate dei campioni. Antonie van Leeuwenhoek ha progettato il primo microscopio ottico composto ...

imaging-biological-samples-with-optical-microscopy

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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

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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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How Light Microscopes Bring Samples Into View | JoVE K12

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How Light Microscopes Bring Samples Into View

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