Microscopy

Introduction to Microscope

Microscopy is the science of using microscopes to observe objects that are too small to be seen with the naked eye. It plays a vital role in a wide array of fields including biology, medicine, materials science, and chemistry.

The Microscope is an optical instrument consisting of a combination of lenses for making enlarged or magnified images of minute objects. The term is derived from the two Greek words ‘micro’ - small, and ‘scope’ - view.  Since microorganisms are very small and invisible to naked eyes, they must be magnified to be clearly seen. The use of a microscope is absolutely indispensable to study microbiology.

Working of lenses

When a ray of light passes from one medium or material to another, refraction occurs, the light ray will bend at the interface.  Refractive index is the measure of how a medium or material slows the velocity of light.  Glass has higher refractive index and air have lower refractive index The direction and magnitude of bending is determined by the refractive indexes of the two media or materials forming the interface. When light passes from air into glass, it is slowed and bent toward the normal, a line perpendicular to the surface and as light leaves glass and returns to air, it accelerates and is bent away from the normal.  As shown in the below image, a prism bends light because of the different refractive indices of glass and air.  

The Bending of Light by a Prism 

(Prescott−Harley−Klein:Microbiology, Fifth Edition)

Lens act like a collection of prisms operating as a unit. When parallel rays of light strike the lens, a convex lens will focus these rays at a specific point termed as the focal point (F). The distance between the centre of the lens and the focal point is called the focal length (f).  Lens strength is related to focal length; a lens with a short focal length will magnify an object more than a weaker lens having a longer focal length.

Lens functions like a collection of prisms 

(Prescott−Harley−Klein:Microbiology, Fifth Edition)

Magnification and resolving power

Magnification and resolving power are important concepts in microscopy and imaging.  Magnification is the process of making an object appear larger than its actual size. Magnification is expressed as the ratio of the object's apparent size to its actual size.  Resolving power or Resolution is the ability to distinguish two objects that are close together. While higher magnification can increase the amount of detail in an image, it doesn't necessarily improve the ability to distinguish fine details. For example, two images may have the same magnification, but one may have a higher resolution and be clearer.

The minimum distance (d) between two objects that reveals them as separate entities is given by the Abbe equation, in which lambda (λ) is the wavelength of light used to illuminate the specimen and n sin θ is the numerical aperture (NA).

With higher resolution, two objects with smaller d could be differentiated as two separate entities.  As d becomes smaller, the resolution increases, and finer detail can be distinguished in the specimen.  Higher resolution is obtained with shortest wavelength light, with light at the blue end (450 to 500 nm) of the visible spectrum.  The wavelength must be shorter than the distance between two objects or they could not be seen clearly.

Numerical aperture

The measure for the resolving powers of a lens is the numerical aperture.  It is n sin θ.  The larger the numerical aperture, then the greater is the resolving power of the lens. Theta is defined as 1/2 the angle of the cone of light entering an objective. Light that strikes the microorganism after passing through a condenser is cone- shaped. When this cone has a narrow angle and tapers to a sharp point, it does not adequately separate images of closely packed objects and thus the resolution will be low. If the cone of light has a very wide angle and spreads out after passing through a specimen, closely packed objects appear widely separated and thus the resolution will be high. 

Numerical Aperture - The angular aperture θ is 1/2 the angle of the cone of light that enters a lens from a specimen, and the numerical aperture is n sin θ. Lens having larger angular and numerical apertures will have higher resolution and its working distance will be smaller (Prescott−Harley−Klein:Microbiology, Fifth Edition)

The angle of the cone of light that enter a lens depends on the refractive index (n) of the medium in which the lens works. The refractive index for air is 1.00. Since sin θ cannot be greater than 1 (the maximum θ is 90° and sin 90° is 1.00), a lens working in air cannot have a numerical aperture greater than 1.00. In order to raise the numerical aperture above 1.00, to achieve higher resolution, the refractive index is to be increased. This could be achieved by using immersion oil, a colorless liquid having the same refractive index as glass (about 1.515). If air is replaced with immersion oil, by adding a drop of oil on the surface of glass slide containing the specimen, light rays that otherwise may not enter the objective due to reflection and refraction will now enter the lens.   So, an increase in numerical aperture and thus increase in resolution results.

Oil immersion objective operating in air and with immersion oil (Prescott−Harley−Klein:Microbiology, Fifth Edition)

Types of Microscopes

A simple microscope consists merely of a single lens or magnifying glass held in a frame, usually adjustable, and will have a stand for holding the object to be viewed and a mirror for reflecting light. A compound microscope consists of two sets of lenses, one known as an objective and the other as an eyepiece.  Compound microscopes give more magnification than simple microscope. 

There are different types of Microscopes. The major being a) Light Microscopes (Optical Microscopes) and b) Electron Microscopes

A. Light Microscope (Optical Microscope):

Light microscopes use visible light and lenses to magnify objects.  Magnification typically ranges from 40x to 1000x.

Compound microscope and its parts (Fundamental Principles of Bacteriology : A.J. Salle)

Parts of a Light Microscope:

Eyepiece (ocular lens): The lens you look through, typically 10x magnification.

Objective Lenses: These are multiple lenses that provide different magnifications (usually 4x, 10x, 40x, 100x).

Stage: The flat surface where the slide with the specimen is placed.

Condenser: Focuses the light onto the specimen.

Diaphragm: Controls the amount of light that passes through the sample.

Coarse and Fine Focus Knobs: Adjust the focus of the image.

Illuminator: A light source, usually a lamp, that illuminates the sample.

Types of Light Microscopy:

Bright-Field Microscopy: The most commonly used technique where light passes through the specimen, and the image appears dark against a bright background.

Dark-Field Microscopy: Light is directed at an angle to the sample, which makes the image appear bright against a dark background.

Phase-Contrast Microscopy: Enhances contrasts in transparent specimens and is useful for studying live cells.

Fluorescence Microscopy: Uses ultraviolet (UV) light to excite fluorophores attached to specimen. When the fluorophores emit light, the specimen appears as brightly coloured on a dark background.

B) Electron Microscopes (EM)

Electron microscopes use electrons instead of light to view specimens at much higher magnifications and resolutions than light microscopes.

Transmission Electron Microscope (TEM):

TEM transmits a beam of electrons through a thin sample. The electrons interact with the sample, and the resulting image is projected onto a screen or film and can achieve magnifications up to 10 million times and capable of Resolution at nanometer scale (less than 1 nm).

Scanning Electron Microscope (SEM):

SEM scans a focused electron beam over the surface of the sample. The electrons interact with the surface and produce signals that are used to form an image. Can achieve magnifications up to 1 million times and resolution of about 1-10 nanometers.

C. Scanning Probe Microscopes (SPM)

Scanning Probe Microscopes work by scanning a sharp probe across the surface of the specimen. The most common type is the Atomic Force Microscope (AFM).  This measures the force between the probe and the surface and help for the construction of a high-resolution topographical image.

D. Other Advanced Microscopy Techniques

Confocal Microscopy uses laser light to scan specimens and produce high-resolution 3D images. Confocal microscopes capture optical sections of the sample, which can then be digitally reconstructed to create a 3D image.

Multiphoton Microscopy is a type of fluorescence microscopy that uses multiple photons to excite fluorescent dyes.

Live-Cell Microscopy is used to observe living cells in real-time, often with the help of fluorescent dyes or proteins.

Super-Resolution Microscopy includes techniques like STED (Stimulated Emission Depletion) and PALM (Photo-Activated Localization Microscopy).  These provide resolutions of less than 100 nm.

The Bright-Field Microscope

The ordinary microscope is called a bright-field microscope because it forms a dark image against a brighter background.

The microscope consists of a sturdy metal body or stand composed of a base and an arm to which the remaining parts are attached.

A light source, either a mirror or an electric illuminator, is located in the base.

Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image.

The stage is positioned about halfway up the arm and holds microscope slides by either simple slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide around smoothly during viewing by use of stage control knobs.

The substage condenser is mounted within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in simpler microscopes but can be adjusted vertically in advanced models.

The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes and are called binocular microscopes.

The nosepiece holds three to five objectives with lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly. Ideally a microscope should be parfocal—that is, the image should remain in focus when objectives are changed. 

The objective lens forms an enlarged real image within the microscope, and the eyepiece lens further magnifies this primary image. The total magnification is calculated by multiplying the objective and eyepiece magnifications together. For example, if a 45X objective is used with a 10X eyepiece, the overall magnification of the specimen will be 450X.

The resolution of a microscope depends upon the numerical aperture of its condenser as well as that of the objective. 

The resolution of a light microscope can be calculated using the Abbe equation. The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical aperture of 1.25) and blue-green light is approximately 0.2 µm.

So, a bright-field microscope can distinguish between two dots which are 0.2 µm apart, the size of a very small bacterium.