Home » What Is Light Microscope and Its Types with Examples

What Is Light Microscope and Its Types with Examples

What is microscopy? Microscopy is a technique for making very small things visible to the unaided eye. An instrument used to make the small things visible to the naked (unaided) eye is called a microscope. There are two fundamentally different types of microscopes: the light microscope and the electron microscope.

History of a light microscope

Microscopy has been around since the early 1600s, when a Dutch scientist named Antonie van Leeuwenhoek used a simple microscope to look at tiny organisms like bacteria and protozoa. The first microscopes were just simple magnifying glasses with only one convex lens. These early microscopes didn’t have much magnification power, so they could only make things look about 30 times bigger than they really were.

Compound microscopes, which had more than one lens to make the image bigger, were made in the 19th century. To magnify the image of the specimen, these microscopes used both objective lenses (which were close to the specimen) and eyepieces (which were close to the viewer).

Microscopy changed a lot in the 20th century when electronic microscopes like the transmission electron microscope (TEM) and the scanning electron microscope (SEM) were made. Instead of using light to make pictures of specimens, these microscopes use a beam of electrons. This makes it possible to get better resolution and magnification than with optical microscopes.

What is a light microscope?

A light microscope is a kind of microscope that uses lenses and visible light to magnify and take pictures of small things. Optical microscopes and optical light microscopes are other names for light microscopes. They are the most common type of microscope and are used in many fields, such as biology, medicine, and materials science.

Light microscopes work by focusing light on the specimen and then using one or more lenses to magnify the image of the specimen. There are two main parts to a light microscope: the objective lens, which is near the sample, and the eyepiece, which is near the viewer. The objective lens gathers light from the object being looked at and forms an image. The eyepiece then makes the image bigger.

Light microscopes are grouped by the type and number of lenses they use, as well as by how much they can magnify and how clear their images are. Simple microscopes, compound microscopes, and stereo microscopes are the most common types of light microscopes. Simple microscopes have only one convex lens that magnifies the image. Compound microscopes, on the other hand, have more than one lens that magnifies and sharpens the image. Stereo microscopes, which are also called “dissecting microscopes,” use two separate lenses to make a 3D image of the object they are looking at.

Principle of a light microscope

The simplest form of light microscope consists of a single lens, a magnifying glass. Microscope made up of more than one glass lens in combination is termed compound microscope. Compound microscope includes condenser lens, the objective lens and the eyepiece lens. Condenser lens focuses the light from the light source at the specimen. 

The one facing the object is called the objective and the one close to the eye is called the eyepiece. The objective has a smaller aperture and smaller focal length than those of the eyepiece (also referred to as the ocular). The objective lens is responsible for producing the magnified image. It is available in different varieties (4x, 10x, 20x, 40x, 60x, 100x). 

The power of a lens is described with a number followed by the letter ‘x’. For example, if through a microscope one can see something 25 times larger than actual size, its magnification power is 25x. The eyepiece works in combination with the objective lens to further magnify the image. A compound microscope with a single eyepiece is said to be monocular and one with two eyepieces is said to be binocular. 

Eyepieces usually magnify by 10x, since an eyepiece of higher magnification merely enlarges the image, with no improvement in resolution. Both living and dead specimens are viewed with a light microscope. The visibility of the magnified specimen depends on contrast and resolution.

The objective lens forms a real and inverted magnified Image of the specimen or object

Figure 1: The objective lens forms a real and inverted magnified Image of the specimen or object (called the real intermediate image) in the focal plane of eyepiece. This image as an object for the eyepiece. The final image is, then, formed at infinity. It is erect with respect to tke first image and hence, inverted with respect to the object. 

How does light microscope work

The magnification or linear magnification of a microscope is defined as the ratio of the image size to the object (specimen) size. If the image and object are in the same medium, then it is just the image distance divided by the object distance. There is a difference in the meaning of the two terms, magnification and magnifying power. Magnifying power or angular magnification is the ratio of the angle subtended by object and image.

The magnification of a compound microscope is the product of the magnification of the objec-tive and the eyepiece. The magnification of the objective Is called the linear magnification, because it is measured In linear dimensions. The magnification of the eyepiece Is called the angular magnification. The overall magnification Is the product of the linear magnification of the objective lens and the angular magnification of the eyepiece with the first Image at the focal length.

Resolving power is the ability of magnifying instrument to distinguish two objects that are close together. The resolving power is inversely related to the limit of resolution. The limit of resolution is defined as the minimum distance between two points that allows for their dis-crimination as two separate points. Thus, the higher the resolving power, the smaller the limit of resolution. 

The limit of resolution of the light microscope depends upon the three factors: the wavelength (λ) of the light used to illuminate the specimen, the angular aperture (α) and the refractive index (n) of the medium surrounding the specimen. The effect of these three variables on the limit of resolution is described quantitatively by the following equation known as the Abbe equation:

Limit of resolution = 0.611 λ / n x sinα

The quantity n x sin α is called the numerical aperture of the objective lens, abbreviated NA. The NA is a measure of the ability of a lens to collect light from the specimen. Lenses with a low NA collect less light than those with a high NA.

Limit of resolution = 0.611 λ / NA

For small value of limit of resolution, the numerator of the equation should be as small as possible and the denominator should be as large as possible. Equation shows that resolution can be improved by shortening the wavelength of the illuminating light, increasing the index of refraction on the objective lens, and increasing sin α. The angle a can be increased either by shortening the distance between the lens and the object or by increasing the diameter of the lens.

The angular aperture of a lens

Figure 2: The angular aperture of a lens. The angular aperture Is the half-angle a of the cone of light entering the objective lens of the microscope from the specimen. It Is, therefore, a measure of how much of the illumination that leaves the specimen actually passes through the lens. The larger the angular aperture, the more information the lens can transmit.

For minimum value of numerator, the wavelength should be small. Thus, for the best resolution specimen is illuminated with blue light of 450 nm. The angular aperture for the best objective lenses is about 70°. Hence, the maximum value for sin α is about 0.94.

The refractive index of air is about 1.0, so for a lens designed for use in air, the maximum numerical aperture is about 0.94. In this situation, the limit of resolution for a glass lens In air Is roughly 300 nm To increase the numerical aperture some microscope lenses are designed to be used with a’ layer of immersion oil between the lens and the specimen.

Immersion oil has a higher refractive index than air therefore, allows the lens to receive more of the light transmitted through the specimen. Since the refractive index of Immersion oil is about 1.5, the maximum numerical aperture for an oil immersion lens is about 1.5 x 0.94 = 1.4.

Thus, the limit of resolution for a microscope that uses visible light Is roughly 300 nm in air and 200 nm with an oil Immersion lens. The limit of resolution of the unaided human eye is 100 um.

Types of a Light Microscope

There are many different kinds of light microscopes. They are grouped by the number and type of lenses they use, as well as by how much they can magnify and how clear their images are. These are the most common kinds of light microscopes:

1. Bright field Light Microscope

2. Dark-Field Light Microscope

3. Phase Contrast Light Microscope

4. Fluorescence Light Microscope

5. Confocal Light Microscope

1. Bright field Light Microscope

Definition

The specimen stands out as a dark spot against the bright background of a bright field microscope. The bright-field microscope, also known as a compound microscope or light microscope, is a common instrument in scientific research facilities.

Under a bright field microscope, samples can be studied in various states, including staining, fixation, and live observation. The components of a bright-field microscope are the eyepiece, objective lens, condenser lens, stage, and light source, all of which are used to gather electromagnetic radiation in the visible range.

Principle

An observational specimen is placed on the stage of a brightfield microscope. From the source, light will pass through the specimen and into the objective lens, where a magnified image of the specimen will be formed. Following this, the light will pass through an oracular lens or eyepiece before finally entering the user’s eyes. The viewer’s sees a black object on a white background. Because transmitted or unscattered light rays are blocked from entering the objective lens of a bright-field microscope, the resulting image appears dark when compared to the background brightfield.

compound microscope drawing with label

Figure 3: Parts of Bright Field Microscope (compound microscope)

Major Parts

The following are parts of the light microscope (bright field microscope) (refer back to my earlier parts of microscope and its function and labelled diagram notes, where we have already discussed each component and its function in detail): Ocular Lens (Eye Piece), Diopter Adjustment, Head, Nose Piece, Objective Lens, Arm (Carrying Handle), Mechanical Stage, Stage Clip, Aperture, Diaphragm, Condenser, Coarse Adjustment, Fine Adjustment, Illuminator (Light Source), Stage Controls, Base, Brightness Adjustment, and Light Switch.

Magnification

The magnification power of a bright field microscope can be calculated by ;

Total Magnification power = Magnification of the objective lens x Magnification of the eyepiece

Ex. If the objective lens has a magnification power of 40x and the eyepiece has a magnification power of 10x, the total magnification power of this bright-field microscope is 40×10 = 400x.

Application

This microscope is used a lot in microbiology. It is used to look at both fixed and living specimens that have been stained with simple dyes. This gives contrast so that things are easy to see under a microscope. Because of this, it can be used to find simple bacteria cells and parasitic protozoans.

2. Dark-Field Light Microscope

Definition

A dark-field microscope is a type of microscope in which objects are illuminated at a very low angle from the side so that the background appears dark and the objects show up against this dark background. It is a technique for improving the contrast of unstained, transparent specimens.

Principle

Darkfield illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted light and collecting only the light scattered by the sample. To view a specimen in a dark field, an opaque disc is placed underneath the condenser lens that blocks light from entering the objective lens directly; light reflected by specimen enters the objective, and the specimen appears light against a black background.

Path of light in Darkfield Microscope

Figure 4: Path of light in Darkfield Microscope (Source: en.wikipedia.org)

Applications

It’s useful for seeing the workings of eukaryotic cells and other larger types of cells. Recognizing bacteria having characteristic cell morphologies.

3. Phase-Contrast microscopy

When light passes through a living cell, the phase of the light wave is changed according the cell’s refractive index: light passing through a relatively thick or dense part of the cell, such as the nucleus, is retarded; its phase, consequently, is shifted relative to light that has passed through an adjacent thinner region of the cytoplasm. The phase-contrast microscope (invented by Frits Zernike) exploits the interference effects produced when these two sets of waves recombine, thereby creating an image of the cell’s structure. The specimen appears as different degrees of brightness and contrast. It is used for the study of live and unstained cells, which are, in general, transparent to light.

Two ways to obtain contrast in light microscopy

Figure 5:  Two ways to obtain contrast in light microscopy. The stained portions of the cell in (A) reduce the amplitude of light waves of particular wavelengths passing through them. A colored image of the cell is, thereby, obtained that is visible in the ordinary way. Light passing through the unstained, living cell (B) undergoes very little change in amplitude, and the structural details cannot be seen even if the im-age is highly magnified. The phase of the light, however, is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope.

4. Fluorescence Light Microscope

In fluorescence microscopy, the specimen itself acts as a light source. The specimens used to study are either fluorescent materials or stained with fluorescent dyes. A chemical is said to be fluorescent if it absorbs light at one wavelength and emits light (fluoresces) at a specific and longer wavelength. Most fluorescent dyes (or fluorochromes) emit visible light, but some emit infrared light. Fluorochromes exhibit distinct excitation and emission spectra that depend on their atomic structure and electron resonance properties. 

Two fluorescent dyes that are commonly used are fluorescein, which emits an intense green fluorescence when excited with blue light, and rhodamine, which emits a deep red fluorescence when excited with green-yellow light. The fluorescence microscope is similar to an ordinary light microscope except that the illuminating light is passed through two sets of filters – one to filter the light before it reaches the specimen (excitation filter) and other to filter the light emitted from the specimen (barrier or emission filter). 

The excitation filter passes only the wavelength that excites the particular fluorescent dye, while the barrier filter blocks out this light and passes only those wavelengths emitted when the dye fluoresces. Only fluorescent light emitted by the fluorescently stained specimen is used to form an image. The wavelength that excites the specimen and induces the fluorescence is not allowed to pass the filters placed between the objective lens and the eye.

The optical system of a fluorescence microscope

Figure 6. The optical system of a fluorescence microscope.

Fluorescence microscopes contain special filters and employ a unique method of illumination to produce images of fluorescent light emitted from excited molecules in a specimen. It contains two essential filters – excitation filter and barrier or emission filter. The diagram shows the orientation of filters. The excitation beam (blue line) passes through the excitation filter and is reflected by the dichroic mirror and directed towards the specimen. The return beam of emitted fluorescence wavelengths (cyan line) passes through the dichrolc mirror and the emission filter to the eye or camera. Excitation wavelengths that manage to pass through the dichroic mirror are blocked by the barrier (emission) filter.

5. Confocal Light Microscope

A confocal microscope creates sharp images of a specimen that would otherwise appear blurred when viewed with a conventional microscope. This is achieved by excluding most of the light from the specimen that is not from the microscope’s focal plane. The image has less haze and better contrast than that of a conventional microscope and represents a thin cross-section of the specimen. Thus, apart from allowing better observation of fine details it is possible to build three-dimensional (3D) reconstructions of a volume of the specimen by assembling a series of thin slices taken along the vertical axis.

Confocal is defined as having the same focus. What this means in the microscope is that the final image has the same focus as or the focus corresponds to the point of focus in the object. The object and its image are confocal. The microscope is able to filter out the out-of-focus light from above and below the point of focus in the object. Normally when an object is imaged in the fluorescence microscope, the signal produced is from the full thickness of the specimen which does not allow most of it to be in focus to the observer.

The confocal microscope eliminates this out-of-focus information by means of a confocal pinhole situated in front of the image plane which acts as a spatial filter and allows only the in-focus portion of the light to be imaged. Light from above and below the plane of focus of the object is eliminated from the final image. The confocal microscope uses a laser beam to illuminate a specimen, usually one that has been fluorescently stained. 

FAQ

FAQs on how does light microscope work

Light microscopes work by focusing light on the specimen and then using one or more lenses to magnify the image of the specimen.

Dr. Hans Janssen

Most people agree that these compound microscopes were invented by the Dutch eyeglass maker Hans Janssen and his son Zacharias.

It is possible to find a wide range in light microscope prices due to the wide variety of available options. While more sophisticated models with high magnification, digital imaging, or specialised optics can cost several thousand dollars, a simple light microscope for educational or hobbyist use can be purchased for as little as $100.

The most common microscope is called a compound light microscope, and it uses an electric lamp or LED for illumination. Specialized light sources, such as lasers or ultraviolet lamps, may be used in microscopes for fluorescence microscopy and other techniques that require the use of short wavelengths of light.

The first known compound light microscope was invented in the early 17th century by Dutch scientist Antonie van Leeuwenhoek.

The intensity of the light is usually controlled by a diaphragm or aperture located between the light source and the stage.