Microscopy-Light microscope (Part-1)

The examination of minute objects by means of a microscope, an instrument which provides an enlarged image of an object not visible with the naked eye.

There are two fundamentally different types of microscope: the light microscope and the electron microscope.

ü  Light microscope (optical microscope) involves the use of optical lenses and light radiations.

Light or optical microscopy involves passing visible light transmitted through or reflected from the specimen through a single lens or multiple lenses to allow a magnified view of the specimen.

The light  microscope creates a magnified image of the specimen which is based on the principle of transmission, adsorption, diffraction, and refraction of light waves.

A simple microscope consists of a single lens, a magnifying glass.

The compound microscope consists of more than one glass lens in combination and various components which gather light and redirects the light path so that a magnified image of the viewed object can be focused within a short distance.

ü  Functions of the major parts of a light microscope-

Light source and condenser- project a parallel beam of light onto the specimen for illumination.

Condenser gathers light and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire view field.

Sample stage with X-Y movement-sample is placed on the stage and different part of the sample (specimen) can be viewed due to the X-Y movement capability.

Fine/focusing knobs-since the distance between objective and eyepiece is fixed, focusing is achieved by moving the sample relative to the objective lens.

Objectives- does the main part of magnification and resolves the fine details on the samples (m_o-4X, 10X, 20X, 40X, 60X, 100X)

Eyepiece- forms a further magnified virtual image which can be observed directly with eyes (m_e̴10)

ü  Magnification

The magnification or linear magnification of a microscope is defined as the ratio of the image size to the specimen size. To visualize any tiny object 0.1mm there is a limit of the unaided human eye. To see microorganisms much smaller than 0.1mm a system required convex lenses. When parallel rays pass through convex lens get converged at a point called focal length (f) of the lens. Hence, a lens with a shorter focal length will have higher magnification power. In compound microscope it will be i.e. 10X, f=16mm; 40X, f=4mm; 100X, f=1.8mm.

The overall magnification is given as the product of the lenses and the distance over which the image is projected:

M= (D*M1*M2)/250mm

Where D=projection (tube) length (usually 250mm), M1 and M2 magnification of objective and ocular lens respectively.

250mm=minimum distance of distinct vision for 20/20 eyes.

ü  Resolution

The ability to distinguish between two closely spaced points in a specimen. The limit of resolution is the closest distance between two points at which the points still can be distinguished as separate entities. Standard light microscopes have a lateral resolution limit of about 0.5µm for routine analysis. In contrast, electron microscopes have a lateral resolution of up to 1 nanometre (nm).

Magnification should be coupled with good resolution to visualize small microorganisms, else magnification alone will produce an inconclusive or blurred image.

Resolution is described mathematically by an equation developed in the 1870s by Ernst Abbe, a German physicist responsible for much of the optical theory underlying microscope design.

The Abbe equation states that the minimal distance (d) between two objects that reveals them as separate entities depend on the wavelength of light (λ) used to illuminate the specimen and on the numerical aperture of the lens (nsinϴ), which is the ability of the lens to gather light.

d= 0.5λ / nsinϴ

As d becomes smaller, the resolution increases, and finer detail can be discerned in a specimen, d becomes smaller as the wavelength of light used decreases and as the numerical aperture increases. Thus the greatest resolution is obtained using a lens with the largest possible numerical aperture and light of the shortest wavelength, the light at the blue end of the visible spectrum (in the range of 450nm to 500nm). The numerical aperture (nsinϴ) of a lens is defined by two components: n is the refractive index of the medium in which the lens works (e.g., air) and ϴ is ½ the angle of the cone of light entering an objective. A cone with a narrow-angle does not adequately separate the rays of light emanating from closely packed objects, and the images are not resolved.

Numerical Aperture in Microscopy- The angular aperture ϴ is ½ the angle of the cone of light that enters a lens from a specimen and the numerical aperture is nsinϴ. In the right-hand illustration, the lens has larger angular and numerical apertures, its resolution is greater and its working distance smaller.

A cone of light with a very wide angle does separate the rays and the closely packed objects appear widely separated and resolved. Some objective lenses work in air, which has a refractive index of 1.00. Since sinϴ cannot be greater than 1 (the maximum ϴ is 90ͦ and sin 90ͦ is 1.00), no lens working in the air can have a numerical aperture greater than 1.00.

The only practical way to raise the numerical aperture above 1.00, and therefore achieve higher resolution, is to increase the refractive index with immersion oil, a colorless liquid with the same refractive index as glass.

ü  Properties of objective lenses

		Objective
Property	Scanning	Low Power	High Power	Oil immersion
Magnification	4X	10X	40X-45X	90X-100X
Numerical aperture	0.10	0.25	0.55-0.65	1.25-1.4
Approximate focal length (f)	40mm	16mm	4mm	1.8mm-2.0mm
Working distance	17mm-20mm	4mm-8mm	0.5mm-0.7mm	0.1mm
Approximate resolving power with the light of 450 nm (blue light)	2.3μm	0.9μm	0.35μm	0.18μm

 

If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so. This results in an increase in numerical aperture and resolution.

The oil immersion objective- An oil immersion objective operating in air and with immersion oil.

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

d= (0.5) (530nm) / 1.25    = 212nm or 0.2μm

At best, a light microscope can distinguish between two dots about 0.2μm apart (the same size as a very small bacterium). Thus the vast majority of viruses cannot be examined with a light microscope.

ü  Bright-Field Microscope: Dark object, Bright background

The most basic model of the light microscope is called bright-field (bright background), which can be achieved with a minimum of optical elements. It is used to examine both stained and unstained specimens. It is called a bright field microscope because it forms a dark image against a brighter background.

The contrast in bright-field images is usually produced by the color or dense sections of the specimen itself. Bright-field is therefore used most often to collect images from pigmented sections of the specimen. However, many microbes are unpigmented and are not clearly visible because there is little difference in contrast between the cells, subcellular structures, and water. One solution to this problem is to stain cells before observation to increase contrast and create variations in color between cell structures. Unfortunately, staining procedures usually kill cells.

Working

·         Bright field microscopy is a technique used in the light microscope which gives a magnified image of the dark specimen with a colorless background. To accomplish the bright field microscopy, transfer the glass slide having a stained specimen onto the microscope stage.

·         The luminous light will come through the source of light or we can say through a source of illumination. The light coming from the illuminator is aimed at the condenser lens, which is present beneath the specimen. The aperture diaphragm helps to focus and control the light coming from the light source illuminator on the specimen.

·         Some of the light will reflect out of the specimen, which is then collected by the objective lens. The objective lens first magnifies the light and then transmits it to the ocular lens. During this whole method, some of the light will get deflected, and some will get absorbed by the stain and the dense areas in the specimen.

·         The intense illumination and can increase the magnification of the image. Therefore, in bright field microscopy, those which have absorbed the part of the light will appear darker and the remaining, i.e. the background will appear brighter.

 

Advantages

·         Bright field microscopy is a simple method to perform, simplicity of setup with only basic equipment required.

·         It can easily produce a magnified image of the fixed specimens and live cells.

Disadvantages

·         The bright field microscopy produces low contrast to the image.

·         The practical limit to magnification with a light microscope is around 1300X. Although higher magnifications are possible, it becomes increasingly difficult to maintain image clarity as to the magnification increases.

·         Low apparent optical resolution due to the blur of out-of-focus material.

Enhancements

·         Reducing and increasing the amount of the light source coming to the specimen by adjusting the iris diaphragm.

·         Use of an oil-immersion objective lens and a special immersion oil placed on a glass cover over the specimen. Immersion oil has the same refraction as glass and improves the resolution of the observed specimen.

·         The use of staining methods adds contrast to the picture.

·         Use of a colored (usually blue) or polarizing filter on the light source to highlight features not visible under white light. The use of filters is especially useful with mineral samples.

 

ü  Dark- field microscope: Bright object, Dark background

The dark-field microscope produces detailed images of living, unstained cells, and organisms by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. The only light that has been reflected or refracted by the specimen forms an image. The field surrounding a specimen appears black, while the object itself is brightly illuminated. The dark-field microscope can reveal considerable internal structure in larger eukaryotic microorganisms. It also used to identify certain bacteria such as the thin and distinctively shaped Treponema pallidum, the causative agent of syphilis.

Working

·         Light enters the microscope for illumination of the specimen. A specially sized disc, the patch stop or dark-field stop, blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification.

·         The condenser lens focuses the light on the specimen. The light enters the specimen. Most are directly transmitted, while some is scattered from the specimen.

·         The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct-illumination block.

·         Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.

 

 

Dark-field microscopy- In dark-field microscopy, a dark-field stop (inset) is placed underneath the condenser lens system. The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen.

Advantages

·         A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light.

·         These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques. But in darkfield, the quality of images obtained is impressive and having high contrast. Darkfield is used to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast, and protozoa as well as some minerals and crystals, thin polymers, and some ceramics.

·         Dark the field is also used in the research of live bacterium, as well as mounted cells and tissues.

·         It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

 

Disadvantages

·         First, dark field images are prone to degradation, distortion, and inaccuracies.

·         The specimen is strongly illuminated, which can cause damage to the specimen.

·         The specimen that is not thin enough or its density differs across the slide, may appear to have artifacts throughout the image.

·         The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image.

·         It need special care that the slide, stage, nose and light source are free from small particles such as dust, like these, will appear as part of the image.

·         Similarly, if need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles. These liquid bubbles will cause images degradation, flare and distortion and even decrease the contrast and details of the specimen.

·         Dark the field needs an intense amount of light to work. This, coupled with the fact that it relies exclusively on scattered light rays, can cause glare and distortion.

·         It is not a reliable tool to obtain accurate measurements of specimens.

·         Finally, numerous problems can arise when adapting and using a dark field microscope. The amount and intensity of light, the position, size, and placement of the condenser and stop need to be correct to avoid any aberrations.

·         However, when employing this technique as part of a research study, need to take into consideration the limitations and knowledge of possible unwanted artifacts.