Study of Microscope

Introduction

Any individual working in laboratory must learn the use and care of laboratory instruments. Most instruments for there efficient use, require some measure of training and skill. Improper use can ruin the equipment leading to loss of time, and money. Instruments are delicate and should be handled carefully.

Microscope
Microscope

Hence it is advisable to read the ‘instruction manual’ carefully before its operation. It provides information regarding operation, maintenance, troubleshooting and essential circuitry for repair jobs. Some of the common instruments used in the microbiology laboratory are described in this chapter.

Microscope

The word microscope is derived from two Greek words ‘micro’ means small and ‘scope’ means to view. In other words a microscope is an instrumental used for visual examination of small objects which can not be properly examined by the unaided eyes.

Optical microscope
Microscope

Components

  • Eyepiece (ocular lens) (1)
  • Objective turret, revolver, or revolving nose piece (to hold multiple objective lenses) (2)
  • Objective lenses (3)
  • Focus knobs (to move the stage)
    • Coarse adjustment (4)
    • Fine adjustment (5)
  • Stage (to hold the specimen) (6)
  • Light source (a light or a mirror) (7)
  • Diaphragm and condenser (8)
  • Mechanical stage (9)

Simple Microscope

The simplest form of microscope is nothing more than a magnifying glass. It consist of two parts : 

  • The optical part or viewing part
  • The mechanical part whose function is to hold the slide or object in the proper position.
Microscope simple diagram

Dissecting microscope is a type of simple microscope which is fitted with mirror to focus bright light and a stage for placing the object.

Compound microscope

It differs from the simple microscope in that, it has two separate lens systems. Component parts of the microscope. their locations and functions are as follows

Microscope compound diagram

Functions of various parts of Compound Microscope

  • Base or stand : the ‘U’ shaped or square foundation which gives stability to the instruments.
  • Arm or handle : by means of which it may be moved or carried, to which are attached the magnifying and adjustment systems.
  • Stage : a platform which provides a surface for the placement of slide with its specimen over the opening. In addition to the fixed stage, most microscopes have a ‘mechanical stage’ that can be moved vertically by means of adjustment knobs.
  • Clips : the set of removable spring dips which held in place the slide with object.
  • Mirrors : the light refracting device placed under stage and employed to illuminate the object on the stage. Modern microscopes have been replaced by in build source of light.
  • Condenser : a system of lenses placed under stage; its function is to focus a strong beam of light upon the object being examined.
  • Iris diaphragm : a device placed beneath the condenser and capable of manipulation so as to adjust the quality of light
  • Body tube : a hollow cylindrical tube of the magnifications system through which light passes from the objective lenses at its bottom to the eyepiece at its top.
  • Nosepiece : a revolving disk at the bottom of the body tube to which the objectives are attached.
  • Objectives : a system of small lenses which constitute primary magnifying mechanism and are attached to the nosepiece. There are usually three objectives, low power, high power, and oil immersion objectives.
  • Eyepiece or ocular : a combination of lenses, placed in the upper portion of the body tube, which magnifies the image formed by the objective lens systems. Two eye-piece 5X and 10X are generally provided.
  • Coarse and fine adjustment : the screw mechanisms by which the body tube and its magnification systems may be raised or lowered quickly over a wide range to bring the objective approximately into focus.

Fine adjustment : moves the body tube slowly, and over a very limited range by means of which the object is brought into sharp a exact focus. (coaxial focus : modern focusing system that has both the coarse and fine focusing knob is mounted on the same axis. Usually the coarse knob is large and on the outside and the fine knob is smaller and on the inside.

Magnification

The total magnification is the product of the separate magnification of the objectives and depends on three factors whose relationship is as follows :

Total magnification = length of the body tube × eyepiece magnification / focal length of objective

MicroscopesOverview
Types of microscopes illustrated by the principles of their beam paths

Resolving power

It is the ability to show two adjacent objects as discrete, entities (separate objects). It is dependent on the wavelength of light used and numerical aperture of the objective lens.

Thus, to have the greater resolving power; the shorter wavelength and the larger numerical aperture values are desirable.

Numerical aperture (NA)

The angle θ subtended by the optical axis and the outermost rays still covered by the objective is the measure of the aperture of the objective; it is the half aperture angle. The magnitude of this angle is expressed as a sine value. The sine value of the half-aperture angle multiplied by the refractive index of the medium filling the space between the front lens and the cover slip gives the numerical aperture :

The use of mineral oil [which has the same refractive index as glass] as the mounting medium increases the amount of light entering the objective and thereby also increase the value of numerical aperture. 

Using Microscope

  • Place the microscope on a stable place on a bench of suitable height free from vibrations.
  • Provide illumination from any of the three sources i.e., built in lamp, external lamp or sunlight.
  • Direct the path of light to pass through diaphragm condenser and the hole of the stage with maximum intensity while setting the mirror keep the condenser at the top position.
  • Place the slide bearing object to be examined between the dips on the mechanical stage.
  • Revolve the nosepiece and align the low power objective 10X to examine the object on the slide (objective must click into place).
  • Look through the eyepiece and not into it. Adjust the illumination to improve the contrast. In case of low power the illumination is cut down to minimum by reducing the aperture size.
  • Put one hand on the focusing knob [course or fine as the case may be], and other on the screw to move the stage. Use the coarse adjustment knob. The low power objective is commonly used to screen the field of the new. Bring the object of interest in the centre.
  • Switch to high power (40X) and increase the illumination as needed. While changing the objective by rotating the nosepiece make-sure that the objective clicks into place. Repeat the process of focusing as describes by using the coarse adjustment knob followed by five adjustment knob. Normally modern microscope being parfocal (once the object is focussed under any objective, it stays in focus for all objectives and no further adjustments are necessary).
Microscope ObjectiveLens
  • After screening under low power and examining under high power oil-immersion objective is used for containing greater details of the object.
  • Place a small drop of oil on the slide, over the path of the light. Increase the illumination to maximum. Turn the nosepiece and set the oil immersion objective in its place.
  • Use fine adjustment, bring the object sharps into focus. It often necessary at times to raise or lower the condenser so that the optimum illumination is secured. This is termed ‘critical illumination’.
  • Use fine adjustment knob to get object in focus. If this fails, use coarse adjustment to focus the slide.
  • Finally when the observation is finished the oil adhering the slide is removed by using xylene and wiping it with tissue paper.

Routine care of Microscope

  • Carry, the microscope by holding its arm or limb with one hand and the other hand under the foot. Never swing the microscope while carrying.
  • Never force on anything.
  • Never touch any optical surface with any thing other than lens tissue.
  • At the end, clean all immersion oil of the lenses. Don’t use organic solvent to remove oil on regular basis. This may dissolve cement which holding the lens.
  • Dust and fungus are the worst enemies of microscope. Growing fungus and scratches caused by dust can ruin the microscope. So store it away from these.
  • When in doubt ask your instructor for assistance.

3D dual color super resolution microscopy

3D Dual Color Super Resolution Microscopy Cremer 2010
3D dual color super resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568 LIMON

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