A compound microscope is a class of optical or light microscope. I have already discussed about basics of a microscope on my previous note “Parts of Microscope with their Functions and Working Principle”, you can check them to get an idea about from which class compound microscope belongs and what is a microscope, and more.
Before Jump into it follow my previous note on Microscope. I have also discussed about Simple Microscope in my previous note “Simple Microscope: Working Principle, Uses, Parts, and their Functions” which is also a class of optical microscope.
Definition of a Compound Microscope
- The first question in your mind will be what is a compound microscope? A compound microscope is a laboratory instrument with high magnification power, which is consists of more than one lenses.
- Compound Microscopes are used for the study of structural details of a cell, tissue, or organ in sections.
- A compound microscope can magnify the image of a tiny object up to 1000.
- The term compound means “multiple” or “complex”.
- The compound microscopes is consists of two lenses includes, the objective lens (typically 4x, 10x, 40x or 100x) in a rotating nosepiece closer to the specimen, and the eyepiece lens (typically 10x) in the binocular eyepieces.
- A compound binocular microscope is more commonly used today.
- Zacharias Jansen created a compound microscope that used collapsing tubes and produced magnifications up to 9X.
- compound microscopes are generally types of bright field microscope.
- Compound microscopes may be categorized as an upright microscope, and Inverted microscope.
- Upright compound microscopes are just like an ordinary microscope which has a lens system, followed by the stage where the specimen is kept, and then the light source.
- Inverted compound microscopes are exactly the reverse replica of the upright microscope with the illumination system first, followed by the stage, and then the lens system.
Types of Compound Microscope
Classification of Compound Microscope
Compound Microscope is classified in two categories;
A. Light Microscope
Light Microscope is further classified into four categories such as;
- Bright-field Microscope
- Dark-Field Microscope.
- Phase-contrast Microscope.
- Fluorescent Microscope.
B. Electron Microscope
Electron Microscope is further classified into three categories such as;
- Scanning Microscope
- Transmission Microscope
- Confocal Microscope
Working Principle of Compound Microscope
The compound microscopes are works on the principle that when a tiny specimen to be magnified is placed just beyond the focus of its objective lens, a virtual, inverted and highly magnified image of the object are formed at the least distance of distinct vision from the eye held close to the eyepiece.
Mechanism of Compound Microscope
Compound microscopes create an image of specimen by these following steps;
- First of all, a specimen is placed between the objective and condenser lens.
- The light emitted from the light source is pointed over the specimen with the help of a condenser lens.
- After that, the light is passed through the specimen and comes towards the objective lens.
- The objective lens captures the light coming from the specimen and creates a magnified image of the specimen, which is called the primary image.
- Then the objective lens passed this image through the body tube to the ocular lens or eyepiece and again magnifies the image.
- At last, the viewer can see a clear and magnified image of the specimen through the eyepiece.
- Occasionally or during the use of a 100x objective lens oil immersion method is used to produce a highly magnified image of the specimen. In this method, a drop of immersion oil is placed between the objective lens and specimen slide.
Magnification Power of Compound Microscope
The total magnification of image formed by the compound microscopes is calculated b this following formula;
m = D/ fo * L/fe
Where, D = Least distance of distinct vision (25 cm)
L = Length of the microscope tube
fo = Focal length of the objective lens
fe = Focal length of the eye-piece lens
Parts of compound microscope and Their functions
- It is located at the top portion of the microscope.
- It contains Eyepiece.
- It is also known as ocular, which is located at the top of a microscope. Viewers see the specimen through it.
- Body Tube:
- It’s a long tube, which connects both eyepiece and objective lenses.
- Nosepiece is located at the bottom portion of body tube.
- Objective lenses are remain attached to it.
- It can rotate to adjust the objective lens.
- Objective lens:
- Compound microscopes contain different types of objective lens (10x, 40x, 100x).
- These are located below the nosepiece.
- These lenses are closest to the specimen.
- The flat metal platform located above the condenser and below the objective lens.
- The slide of the test specimen is placed over it.
- Stage Clips:
- It is above the stage.
- It holds the slide.
- It supports all the components of the microscope.
- It connects the body tube and base of the microscope.
- An illuminator is the light source of compound microscopes.
- It is a low voltage bulb, which is located below the stage.
- It is a small hole in the middle of stage.
- It pass the light from the Illuminator to the specimen slide.
- It is located below the stage.
- It gathers and focuses light from the illuminator onto the specimen being viewed.
- Iris diaphragm:
- It adjusts the amount of light that reaches the specimen.
- On/off switch:
- It is located at the base.
- This switch turns the illuminator off and on.
- Stage Controller:
- These knobs move the stage in left and right or up and down.
- Brightness Adjustment:
- It located at the base.
- It adjust the brightness of Illuminator.
- It is a five holed disk placed under the stage.
17. Fine adjustment: Fine adjustments can be used to adjust the focus and increase the detail of the specimen.
18. Coarse adjustment: Focuses on the specimen.
19. Diopter Adjustment: Diopter Adjustment is a tool that allows you to adjust the focus of one eyepiece to correct any differences in vision between your eyes.
20. Slide or specimen: A specimen is an object that has been examined. Most specimens are mounted on slides. These flat rectangular pieces of thin glass are used for mounting.
A cover slip is placed above the specimen and the specimen is placed on top of the glass. This covers the microscope so that the slide can be easily removed or inserted. This allows for the specimen to be easily labeled and transported without damage.
Magnification of the Object Image by Compound Microscope
Bright-field and compound microscopes are used to magnify or enlarge the image of an object being viewed. This is not possible with the naked eye. Magnification can be described as the extent of magnifying an object’s image using a microscope.
Magnification of a microscope depends on the individual magnifying abilities of the objectives and the oculars. If the objective is 40X and the ocular 10X, then the specimen will be magnified 400x. The specimen will be magnified 1000x if an oil immersion objective (100X), is used with the 10X ocular.
Factors play an important role in magnification
- Length of optical tube.
- The focal length of the objective lens.
- Magnifying power of the ocular.
Compound Microscope Resolution Power (Resolving power)
A compound or bright-field microscope’s resolution power (resolving ability) is its ability to distinguish between very close particles. A magnified image should show the object in a larger size. However, it should still have clear details.
This can be achieved when the microscope is able to see two points very close together as two distinct entities. The resolution power is the distance between two objects that can be clearly seen as separate structures in a magnified image.
This explanation is easily understood by comparing it to the human eye. The human eye works on the same principle as a light microscope or bright-field, meaning that objects can be seen by reflecting light.
The human eye has a resolution power of 0.25mm. Two dots placed 0.25mm apart (or more) can be seen only as two dots. Anything closer will appear as a single dot.
Factors that determine Resolution Power
Two factors affect the resolution power of a bright field (light) microscope:
- Wave length of light
- Numerical aperature of the objective.
Wave length of the light
The visible wavelength of light used to illuminate light microscopes (bright field) falls within the visible range (400-750nm). The resolution will increase if the light used is shorter in this range. Blue light, for example, has a shorter wavelength that red light. A blue light can be used as an illumination source to achieve greater resolution than a red one.
The Objective’s Numerical Aperture (NA)
The property of a lens which determines how much light can enter it is called numerical aperture (NA). It is dependent on two factors.
- The medium’s refractive index is the area between the specimen, the front of an objective lens.
- The angle between the divergent rays that pass through the lens and the optical axis is called the angular aperture. The resolution power is greater if the objective can accept more divergent or oblique light.
Numerical aperture (NA) can be mathematically calculated with the help of following formula.
NA = n sin f
Where, n = refractive index of the medium
f = angular aperature
Calculation of Resolution Power
The resolution power of a bright-field microscope can be calculated using the following formula:
Resolution (resolving) power (RP) = Wave length of light used for illumination/2 x Numerical aperture (NA)
For convenience, if yellow light of wave length of 580 nm with numerical aperture (NA) of 1.0 is used in the microscope, the resolution power (RP) of the microscope will be:
Resolution power (RP) = 580/2 x 1 = 290 nm
Object Size Measurement by using Compound Microscope
A micrometer can accurately determine the size of objects when they are viewed under a compound microscope. This latter is made up of two scales: the eyepiece scale (also known as ‘graticule or ‘ocular’), and the stage microscope scale. The stage micrometer calibrates the eyepiece scale and then the latter is used to measure.
The microscope eyepiece scale is located inside the microscope eyepiece. The stage micrometer is on the microscope stage. The scale is approximately 1 mm in length and is divided into 100 divisions. Each division is 10 um. The stage micrometer is used for calibrating the eyepiece scale, as mentioned earlier.
- It is important to note first which objective lens has been used on the microscope.
- The stage micrometer is placed so that it is visible from the field of vision.
- Rotating the eyepiece so that the two scales, either the eyepiece or the ocular scale, and the stage micrometer, are parallel is a good idea.
- Now, the stage micrometer must be moved so that both scales’ first division marks are in line.
It is now possible to see which divisions of the eyepiece and stage micrometer scale correspond with each other. One division on the stage microscope equals 10 um. This allows one to calculate the value of the eyepiece scale.
The four divisions of the eyepiece stage scale equal 10 divisions (i.e. 100 um) on the stage micrometer scale. 1 division on this eyepiece scale = 25nm for the specific objective lens in this case.
These positions can be repeated with objective lenses. The following information’s are recorded onto an adhesive label. For future reference, adhesive labels are stuck to the base and sides of the microscope.
After calibrating the eyepiece scales for all objective lenses, the microscope can be used to measure the dimensions and morphology of cells and sub-cellular structures.
Working Mechanism of The Compound Microscope
- Look into the eyepiece. Adjust the mirror so that enough light can pass through the microscope.
- Clean the mirrors, lenses, stage and slides.
- Place the slide in central position on the stage.
- Secure the slide securely with clips along the edges to stop it moving.
- The nose piece can be adjusted so that the low power objective aligns with the object of focal on the slide.
- You can adjust the coarse adjustment knob upwards or downwards so that the slide remains in focus.
- To get a sharp and clear image of the object in focus, turn the fine adjustment knob upwards or downwards.
- Under low power objectives, all details are captured. The diagrams that are necessary are drawn.
- To align the high power objective with the object, the nose piece must be turned. To get a clear and sharp view of the object, the fine adjustment knob must be adjusted as much as possible.
- High power can be used to observe the details of an object. Make the diagrams. When the object is being examined at high power, the coarse adjustment knob should be avoided as it could crush the slide.
- Before using, clean the eyepiece and objective lenses with a silk cloth and some cleaning liquid.
- When using the microscope, it should not be tilted.
- Focus on the lowest power object first, then move on to the higher power.
- After all observations have been completed, the lower power must be retained.
- Focusing requires that you ensure the objective lens does not touch the slide or stage.
- When the high power objective needs to be achieved, the fine adjustment knob is the only thing that should be used.
- Always use a cover slip to cover well-mount preparations prior to observation under the microscope.
- Do not take apart the microscope.
- Always use both hands when carrying the microscope
- After using the microscope, place it in a container.
- Dimming the light should be used on the concave portion of your mirror.
- It is not recommended to use oil immersion lenses without oil.
Use of Compound microscope
- Compound Microscopes used for blood analysis in pathological labs.
- In forensic laboratories, the compound microscopes are used for examining the human cells, paper, etc. Which are related to the crime scene.
- Used for the detection of drugs, by viewing their particles under compound microscopes.
- In university and college laboratories students use compound microscopes for studying the fungi, bacteria, plant cells, animal cells, etc.
Advantages of Compound Microscopes
- It is not very expensive.
- Can look at live samples
- Can magnify up to 2000 times
- This microscope is easy to use.
- These microscopes are easily transferable due to their compact size.
- It can produce a clear image as compared to a simple microscope.
Disadvantages Compound Microscope
- Compound Microscopes Can’t magnify more than 2000 times