Microscopy, different types of microscopes, microbiology notes for PCI syllabus
MICROSCOPY
Compound microscope
| Principle | When an object is placed just beyond the focus of the objective lens, a magnified, virtual and inverted image is formed just at the distinct vision of eye held close to eye piece. |
| Maximum magnification | 1000 times |
| Working | Light enters from the base and passes through the diaphragm. The specimen is placed on the stage. The objective lens magnifies the image before it enters into the ocular tube, from where the eye piece further magnifies the image and produces and inverted, virtual and magnified image of the specimen. |
| Advantages | · Simple and easy to use
· Does not require professionals to handle · Can be stored easily · Does not require artificial light sources
|
| Disadvantages | · Have a limited range of magnification
· Can only be used under visible light range · Lenses got blurry overt-time |
| Uses | · Used in pathology labs
· Used in forensic labs · Used for showing specimens in colleges and schools · For studying the movements of cell |
| Other information | · The microscope falls under the category of bright field microscopy
· Focus can be adjusted by using course and fine knobs present on the microscope. |
The above image is taken only for educational purpose from microscope.com
Dark field microscopy
| Principle | Only the light that is scattered by the object placed on the slide reaches the eye due to presence of an opaque disk in microscope. |
| Working | It has a dark field condenser instead of simple condenser and has an opaque disk. Light which should directly enter into condenser is blocked by the opaque disk and only the light which is reflected back from the specimen is entered into condenser. In this way, a light image is formed under dark background. |
| Advantages | Microbes that cannot be stained or have same refractive indices can be viewed undead under this microscope.
Microscope is simple and effective. |
| Disadvantages | · Final image formed have very low light levels.
· Specimens must be highly illuminated which may damage the specimen |
| Uses | · Microbes that cannot be stained or have same refractive indices can be viewed undead under this microscope.
· Imaging of bacteria suspended in liquid · Study of internal structure of eukaryotic microorganisms · Imaging of specific and highly thin spirochaetes. · On combining with hyperspecteral imaging, dark field microscopes can be used for imaging nanoparticles embedded in the cells. · Technique is also used in modern mouse pointers. |
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Phase contrast microscope
| Principle | By using special objective lenses and condensers, the small differences of the refractive index of different structures within the cell is accentuated. The changes in the speed of light can be seen as different level of brightness. |
| Maximum magnification | 1000 times |
| Working | Light when passes through different structure which are having different refractive indexes in the cell, the speed of light varies with structures. On passing through the special condenser and objective lenses, light is accentuated according to its speed. Rays which are not diffracted are directly passes the phase ring while the speed of rays which are diffracted is reduced. Undiffracted rays appears red while diffracted rays appears golden. |
| Advantages | · Better resolution
· Live microbes can be seen · Movements of internal matter of the cell can be seen · Staining is not required · Saves time · Structures can be visible with better sharpness · Cells in their original volume can be seen |
| Disadvantages | · Expensive
· Complicated · Numerical aperture size is limited |
| Uses | · Imaging the transparent microorganisms
· Observing internal structures of cells · Observing living cells without killing or using dyes
|
The above image is taken only for educational purpose from easybiologyclass.com
Nomarski microscope (differential interference contrast microscope)
| Principle | Same as phase contrast microscope. |
| Maximum magnification | 100 times |
| Working | Same as phase contrast microscope, just difference is, the microscope used is a differential interference contrast microscope. In this, two plane polarized light beams held at right angles to each other is passed through the specimen and the clear zone in the slide. The emerging beam is combined and actual inference so produced forms the image |
| Advantages | · Live microbes can be used
· No need of staining · 3-dimensional image is formed · Highly colored images are formed · Clear and distinct images of each structure of cell |
| Disadvantages | · 3-D images may not be accurate
· Image formed may be distorted · Expensive · Complicated |
| Uses | · Study of internal structures like cell wall, nucleus, mitochondria, etc of eukaryotic microorganisms
· Producing 3-D images of cell structures |
The above image is taken only for educational purpose from slideshare.net
Fluorescence microscope
| Principle | When light is projected on any substance, they store the light in form of energy and then they again emit lack this entrapped energy in the form of light. Different substances emits back different wavelength of light and thus, can be distinguished by using fluorescent microscopy. |
| Maximum magnification | 100 times |
| Working | In this, a very high intensity light is illuminated on the sample. Due to this, the fluorescence species of the specimen emits light of longer wavelengths. This emitted light is then used for the formation of image. |
| Advantages | · Can precisely distinguish between individual antigens
· Can detect few molecules per cubic micron · Possibility to track different molecules by using different colors · Observing colocolization is possible |
| Disadvantages | · Photobleaching
· Cannot focus on specimen for longer duration at higher magnification · Sophisticated instrumentation · Experimental optimization required |
| Uses | · Studying structural components of small specimens
· Studying viability on cell populations · Studying the genetic material within the cell · Imaging specific cells within large population |
| Other information | · Very few microbes are there which can do fluoresce naturally hen subjected to special lights. Dye known as fluorochromes must be used for the other microbes undergoing fluorescence investigations. They can be viewed under UV or near-UV light range as the bright objects under dark background |
The above image is taken only for educational purpose from microbenotes.com
Transmission electron microscope (TEM)
| Principle | Principle of TEM is same as that of simple microscopes, but here, instead of light, electrons are used. Since electrons have very small wavelength, TEM images are far better in magnitude than simple microscopes. |
| Maximum magnification | 500,000 times |
| Working | Preheated tungsten filaments are used as an electron gun. Electrons coming from electric gun is focused on the specimen by using special electromagnetic objective lenses which are doughnut in shape. Whole column is maintained under vacuum. Scattering of electrons takes place when it passed near the specimen. These scattered electrons are then focused by help of electromagnetic projector to the fluorescent screen. Images so formed on the fluorescent screen can be converted into photographic plates for permanent impression of the specimen. |
| Advantages | · Magnification to higher degree
· Study of extremely small particles is possible · Provide better view of internal structure of microbes
|
| Disadvantages | · Very expensive
· Very complicated · Preparation of specimen required much professional knowledge and experience · Sensitive to vibration and electromagnetic waves · Constant supply of currents, voltage and cool water is required during operation · Images are black and white · Requires special housing and maintenance |
| Uses | · Study of crystals and metals
· Semiconductor analysis · Identification of flaws, fractures and damages in micro-sized objects · For research purposes
|
| Other information | · For the preparation of the specimen to be observed under TEM, specimen is first embedded in the plastic block ad then it is cut with the help of diamond or glass knife to produce very thin slices. |
The above image is taken only for educational purpose from biosciencenotes.com
Scanning electron microscope (SEM)
| Principle | The beam of electrons which is scattered back by the metal coated cells, results in increase in the electric current, which then can be converted into 3D images. |
| Maximum magnification | 50,000 times |
| Working | Specimen covered by the metal layer is mounted on the microscope. A narrow beam of electron is focused on the specimen which results in the showering back of the secondary electrons from the specimen. These electrons are recorded by the detector which further strikes a scintilator which emits light flashes. These light flashes increases the current by using photomultiplier tube. The current is then amplified and sent to cathode-ray tube to form sharp images of the specimen. |
| Advantages | · Highly magnified images can be produces.
· 3-dimensional imaging is possible · Vey fast working
|
| Disadvantages | · Very expensive
· Requires special housing area · Special training and experience required for proper uses · Limited to solid, inorganic samples which can fit inside vacuum tubes · Risk of radiation exposure |
| Uses | · Analyzing surface fractures
· Studying microstructures · Examination of surface contamination · Qualitative chemical analysis · Identification of crystalline structure · Research tool for microbiology lab · Semiconductor inspection |
| Other information | · Specimen can be prepared by coating with heavy metals like gold and platinum. |
The above image is taken only for educational purpose from biosciencenotes.com
MCQs
1. Maximum magnification of compound microscope is?
a. 10 X
b. 100 X
c. 1000 X
d. 10000 X
2. An opaque disk is used in which microscope?
a. Compound microscope
b. Fluorescence microscope
c. SEM
d. Dark field microscope
3. SEM can resolve image upto?
a. 100 times
b. 5000 times
c. 10000 times
d. 50000 times
4. 3D microscopic images are possible from?
a. Scanning electron microscope
b. Compound microscope
c. Dark field microscope
d. 3-D imaging is not possible
5.Type of light used in Nomarski microscope is?
a. Visible light source
b. Sunlight
c. No light should be there
d. Plane polarized light
6. Which of the following statements are correct related with Microscopy?
I. Photobleaching is a disadvantage of compound microscope
II. Pre-heated tungsten filaments are used as an electron gun in TEM
III. Dark image of specimen is formed on bright background in dark field microscopy.
IV. Most simple form of microscope is TEM.
a. II
b. II, III, IV
c. I, IV
D, I, II, IV
7. Match the following microscopes with their correct applications:
| I. Compound microscope | A. To study the Microbes that cannot be stained or have same refractive indices |
| II. SEM | B. Identification of flaws, fractures and damages in micro-sized objects
|
| III. TEM | C. 3-D imaging of microbial structures |
| IV. Dark-field microscope | D. For studying the movement of cell |
a. I-A, II-B, III-C, IV-D
b. I-D, II-C, III-B, IV-A
c. I-B, II-C, III-A, IV-D
d. I-D, II-A, III-B, IV-C
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ANSWERS
1-c
2-d
3-d
4-a
5-d
6-b
7-a
REFERENCES
[1] Black JG, Black LJ. Microbiology: principles and explorations. John Wiley & Sons; 2018 Jan 4.