Microbiologist Microscopy question discussion

Microscopy related mcq discussion

1. Microbiologist Kerala Water Authority Topic: Microscopy

2. Question No 1

3. Explanation The total magnification is the product of the magnifications of two lens systems. Hence, the microscope which has a 10X ocular lens and 20X objective lens, it would magnify the object by 10 × 20 = 200 times. The power of a microscope lens is the amount of magnification the lens gives.

4. Question No 2

5. Explanation The resolving power of the microscope increases with the decrease in wavelength of light and an increase in the numerical aperture. Resolving power of the compound microscope is the ability of the compound microscope to form a separate image of two close objects which are placed very near to each other. This is given by the famous Abbe's criterion given by Ernst Abbe in 187 . -Resolving power can be measured by, ResolvingPower =0. 5 λ/NA

6. -The minimum distance between two objects can be explained as-  d=0. 5 λ/ nsinθ -And the resolving power is inversely proportional to the minimum distance between the objects so, Resolving power= 1/d Resolving power= nsinθ /0. 5 λ Where lambda is the wavelength of light and  nsinθ  is the numerical aperture. It can be observed from the formula that the resolving power is directly proportional to the numerical aperture but is indirectly proportional to the wavelength of the light.

7. Question 3

8. Explanation Scanning electron microscope

9. SEM Best for: Looking at surfaces of objects. Looking at objects in 3D. Disadvantages: Resolution often not as high as the transmission electron microscope. Can’t be used to look at living things (samples need to be dried and coated in metal before visualising ). Costly to run.

10. Question 4

11. Explanation Refractive index is defined as the ratio of the speed of light in a vacuum to its speed in a specific medium. Refractive index is also referred to as refraction index or index of refraction.  The speed of light in a medium depends on the properties of the medium.  The refractive index for air very close to 1 , on average, is approximately 1.0003. So, light travels 1.0003 times faster in a vacuum than in air.

12. Question 5

13. Explanation An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects.

14. Question 6

15. Explanation Ernst Ruska and Max Knoll developed the first electron microscope in 1931, and the first commercial, mass-produced instrument became available in 1939. Afterward, electron microscopes improved rapidly in the 1950s, when developing scanning electron microscope in Charles Oatley’s laboratory at Cambridge University led to the first commercial scanning electron microscope by Cambridge Instruments in 1965.

16. Question 7

17. Explanation The ability of the eye to differentiate between two points close to each other is called its resolving power. The limit of resolution of the human eye is about 0.1 mm, or 100 microns. Objects that are smaller than this cannot be seen clearly without magnification. Since most cells are much smaller than 100 microns, we need to use microscopes to see them.

18. Question 8

19. Explanation The condenser on a microscope is an optical component between the light source and the specimen, which captures and concentrates light rays in the plane of the microscopic field, allowing the specimen to be seen.

20. Question 9

21. Explanation When we see a distant object, the parallel rays of light falling on our eye are focused by the lens on the retina. Therefore, the object is seen distinctly. So, when we see a nearby object, the muscles in the eye contract to increase the curvature of the lens. Hence, the focal length of the lens decreases and a clear image of the object is formed at the retina. But there is a limit to this accommodation. The nearest distance up to which the eye can see clearly is called the ‘least distance of distinct vision’ For normal eyes it is 25cm. The point at this distance is called the ‘near point’ of the eye. If an object is placed at a distance less than this, from the eye, it will not be seen distinctly. Therefore, for a normal eye, in case of an adult, the least distance of distinct vision is 20 to 25 cm.

22. Question 10

23. Explanation Most instruments that use fluorescence, including fluorescence microscopes, rely on optical filters. A typical microscope has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or dichroic mirror), and an emission filter (or emitter). These three filters form what is referred to as a “filter set” and are often housed in a special assembly called a “filter cube” that can be quickly mounted in a microscope.

24. The exciter filter removes all light except blue light in fluorescence microscopy. The barrier filter stops blue light but enables any other light generated by the fluorescing material to pass through and reach the eye. A dichroic mirror allows light of a certain wavelength to pass through, while light of other wavelengths is reflected.

25. Question 11

26. Explanation The part of a microscope that allows you to adjust the amount of light that shines through is called the diaphragm or iris diaphragm. Diaphragm which opens and closes to permit more or less light into the viewing area. It is located beneath the stage of the microscope and can be adjusted to control the intensity and size of the light beam that passes through the specimen. This adjustment helps in achieving the desired illumination for observing the specimen.

27. Question 12

28. Explanation Most staining procedures destroy cells, cells are sometimes stabilised before staining. Osmic acid and, in particular, glutaraldehyde is often used as chemical fixatives. However, heat is the most widely employed fixative in light microscopy. Heat fixation is used to kill, adhere, and alter the specimen so it accepts stains. 

29. Question 13

30. Explanation

31. Question 14

32. Explanation The disadvantages of a scanning electron microscope start with the size and cost. SEMs are expensive, large and must be housed in an area free of any possible electric, magnetic or vibration interference. The maintenance involves keeping a steady voltage, currents to electromagnetic coils and circulation of cool water.

33. Question 15

34. Explanation In TEM, the image is obtained on a phosphorescent screen but in SEM the magnified image of the surface topography of the specimen is obtained on the cathode ray tube. The electronic signals generated scan the specimen in a raster pattern in the manner of a television system to produce an image on a cathode ray tube.

35. Question 16

36. Explanation Numerous techniques are available for use with electron microscopy which extends its usefulness in characterizing cellular structure.  Some of them are Negative-Staining (which increases the electron opacity of surrounding), Shadow Casting (helps in producing three-dimensional structure of the object), Ultrathin Sectioning and Freeze-Etching.

37. Question 17

38. Explanation SEM samples require little or no effort for sample preparation and can be directly imaged by mounting them on an aluminum stub. In contrast, TEM sample preparation is a quite complex and tedious procedure that only trained and experienced users can follow successfully.

39. TEM Sample Preparation

40. Question 18

41. Explanation  The primary function of the objective lens is to gather light from the specimen and form the initial magnified image. The objective lens has a short focal length and is positioned close to the specimen. It produces a magnified, inverted real image of the specimen. The eyepiece is responsible for further magnifying the real image formed by the objective lens. It has a longer focal length and is positioned close to the observer's eye. The eyepiece magnifies the real image produced by the objective lens, creating a virtual image that is seen by the observer. The magnification provided by the objective lens is generally higher than that of the eyepiece. For example, a typical objective lens might have a magnification of 10x, 20x, or higher, while the eyepiece commonly has a magnification of 10x. Therefore, the objective lens contributes more significantly to the overall magnification in a compound microscope.

42. Question 19

43. Explanation The maximum magnification of a compound microscope is roughly 1000x. This is a result of a maximum ocular magnification of 10x and a maximum objective lens magnification of 100x. Typical magnification ranges between 40x and 1000x to create two-dimensional images with a compound light microscope. 

44. Question 20

45. Explanation The tungsten hairpin was the source used for the first generation of electron microscopes and remains the most common source used to this day. Tungsten sources are relatively cheap, designed to be replaced quickly and easily during regular usage of the SEM. Of all metals in pure form, Tungsten has the highest melting point, the lowest vapor pressure, the lowest thermal expansion, and a very high tensile strength, which are all ideal properties for making an electron source. Tungsten filaments are commonly used in scanning electron microscopes (SEM) as electron sources.

46. Question 21

47. Explanation The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination.

48. Question 22

49. Explanation

50. The confocal microscope uses fluorescence optics. Instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. A laser beam is scanned across the sample to form the image, which is stored on a computer as a digital file. Confocal microscopy is a powerful tool for visualizing fluorescent specimens. The principal advantage of confocal microscopy over conventional wide-field microscopy is that it can reveal the three-dimensional structure of the specimen.

51. Question 23

52. Explanation Many fluorescent stains have been designed for a range of biological molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains such as DAPI and Hoechst (excited by UV wavelength light) and DRAQ5 and DRAQ7 (optimally excited by red light) which all bind the minor groove of DNA, thus labeling the nuclei of cells.  Major examples of nucleic acid stains like DAPI and Hoechst, phalloidin which is used to stain actin fibers in mammalian cells.

53. Question 24

54. Explanation

55. Question 25

56. Explanation

57. Thank you