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V. Micro 151 > 4 The Study of Microbial Structure: Microscopy > Flashcards

4 The Study of Microbial Structure: Microscopy Flashcards

1
Q

-are credited with creating one of the first compound microscopes around 1590.

-Their microscope used two lenses, an objective lens near the object and an eyepiece lens through which the user viewed the magnified image.

-This device could magnify objects up to 10x to 30x, which was revolutionary at the time.

A

Hans and Zacharias Janssen (Late 16th Century)

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2
Q

an Italian scientist, known
for his work in astronomy, also made significant
contributions to microscopy

A

Galileo Galilei (1609-1610)

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3
Q

a simple microscope of his own design, which was more of a powerful magnifying glass than a compound microscope, he was the first to observe and describe single-celled organisms, including bacteria, sperm cells, and blood cells.

A

Antonie van Leeuwenhoek (1670s-1720s)

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4
Q

an English scientist, published his
landmark book Micrographia in 1665.
He used an improved compound microscope to observe various objects, including a thin slice of cork, where he first described the “cells” that make up plant tissue

A

Robert Hooke (1665)

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5
Q

developed the first transmission electron microscope (TEM), allowing scientists to observe specimens at a much higher resolution than light microscopes.

A

Ernst Ruska and Max Knoll

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6
Q

invented the phase-contrast microscope, enabling the visualization of transparent biological specimens without staining

∙ 1981: The scanning tunneling microscope (STM) was developed, allowing for the imaging of surfaces at the atomic level.

A

Frits Zernike

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7
Q

Purpose of microscope (7)

A
  • Microscopy is the technology of making very small things visible to the human eye.
  • Used in the diagnosis of many diseases.
  • Used for soil, insects and rocks.
  • Used in pathological laboratories and medical.
  • Industrial microscope : Used for metals and various kinds of measurement .
  • Educational microscope.
  • Used for research.
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8
Q

*General Principles of Microscopy

refers to the distance between successive peaks of a wave, typically measured in nanometers (nm) for light waves. (visible light spectrum ranges from 400-700 nm)

A

Wavelength of radiation

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9
Q

*General Principles of Microscopy

  • is the ability of a microscope to
    enlarge the image of a specimen
A

Magnification

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10
Q

*General Principles of Microscopy

is the ability to distinguish between
two closely spaced objects as separate entities

A

Resolution

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11
Q

*General Principles of Microscopy

Differences in intensity between two objects, or between an object and background
– Important in determining resolution

A

Contrast

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12
Q

4 main parts of a microscope

A

– Frame work

– Adjustment system

– Magnification

– Lighting System

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13
Q

the framework of the microscope includes three parts:

A
  1. Base
  2. Arm
  3. Mechanical stage
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14
Q

● Stage - the location of the specimen to be viewed
● Clips - utilized in holding the specimen in place

which part of the framework can these be seen?

A

Mechanical Stage

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15
Q

*Stage Knobs control

is to move backward and forward

A

Top knob

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16
Q

*Stage Knobs control

is to move the stage right and left.

A

Bottom knob

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17
Q

Adjustment system consists of the
following parts:

A
  1. Optical tube.
  2. Coarse adjustment.
  3. Fine adjustment.
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18
Q

*Focus and Resolution Parts

is the larger of the two knobs. It is used in bringing the object into quick focus.

A

Course-adjustment knob

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19
Q

*Focus and Resolution Parts

is used for improving the clarity especially under high of the image, when viewing high power.

A

Fine-adjustment knob

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20
Q

To control the distance between the ocular lenses to adapt the distance between viewers eyes so, the eyepiece lenses will spread apart or get closer together to fit each individual.

A

Interpupillary adjustment

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21
Q
  • an adjustable component located on one or both eyepieces of a binocular microscope.
  • It allows the user to fine-tune the focus of one eyepiece independently of the other
A

Diopter ring adjustment

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22
Q

Microscope has two sets of lenses which are :

A
  1. Ocular lens (eyepiece).
  2. Objective lens.
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23
Q

are fundamental concepts in microscopy that significantly influence how images are formed and the quality of observations made through microscopes

A

Refraction

Refractive Index

24
Q

— distance between the front surface of lens and surface of cover glass or specimen

A

*working distance

25
Q

-measurement of the extent that the substance bend light.

– a measure of how greatly a substance slows the velocity of light

A

refractive index

26
Q

-bending

-light is refracted (bent) when passing from one medium to another

A

refraction

27
Q

Microscope lens that is used for viewing

A

Ocular lens or eyepiece

28
Q

contains objective lenses that are used to magnify the image in combination with the ocular lens.

A

Revolving nosepiece

29
Q

It is the ability of microscope, at specific magnification to distinguish two separate objects situated close to one another and the ability of the lens to reveal fine details.

*The smaller the distance between the two specific objects that can be distinguished apart, the greater the resolution power of the microscope.

A

Resolution

30
Q
  • As magnification increased the resolution also ____
  • To achieve high magnification with good resolution the objective lens must be ____

-Shorter wavelengths of light provide ____ resolution.

A

increased

small

greater

31
Q

This is a number that expresses the ability of a lens to resolve fine detail in an object being observed. It is derived by a complex mathematical formula and is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen.

A

Numerical Aperture (N.A.)

32
Q

Lighting system consists of modern microscopes of the following parts: (5)

A

i. Power supply

ii. Transformer

iii. light source

iv. sub stage condenser

v. Aperture diaphragm

33
Q
  • typically a light source underneath the stage
A

Lamp

34
Q

controls the amount of light allowed to pass through the specimen

A

Diaphragm

35
Q

Factors that influence the quality and size of image: (5)

A

● Quality of Microscope and lenses.

● Size of sample.

● Type of sample.

● Amount of light on the sample.

●Quality of sample

36
Q

Types of
Microscope

A
  1. Light Microscopes (LM)
  2. Electron Microscope (EM)
  3. Probe microscopes
37
Q

*Many types:
– Bright-field microscope
– Dark-field microscope
– Phase-contrast microscope
– Fluorescence microscope
– Confocal microscope
*are compound microscopes
– image formed by action of ≥2 lenses

A

The Light Microscope

38
Q

*The most common general use microscopes
*produces a dark image of the specimen against a brighter background
*has several objective lenses
– parfocal microscopes – image remain in focus when objectives are changed
*total magnification

-Simple design
-Light directed at specimen is absorbed to form image
-Unstained specimens have poor contrast
-Stained specimens show excellent contrast
-Ideal for stained bacteria, cells, tissues
-High N.A., good resolution
-Bright background, dark specimen
-tungsten or halogen light source

A

Bright-Field Microscope

39
Q

–Best for observing pale objects
–Only light rays scattered by specimen enter objective lens
–Specimen appears light against dark background
– Increases contrast and enables observation of more details
– for living, unstained cells and organisms
– for ID of spirochetes e.g Treponema pallidum, causative agent for syphilis; Leptospira, Borrelia

*The central portion of the light is blocked so that only oblique light strikes the specimen,

-A method from the 19th century
-Bright specimen, dark background
- Light not scattered bythe specimen bypasses the objective, therefore making the “field” dark.
-Can see very small objects but resolution is variable
-High contrast, good for unstained, live, and motile Specimens

A

Dark-field microscopes

40
Q

–Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining
– Light rays in phase produce brighter image, while light rays out of phase produce darker image
–Contrast is created because light waves are out of phase
–Two types::
–Phase-contrast microscope
–Differential interference contrast microscope

A

Phase-contrast microscopes

41
Q

-technology from 1940’s
-provides high contrast, good resolution
-good for bacteria, flagella, cilia, organelles such as mitochondria
-good for unstained or live mounts
-phase halos (artifacts) occur

A

Phase-contrast microscope

42
Q

*also called Normarski optics
*this is similar to the phase contrast microscope
*it creates an image by detecting differences in refractive indices and thickness
*Two beams of plane polarized light at right angles to each other are generated by prisms.
-produces 3-D images
-excellent resolution, high NA, high contrast
-good for unstained specimens, live mounts; can see membranes within cells

A

Differential Interference Contrast (DIC) Microscope

43
Q

–Direct UV light source at specimen
–Specimen radiates energy back as a longer, visible wavelength
–UV light increases resolution and contrast
–Some cells are naturally fluorescent; others must be stained
–Used in immunofluorescence to identify pathogens and to make visible a variety of proteins

-specimens are stained with dye molecules, fluorochromes, that fluoresce brightly upon exposure to light of a specific wavelength, but some microorganisms are autofluorescing.

A

Fluorescence microscopes

44
Q

–Use fluorescent dyes
–Use UV lasers to illuminate fluorescent chemicals in a single plane
–Resolution increased because emitted light passes through pinhole aperture
–Computer constructs 3-D image from digitized images

A

Confocal microscopes

45
Q
  • A laser is focused at a plane microscopy in the specimen and scans the specimen in a horizontal (XY) plane.
  • Only light from the plane of focus reaches the detector.
  • The scanned image (an optical section) is digitally recorded.
  • Images from consecutive focal planes can be recorded, and composite or 3-D images can be digitally created.

*The higher the magnification the less “depth of field” - only a narrow slice is in focus
*this icroscope gets around this problem
*Lasers scan the specimen at different depths
*A computer reconstructs a clear 3D image
*Usually used with fluorescent specimens
-krypton/argon laser
-high resolution, sharp image
-high sensitivity

A

Confocal Laser Scanning Microscope (CLSM)

46
Q

– have greater resolving power and magnification
– Magnifies objects 10,000X to 100,000X
– Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms
– Two types:: 1. Transmission Electron Microscope (TEM) 2.. Scanning Electron Microscope (SEM)

*beams of electrons are used to produce images
*wavelength of electron beam is much shorter than light, resulting in much higher resolution

A

Electron Microscopy

47
Q
  • Fixed, dehydrated specimens are mounted on stubs and surface-coated with gold, palladium or rhodium.
  • The specimen is placed in a vacuum and an electron beam scans back and forth over it.
  • An electron beam is “bounced off” the specimen to a detector, instead of being passed through it
  • Electrons that bounce off the metal-coated specimen surface are collected, converted to a digital image and displayed on a TV-like monitor.
  • It produces a detailed image of the surface of the specimen, but not its internal structure
A

Scanning Electron Microscope (SEM):

48
Q

– a powerful imaging technique that allows for the visualization and manipulation of surfaces at the nanoscale
– Magnifies more than 100,000,000 times
– Two types::
–Scanning tunneling microscopes (STM)
–Atomic force microscopes (AFM)

A

Probe Microscopy

49
Q

*used to visualize the electronic properties of surfaces, study surface defects, and investigate the arrangement of atoms in materials
– applications in nanotechnology, semiconductor research, and surface chemistry
– provides atomic resolution, allowing researchers to observe individual atoms and molecules on surfaces

A

Scanning Tunneling Microscopy (STM)

50
Q

*used to study a wide range of materials, including polymers, biological samples, and nanostructures
– It provides high-resolution images and can operate in various environments, including air, vacuum, and liquid
– this microscope typically has lower resolution than STM, although it can still achieve nanometer-scale imaging

A

Atomic Force Microscopy (AFM)

51
Q

How to use Microscope (7)

A
  1. Plug in microscope and turn on illuminator. Rotate nosepiece to lock 4X objective in place
  2. Place smear on stage and center it under the 4X objective.
  3. Using the course adjustment knob, move the objective lens to its lowest point. Look through the ocular and focus upward with the coarse adjustment until an image comes into view.
  4. Rotate nosepiece to obtain the next objective lens 10x and repeat step 3.
  5. Rotate nosepiece to obtain the next objective lens 40x .
  6. Look through the ocular and focus upward with the fine adjustment until an image comes into view.
  7. When using oil immersion lens put a drop of oil on the slide and rotate nosepiece and repeat step 6
52
Q

Microscope Cleaning Process (5)

A
  • Cleaning the Eyepiece,
  • Cleaning the Objectives,
  • Cleaning the Microscope Stage,
  • Cleaning the Microscope Body,
  • Cleaning the Condenser
53
Q

Cleaning Solutions and Solvents

A
  • Soap solution for cleaning of body and stage.
  • Ether-Alcohol, Alcohol, or Lens Cleaner Solution for cleaning of lenses.

organic sovents

54
Q

Cleaning Materials (4)

A
  • Lint-free cotton gauze pads
  • Lint-free cotton swabs
  • Lens paper
  • Commercial lens tissue for optics

Caution: Do not use paper towels or other rough paper products

55
Q

alternative for the cleaning materials (3)

A

– Fine quality tissue paper
– Muslin cloth
– Silk

V. Micro 151 (10 decks)

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