UNIT II C- PRINCIPLES OF MICROSCOPY

Question 1

Electron Microscopy

Answer 1

Electron beam replaces

light to form the image

Question 2

Fluorescence

Microscopy

Answer 2

Fluorescent materials
emit visible light when
they are irradiated with
ultraviolet (UV rays)

Question 3

Immune Electron

Microscopy

Answer 3

Electron microscopy of
biological specimens to which a specific antibody
has been bound

Question 4

Immunofluorescence

Microscopy

Answer 4

Antibodies labeled with a 
fluorescing substance and 
a fluorescence 
microscope to detect the 
binding of the antibody 
through the production of 
a characteristic visible 
light under UV light

Question 5

Nomarski

Microscopy

Answer 5

An unusual type of 
microscopy requiring a 
special optical system, the 
Nomarski optics, to do 
“differential interference 
contrast microscopy”

Question 6

Time-lapse

Microscopy

Answer 6

The same object is 
photographed at regular 
intervals over time to, for 
example, observe a cell 
go through division

Question 7

Stereo microscope

Answer 7

• Dissecting Microscope
• has two optical paths at slightly different
  angles allowing the image to be viewed
  three-dimensionally under the lenses
• magnify at low power, typically between 10X
  and 200X, generally below 100x
• either fixed or zoom variety; inexpensive
  Uses: Looking at surfaces, microsurgery watchmaking, building and expecting circuit boards
• can be used to view almost anything fitted

Question 8

Digital microscope

Answer 8

• microscope invented in Japan in 1986
• uses the power of the computer to view
  objects not visible to the naked eye
• this kind can be found with or without
  eyepieces to peer into
• connects to a computer via USB cable
• the computer software allows the display of
  magnified specimen
• moving images and single images can be
  recorded
  Advantage: ability to email images, comfortably
  watch moving images for long periods

Question 9

USB computer microscope

Answer 9

• not well-suited to the same scientific
  applications as other light microscopes
• used on almost any object and requires no
  preparation of the specimen
• essentially a macro lens used to examine
  images on a computer screen plugged into
  its USB port
• magnification is restricted at only 200X with
  a relatively small depth of field
• can be for kids and students
• inexpensive

Question 10

Pocket microscope

Answer 10

• small and durable
• for hand-held imaging of a variety of
  specimens/objects in the field or in the
  laboratory
• portable with a magnification ranging from
  25x to 100x

Question 11

Electron microscope

Answer 11

• a powerful microscope available in use
  today
• to view a specimen at nanometer size
  1. Transmission Electron Microscope (TEM)
• the first type of EM
• is capable of producing images 1 nanometer
  in size
• a popular choice for nanotechnology and
  semi-conductor analysis and production
  2. Scanning Electron microscope (SEM)
• are approximately 10 times less powerful
  than TEMs
• produce high-resolution, sharp, black and
  white 3D images
  Practical Applications:
  • Biology, Chemistry, and other science fields
  • To provide information on the topography,
  morphology, composition, and crystal
  graphic data of sample

Question 12

Scanning probe microscope

Answer 12

• used in academic and industrial setting,
  sectors involving physics, biology and
  chemistry
• used in research and development as
  standard analysis tools
• Images are highly magnified
• observed as three-dimensional-shapedspecimens in real time
• employ a delicate probe to scan the surface
  of the specimen eliminating the limitations
  that are found in electron and light
  microscopy

Question 13

Acoustic micrsoscope

Answer 13

• less about resolution
• more about finding faults, cracks or errors
  from samples during the manufacturing
  process
• Scanning acoustic microscopy, or SAM,
  is the most current type of acoustic
  microscopy
• view a sample internally without staining it or
  causing it any damage

Question 14

PREPARING SPECIMENS FOR MICROSCOPIC

EXAMINATION: Light microscopy: 1.Wet mounts

Answer 14

• simplest type of preparation
• specimen is placed on the slide in a drop of
  liquid
• some specimens in a drop of urine are
  already in a liquid form can be deposited
  on the slide immediately using a dropper
• solid specimens, such as skin scraping,
  can be placed on the slide before adding a
  drop if liquid to prepare the wet mount
• liquids used can be water but often stains
  are added to enhance contrast
• once the liquid has been added to the slide,
  a cover slip is placed on top and the
  specimen is examined under the
  microscope

Question 15

PREPARING SPECIMENS FOR MICROSCOPIC

EXAMINATION: Light microscopy: 2. fixed specimens

Answer 15

• attaching the specimen to the slide, kills
  microorganisms in the specimen, stopping
  their movement and metabolism while
  preserving the integrity of their cellular
  components for observation
• Fixation is often achieved by heat-fixing,
  chemically treating the specimen
  For example: A preparation of smear

Question 16

PREPARING SPECIMENS FOR MICROSCOPIC

EXAMINATION: Light microscopy: 3. Sectioning

Answer 16

• Used to cut and cross-section the sample
• Performed using microtomy or cryotomy
• Cut them in thin cross-section to observe
  every detail in the sample
• important step for the preparation of slides
  as it ensures a proper observation of the
  sample by microscopy
  • Paraffin-embedded samples are cut by
  cross section, using a microtome
  • Frozen samples are cut using a cryostat

Question 17

PREPARING SPECIMENS FOR MICROSCOPIC

EXAMINATION: Light microscopy: 4. Staining and immunolabeling

Answer 17

• Staining increases contrasts in order to
  recognize and differentiate the different
  components of the biological material. The
  sample is first deparaffinized and
  rehydrated so that polar dyes can
  impregnate the tissues. The different dyes
  can thus interact with the components to be
  stained according to their affinities. Once
  staining is completed, the slide is rinsed and
  dehydrated for the mounting step.
• Immunolabeling detects the particular cell
  and tissue components (antigens) by using
  polyclonal or monoclonal antibodies. A
  solution, which contains the antibody, is
  deposited onto the sample, which is then
  washed in order to remove the unbound
  antibodies. Direct or indirect approach
  can be used by using antibodies coupled
  with fluorochromes (immunofluorescence)
  or by using enzymes that react to
  chromogens (immunochemistry)

Question 18

PREPARING SPECIMENS FOR MICROSCOPIC

EXAMINATION: Light microscopy: 5. mounting

Answer 18

- Sections are mounted between slides and 
coverslips using a product to ensure 
adhesion. The slides are then ready for 
storage or observation.
- For fluorescence microscopy 
observations, a mounting medium is used 
in order to temporarily decrease the 
fluorescence loss.