UNIT II C- PRINCIPLES OF MICROSCOPY Flashcards
1
Q
Electron Microscopy
A
Electron beam replaces
light to form the image
2
Q
Fluorescence
Microscopy
A
Fluorescent materials
emit visible light when
they are irradiated with
ultraviolet (UV rays)
3
Q
Immune Electron
Microscopy
A
Electron microscopy of
biological specimens to which a specific antibody
has been bound
4
Q
Immunofluorescence
Microscopy
A
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
5
Q
Nomarski
Microscopy
A
An unusual type of microscopy requiring a special optical system, the Nomarski optics, to do “differential interference contrast microscopy”
6
Q
Time-lapse
Microscopy
A
The same object is photographed at regular intervals over time to, for example, observe a cell go through division
7
Q
Stereo microscope
A
- 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
8
Q
Digital microscope
A
- 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
9
Q
USB computer microscope
A
- 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
10
Q
Pocket microscope
A
- 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
11
Q
Electron microscope
A
- 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
12
Q
Scanning probe microscope
A
- 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
13
Q
Acoustic micrsoscope
A
- 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
14
Q
PREPARING SPECIMENS FOR MICROSCOPIC
EXAMINATION: Light microscopy: 1.Wet mounts
A
- 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
15
Q
PREPARING SPECIMENS FOR MICROSCOPIC
EXAMINATION: Light microscopy: 2. fixed specimens
A
- 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
16
Q
PREPARING SPECIMENS FOR MICROSCOPIC
EXAMINATION: Light microscopy: 3. Sectioning
A
- 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
17
Q
PREPARING SPECIMENS FOR MICROSCOPIC
EXAMINATION: Light microscopy: 4. Staining and immunolabeling
A
- 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)
18
Q
PREPARING SPECIMENS FOR MICROSCOPIC
EXAMINATION: Light microscopy: 5. mounting
A
- 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.
