microscope histo Flashcards
an instrument composed of several lenses used in magnifying objects too small to be seen by the naked eye
microscope
inventor of the microscope
zacharias jansen
improved the microscope; Father of Microbiology
Anton van Leeuwenhoek –
USES VISIBLE LIGHT FOR ILLUMINATION
light microscope
– uses visible light as light source; shows internal structure and outline of the transparent sheath
BRIGHT FIELD MICROSCOPE
- examination of live microorganisms not seen under an ordinary light microscope
- possess opaque disks
- suitable for structures that cannot be stained or easily distorted
- useful for detection of Spirochetes
DARKFIELD MICROSCOPE
- used to facilitate detailed examination of the internal structures of living specimens
- especially for transparent objects
PHASE CONTRAST MICROSCOPE
- Principle in phase contrast microscope
Light Changes its speed when passing through cellular and extracellular structures with different refractive indices
- produces images with a 3-D aspect as compared to phase-contrast microscopes
- objects appear bright against a dark background
DIFFERENTIAL INTERFERENCE MICROSCOPE
two types of DIFFERENTIAL INTERFERENCE MICROSCOPE
: Hoffman (modulation contrast)
Nomarski (differential contrast)
- Uses a strong UV Light Source and special filters that select rays of different wavelength
- Use of fluorescent compounds such as Acridine Orange, Rhodamine
FLUORESCENCE MICROSCOPY
Principle of FLUORESCENCE MICROSCOPY
: Substances are exposed to light (radiation) at a wavelength, these emit light with a longer wavelength which is visible
- more advantageous than regular bright field microscopes
- Uses small point of high intensity light (laser) and a pinhole aperture
CONFOCAL MICROSCOPE
- Advantage of CONFOCAL MICROSCOPE
avoids stray light; produces more resolution
- used for viewing highly organized molecules (crystals and lipids, cellulose, collagen, microtubules, microfilaments)
- uses the principle of polarization wherein light can be rotated in one direction or another (“birefringence”)
POLARIZING MICROSCOPE
Uses beams of electrons to produce a magnified image
Has greater resolving power as compared to light microscopes; Has higher resolution (1000fold increase)
electron microscopy
- Allows magnifications of up to 400,000 times
- Requires very thin sections (40-90nm)
- Freeze Fracture Techniques are usually combined in TEM
- Used to Examine Viruses or Internal UltraStructures of cells
- Structures appear as black/gray or white
TEM
- Studies the surface features of cells and viruses
- Provides a 3-D image of the surface
SCANNING ELECTRON MICROSCOPY
It is particularly useful for evidence not requiring very high magnification (10x–125x).
Its large working distance makes it quite applicable for the microscopic examination of big, bulky items.
Stereo/Dissecting Microscopes
: ability of the lenses to distinguish fine detail and structure
refers to the ability of the lenses to distinguish between two points a specified distance apart
Resolution
—— wavelength: the —— the resolution
shorter; greater
resolution is dependent on the —— used
Dependent on the Objective Used
ability of the microscope to distinguish/separate 2 points distinctly
Resolving Power:
a measure of the light bending ability of a medium (substances through which light passes)
Refractive Index
distance between the objective and object being focused (slide)
Working distance:
Magnification: Formula
magnification of ocular x objective magnification
function of the wavelength and characteristic of the lenses
gives the idea of how much light can enter the objective
Measures light gathering ability of the microscope
Numerical Aperture:
only slight adjustment is needed to focus the object being studied when the objective is shifted from one objective to another.
Parfocal:
used to preserve the direction of light rays at the highest magnification.
Immersion Oil:
See to it that the________objective is in position when the microscope is not in use
LPO
Use ————– in cleaning the objectives and stage
alcohol or xylol
Do not apply too much xylol for it may ___________
loosen the screws
Dry with ___________ immediately after application of xylol
soft tissue paper
Clean the______ first with soft tissue paper ;
OCULAR
see to it that the stage and objectives are free from __
OIL
Invented the compound microscope
zacharias jansen
optical system of bright field microscope
condenser
objective
eyepice
Resolution of TEM
2.5 nm
– collects and focuses a cone of light that illuminates the object to be observed
a. Condenser
Resolution of SEM
2.0 nm
enlarges and projects the image of the object in the direction of the eyepiece
b. Objective –
Pathway of electron of TEM
Passes thru the specimen
- further magnifies this image and projects it onto the viewer’s retina or a charge-coupled device
c. Eyepiece
Pathway of electron of SEM
Reflected by a metal
enlarges only the image obtained by the objective; does not improve resolution
-eyepiece
Magnification of stereo/dissecting microscope
10x-125x
-maximal resolving power:
~0.2 micrometer
Numerical aperture of scanning
0.10
-objects smaller than 0.2 micrometer ——-
cannot be distinguished
Numerical aperture of LPO
0.25
-2 structures will be seen as __________ if they are separated by less than 0.2 micrometer
only one object
Numerical aperture of HPO
0.65
principle – when certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength
*Fluorescence
Numerical aperture of OIO
1.25
Acridine Orange, DAPI, Hoechst stain
*Fluorescent compounds:
type of light microscope
Produces an image only of material having repetitive, periodic macromolecular structure; features without such structures are not seen
polarizing
-feature of crystalline substances or those with highly oriented molecules (cellulose, collagen, microtubules, actin filaments)
birefringence
-areas through which electron passed readily: appear _____
brighter or electron lucent
-areas where electrons were absorbed or deflected: appear _______
darker or more electron dense
- added to the fixative or dehydrating solutions
- used to improve contrast and resolution in TEM
*Compounds with heavy metal ions
-osmium tetroxide, lead citrate, uranyl compounds
*Compounds with heavy metal ions
techniques that allow TEM study of cells without fixation or embedding
Cryofracture and freeze etching –
– useful in the study of membrane structure
- very small tissue specimens are rapidly frozen in liquid nitrogen and either fractured or cut with a knife
- replica is produced
Cryofracture
Type of EM
surface of the specimen is first dried and spray coated with very thin layer of heavy metal (often gold) through which electrons do not pass readily
SEM
Type of EM
images are usually easy to interpret because they present a 3D view that appears to be illuminated from above
SEM
method of localizing newly synthesized macromolecules (DNA, RNA, protein, glycoproteins, polysaccharides)
AUTORADIOGRAPHY –
This will help protect the objective lenses if they touch the slide.
Coverslip or cover glass
When you move the slide to the right, the image goes to the
left!
number of times the image was enlarged
magnification
the higher the magnification the ________- working distance
LOWER
Side of mirror when using natural light
plane
side of mirror when using artificial light
concave
