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Flashcards in microscope histo Deck (72):
1

an instrument composed of several lenses used in magnifying objects too small to be seen by the naked eye

microscope

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inventor of the microscope

zacharias jansen

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improved the microscope; Father of Microbiology

Anton van Leeuwenhoek –

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USES VISIBLE LIGHT FOR ILLUMINATION

light microscope

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– uses visible light as light source; shows internal structure and outline of the transparent sheath

BRIGHT FIELD MICROSCOPE

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- 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

7


- used to facilitate detailed examination of the internal structures of living specimens
-especially for transparent objects

PHASE CONTRAST MICROSCOPE

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- Principle in phase contrast microscope

Light Changes its speed when passing through cellular and extracellular structures with different refractive indices

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- produces images with a 3-D aspect as compared to phase-contrast microscopes
- objects appear bright against a dark background

DIFFERENTIAL INTERFERENCE MICROSCOPE

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two types of DIFFERENTIAL INTERFERENCE MICROSCOPE

: Hoffman (modulation contrast)
Nomarski (differential contrast)

11


-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

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Principle of FLUORESCENCE MICROSCOPY

: Substances are exposed to light (radiation) at a wavelength, these emit light with a longer wavelength which is visible

13

- more advantageous than regular bright field microscopes
-Uses small point of high intensity light (laser) and a pinhole aperture

CONFOCAL MICROSCOPE

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- Advantage of CONFOCAL MICROSCOPE

avoids stray light; produces more resolution

15

- 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

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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

17

-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

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- Studies the surface features of cells and viruses
- Provides a 3-D image of the surface

SCANNING ELECTRON MICROSCOPY

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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

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: 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

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------ wavelength: the ------ the resolution

shorter; greater

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resolution is dependent on the ------ used

Dependent on the Objective Used

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ability of the microscope to distinguish/separate 2 points distinctly

Resolving Power:

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a measure of the light bending ability of a medium (substances through which light passes)

Refractive Index

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distance between the objective and object being focused (slide)

Working distance:

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Magnification: Formula

magnification of ocular x objective magnification

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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:

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only slight adjustment is needed to focus the object being studied when the objective is shifted from one objective to another.

Parfocal:

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used to preserve the direction of light rays at the highest magnification.

Immersion Oil:

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See to it that the________objective is in position when the microscope is not in use

LPO

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Use -------------- in cleaning the objectives and stage

alcohol or xylol

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Do not apply too much xylol for it may ___________

loosen the screws

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Dry with ___________ immediately after application of xylol

soft tissue paper

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Clean the______ first with soft tissue paper ;

OCULAR

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Invented the compound microscope

zacharias jansen

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Resolution of TEM

2.5 nm

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Resolution of SEM

2.0 nm

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Pathway of electron of TEM

Passes thru the specimen

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Pathway of electron of SEM

Reflected by a metal

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Magnification of stereo/dissecting microscope

10x-125x

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Numerical aperture of scanning

0.10

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Numerical aperture of LPO

0.25

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Numerical aperture of HPO

0.65

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Numerical aperture of OIO

1.25

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Acridine Orange, DAPI, Hoechst stain

*Fluorescent compounds:

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type of light microscope
Produces an image only of material having repetitive, periodic macromolecular structure; features without such structures are not seen

polarizing

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-feature of crystalline substances or those with highly oriented molecules (cellulose, collagen, microtubules, actin filaments)

birefringence

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-areas through which electron passed readily: appear _____

brighter or electron lucent

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-areas where electrons were absorbed or deflected: appear _______

darker or more electron dense

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- added to the fixative or dehydrating solutions
-used to improve contrast and resolution in TEM

*Compounds with heavy metal ions

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-osmium tetroxide, lead citrate, uranyl compounds

*Compounds with heavy metal ions

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techniques that allow TEM study of cells without fixation or embedding

Cryofracture and freeze etching –

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– 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

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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

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Type of EM
images are usually easy to interpret because they present a 3D view that appears to be illuminated from above

SEM

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method of localizing newly synthesized macromolecules (DNA, RNA, protein, glycoproteins, polysaccharides)

AUTORADIOGRAPHY –

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This will help protect the objective lenses if they touch the slide.

Coverslip or cover glass

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When you move the slide to the right, the image goes to the

left!

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number of times the image was enlarged

magnification

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the higher the magnification the ________- working distance

LOWER

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Side of mirror when using natural light

plane

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side of mirror when using artificial light

concave

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principle – when certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength

*Fluorescence

64

-2 structures will be seen as __________ if they are separated by less than 0.2 micrometer

only one object

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-objects smaller than 0.2 micrometer -------

cannot be distinguished

66

-maximal resolving power:

~0.2 micrometer

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enlarges only the image obtained by the objective; does not improve resolution

-eyepiece

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- further magnifies this image and projects it onto the viewer’s retina or a charge-coupled device

c. Eyepiece

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enlarges and projects the image of the object in the direction of the eyepiece

b. Objective –

70

– collects and focuses a cone of light that illuminates the object to be observed

a. Condenser

71

optical system of bright field microscope

condenser
objective
eyepice

72

see to it that the stage and objectives are free from __

OIL

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