Download parts of microscope

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Al Iraqia university
College of medicine
Y1 L1
Biology
Prof. Dr. Samia Al shahwani
7 -11- 2016
Light microscope L.M.
Objectives; by the end of this session the student should be able to:
Recognize importance of microscopy for biology.
Recognize terminology concerning microscopy.
Identify microscope parts & their function
Recognize microscope types.
Microscopy; Science of investigating small objects using microscope.
Microscopic means invisible to eye unless aided by a microscope.
Microscopy development revolutionized biology.
All biology branches use microscopy; let us have a look at biology
Biology, named the doctrine of life (Life Sciences):The scientific study of
all organisms, (from sub atomic structure in a single cell & life origin to
human & entire ecosystem), regarding, morphology (structure & shape),
classification (taxonomy), genetics, physiology (function), Anatomy
(structure, form &function), growth, distribution, evolution, origin &
ecology. It is a base level study that leads to different areas. Scientists,
students, use microscopy to study, organisms the world over.
Studied living beings are classified, & given a scientific name it is
obtained from the genus & species. e.g. human beings belongs to:
 Domain; Eukaryote
 Kingdom; Animalia
 Phylum; Chordates
 Sub- phylum; Vertebrates
 Class;
Mammals
 Order; Primates
 Genus: Homo
 Species: Sapiens
Scientific name is Homo sapiens, Homo is the genus & sapiens is species.
It must be written in Italic or underlined & the first letter capital.
Biology is very broad field, it includes disciplines & sub disciplines,
biology Layer cake may be divided “horizontally" to "basic “divisions
(fig1) concerned with fundamentals common to all life, not restricted to a
particular organism, e.g., molecular biology, cell biology, genetics,
histology, physiology, anatomy, morphology, ecology, evolution…etc. &
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may be divided vertically to taxonomic divisions which deals with basic
being studied: e.g. Zoology, Entomology, botany, human biology…. etc.
Also biology sub disciplines are recognized on bases of the scale at which
the organism are studied & methods used to study them
Life is studied over a wide range of scales, at levels of:
-Atomic & molecular scale: molecular biology, biochemistry, mol.genetics
 The cell: cell biology.
 Multi cellular: anatomy, physiology, & histology.
 Individual organism development: developmental biology.
 More than one organism: genetics.
 Entire population: population genetics.
 Interdependent populations & their habitats: ecology.
L.M. is useful tool & essential technique in life sciences, Most common
microscope (first invented) is optical or L.M. it furthered medical
research, helped to solve crimes, unlock secrets of microscopic world.
Enjoy learning microscopy, in this microscopic trip.
Microscope; tool magnify image of very small organisms & ‘resolves’ it
better. Microscopy, gave rise to histology, the common procedure to study
tissue samples, is preparation to be studied using a microscope by,
embedding in supporting medium, sectioning (cut to thin sections by
microtome) & fixation which makes cells permeable to staining. Specific
molecule (e.g. Protein) seen by fluorescence microscopy using fluorescent
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dye. Living cells seen in phase-contrast. Fixed cells (dead) examined by
L.M. images analyzed by Electronic M. (TEM & SEM). 3-D objects
imaging is possible with con-focal scanning L.M.
Microscopy branches: light, Electron M. & Scanning probe. types (table1)
First L.M. constructed by Anton Van Leeuwenhoek, Father of microscopy
(1632-1723). It consisted of single biconvex lens fitted in window of
“board " the object viewed through, it is referred as simple, with it he
discovered microorganisms thereby founding microbiology & provided
basis of disease germ theory. Compound M was developed using 2 lenses.
Microscope types are based on, principle to make image, application area,
cost, structure, versatility or other aspect.
Microscopic biology began1665, Robert Hooke (1635-1703) discovered
organisms are made of cells, Matthias Schleiden &Theodor Schwann,
recognized similarities between plant & animal cells & further expanded
cell study 1830s. In early 20th century alternative to L.M. was developed,
Ernst Ruska developed first Electron M. in 1931, transmission (TEM),
works on same principle as L.M. but uses electrons instead of light (this
allow higher resolution) & electromagnets instead of glass lenses. TEM
followed 1935 by scanning (SEM) development by Max Knoll.
Microscope improvements continue, newer & newer microscopes are
designed & still being developed.
Terminology concerning microscopy:
Resolution: is the feature which makes it possible to differentiate between
2 points close together in the objects being viewed, it is the magnifying
power, or the smallest distance between two objects so that they still
appear distinctly separate in the microscope picture
Magnification: Ratio of image size to viewed object, or factor by which
an image is enlarged, different according to M type.
Magnification power x = magnification of, objective lens × eye lens
Resolving Power: The smallest distance between 2 particles at which they
can be seen as separate objects. Maximal resolving power of L.M. is
about 0.2 (µm) it permits good images magnified 1000-1500 times.
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*The critical factor in obtaining a crisp detailed image with L. M. its
resolving power. ability to, show 2 closely lying points as 2 distinct
points, or perceive 2 points near each other as distinct points e.g. If 2
points are really close to each other, you might see them as a single point
rather than 2. However with high resolving power you will see them as 2
points. Resolution/magnification if without good resolving power will just
cause the image to become blurred. Resolution, resolving power, & limit
of resolution all mean the same thing. They refer to the ability of a
microscope to distinguish between two objects.
Measurements seen by eye, light microscope, electron microscope
Logarithmic Scale of Cell Measurement
Light microscope (L.M)
Parts & use
Simple M.: 1lens, original design, does not provide high magnification
Compound L M; 2 lens systems, lens to collect light from sample
(objective) & lens to focus light to eye or camera, ocular (eye piece).
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Maximum magnification 1000x. C. L.M. Are expensive, larger, heavier
due to increased lenses number, makes more advanced illumination
setups, as contrast, furthered medical researches, and helped to solve
crimes, unlock secrets of microscopic world. Essentially a compound
light microscope consists of basic structural (head, base & arm)
&optical components.
L.M . Components
1. Optical system; eye piece, revolving nose piece& objective lenses
2. Mechanical system; stage, control knobs, coarse & fine, arm.
3. Illumination systems. Lamp, condenser
Depending on manufacturer, there are different parts but basic parts,
present in every microscope, are nine:1-Eyepiece.
2-Turret
3-Objective lenses
4-Focus knobs
5-Stage
6-Illuminator
7-Condenser
8-Diaphragm
9-Base
Further will be described parts of microscope used in this lab, that
you should be familiar with. Fig 1
- Ocular lens (eyepiece): Portion, lens at the beginning, where the user
looks through, a cylinder contains 2 or more lenses. Its function to bring
image into focus for eye. According to number of eyepiece lenses, there
are monocular, binocular or trinocular microscopes. Simple M, has 1,
while compound have binocular, ocular lens is 10x in magnification
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-Diopter Adjustment: to change focus on one eyepiece so as to correct
any difference in vision between the two eyes.
-Body tube (Head): Connects the eyepiece to objective lenses. It houses
optical parts in upper part of microscope
-Revolving nose piece or turret or Revolver; rotating part where objective
lens are installed& allows switching between them
Fig.1 Binuclear compound light microscope
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-Objective
lenses: close to sample, in cylinder housing containing glass
single or multi-element compound lens. There are multiple objectives of
varying magnifications, its function to collect light from sample;
1.
2.
3.
4.
Very or extra, low power or scanning 4x
Low power objective
10 x
High power objective
40x
Oil immersion objective lens
100×; used for greater resolution
at high magnification. With immersion oil & cover slip between
objective lens &sample. larger numerical aperture allows collection of
more light making detailed observation of smaller details possible.
-Mechanical stage: Square platform with hole (stage aperture) in center,
below objectives to place slides on, moves in 4 directions by rotating
stage control knobs. Y (top) to move stage forward - back, and X move
stage from right- left,
the stage provided with metal clips that hold
slide in place. Light passes to illuminate specimen through the aperture,
-Coarse adjustment: large on side, arm, move stage up & down, to find
specimen & roughly focus.Always begin focusing with coarse adjustment
-Fine adjustment; small, its function to focus specifically on the specimen
Frame: Optical assembly attached to rigid arm in turn to a robust U
shaped foot to provide rigidity.
- Arm: Handle or portion, connects body tube, eye piece (head) to the
base. It supports microscope head, & used to carry the microscope.
-Base or foot; Structure on which microscope stands, or mechanism that
keeps all parts together. It supports microscope & houses the illuminator.
-On/off switch: On microscope base it turns illuminator off /on.
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-Illuminator; Lamp is integral base part, points up toward stage, sources,
light or mirror: Many, daylight directed via mirror. Most have controlled
light source halogen lamp, function; provides light for image viewing.
-Condenser; part of illumination system below stage, lever projects to
change iris diaphragm, part or lens for collecting focusing light from
illuminator to sample, it may also include other features, as Diaphragm
&/or filters: to manage the quality and intensity of the illumination
The diaphragm is under stage, allow to vary light, shining on image. It
regulates the amount of light reaching the sample.
Lenses & Light Bending
Light is refracted (bent) when passing from one medium to another.
Focus light rays at a specific place called the focal point.
Distance between center of lens and focal point is the focal length.
Short focal length more magnification
How Does a Microscope Works?
-All parts of microscope work together - light from illuminator passes
through, aperture, slide, objectives, where the image of the specimen is
magnified. Magnified image continues up through body tube to eyepiece,
which further magnifies image the viewer then see. Next step
- Use & adjust microscope
- Practice cleaning microscope.
The parts of microscope work together in hospitals & forensic labs for,
scientists, students, bacteriologists, biologists so that they view, bacteria,
plant & animal cells, tissues, various microorganisms the world over.
- All microscopes are similar in the way lenses work.
-Magnification is a function of the number of lenses.
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-Resolution is a function of the ability of a lens to gather light.
-Apertures used to affect resolution & depth of field.
Contrast & Illumination
-Brightness contrast arises from different degrees of light absorption at
different points in the specimen. Color contrast can also arise from
absorption when degree of absorption depends on wavelength & varies
from point to point in specimen.
-Phase contrast arises from a shift in the phase of light as a result of
interaction with the specimen.
-Polarization-dependent phase contrast arises when phase shift depends on
plane of polarization of incident light.
- Fluorescence contrast arises when the incident light is absorbed and
partially reemitted at a different wavelength.
Basic L.M. principles.
Magnification power, resolution, contrasts.
Contrast; is the differences in light intensity, measured according to
If contrast Zero you cannot see the sample
If contrast equals a positive amount you can see sample
If contrast equals negative amount you cannot see sample
Table1Descripe microscopes within optical, electron, &scanning probe.
Category
Light
(Optical)
Type
Description
Binocular
stereoscopic
Allows easy observation of 3D
objects at low magnification.
Bright field
microscope
TypicalM. Use transmitted light to
observe at high magnification
Polarizing
microscope
Use different light transmission
characteristics of materials, as
crystalline structures, for image
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Phase contrast
Visualizes surface irregularity
using light interference,observe
living cells without staining
Differential
interference
contrast M.
Similar to phase contrast, used to
observe minute surface
irregularities at a higher resolution.
However, use of polarized light
limits variety of observable
specimen containers.
Fluorescence
microscope
Biological.M. observes
fluorescence emitted by samples
by light sources as mercury lamps.
When combined with additional
equipment, bright field microscope
can also perform fluorescence
imaging.
Total internal
reflection
fluorescence
microscope
Fluorescence M. use evanescent
wave to illuminate near specimen
surface. Region viewed is v. thin
compared to conventional
M.observ. is possible in molecular
units due to reduced background
light.
Laser
microscop
(Laser scanning
Clear observation of thick samples
with different focal distances.
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confocal)
Multiphoton
excitation
microscope
The use of multiple excitation
lasers reduce damage to cells &
allows high-resolution, observation
of deep areas. This microscope
used to observe nerve cells &
blood flow in brain.
Structured
illumination M.
A high-resolution M.with
advanced technology to overcome
limited resolution found in LM
caused by the diffraction of light.
Electron
microscope
EM
Transmission
EM(TEM
-Scanning EM
(SEM)
-TEM was first developed
Emit electron beams, not light
toward targets to magnify them.
Scanning
probe M.
(SPM)
Atomic Force
M(AFM)
-Scanning Nearfield Optical
SNOM
Scans sample surface with a probe
& this interaction is used to
measure fine surface shapes or
properties.
Others
-X-ray
ultrasonic
In addition to the above, Light microscopes can be classified as follows:
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Classification by application
-Magnificatio n (50x - 1,500x)
Biological microscope
- Use sliced samples that are fixed onto
slides for observation.
Allow 3D observation of samples, as
insects or minerals, in their natural state
without need to be sliced.
(Binocular) stereoscopic
microscope
- Magnification (10x to 50x)
Classification by structure
-Observes target from above
Upright microscope -Used to observe specimen on slide
-Observes target from below
Inverted microscope -Used to observe, e.g., cells soaked with culture in a
dish.
L.M Applications:
-Microelectronics
- Microbiology
-Nanophysics
- Medical diagnosis
-Biotechnology
-Smear test
-Pharmaceutical research
- Industrial
-Mineralogy
-Histopathology
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Thank you…. Samia