Microscopy & Resolution FOR... · • •OU NanoLab/NSF NUE/Bumm & Johnson. The History •Hans and...
Transcript of Microscopy & Resolution FOR... · • •OU NanoLab/NSF NUE/Bumm & Johnson. The History •Hans and...
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Microscope
Dr. Leena Barhate
Department of Microbiology
M.J.College, Jalgaon
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Acknowledgement
• http://www.cerebromente.org.br/n17/histor
y/neurons1_i.htm
• Google Images
• http://science.howstuffworks.com/light-
microscope1.htm
• www.worldofteaching.com
• OU NanoLab/NSF NUE/Bumm & Johnson
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The History
• Hans and Zacharias Janssen of Holland
in the 1590’s created the “first”
compound microscope
• Anthony van Leeuwenhoek and Robert
Hooke made improvements by working
on the lenses
Anthony van Leeuwenhoek
1632-1723
Robert Hooke
1635-1703 Hooke Microscope
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Light Microscope
• Microscope utilizing light as a source of
illumination is called as light microscopes.
• Type of light microscopes
– Bright field microscope
– Dark field microscope
– Phase contrast microscope
– Fluorescence microscope
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Bright field microscope
• Microscope that forms dark image against
bright background is called as bright field
microscope
• Widely used bright field microscope for
basic study of microbes is the compound
microscope
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How a Microscope Works
Convex Lenses are
curved glass used to
make microscopes
(and glasses etc.)
Convex Lenses bend
light and focus it in
one spot.
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The History
Zacharias Jansen
1588-1631
The “First” Microscope
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How a Microscope Works
Ocular Lens
(Magnifies Image)Objective Lens
(Gathers Light,
Magnifies
And Focuses Image
Inside Body Tube)Body Tube
(Image Focuses)
•Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again).
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Body Tube
Nose Piece
Objective
Lenses
Stage
Clips
Diaphragm
Light Source
Ocular Lens
Arm
Stage
Coarse Adj.
Fine Adjustment
Base
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Body Tube
• The body tube holds the objective lenses
and the ocular lens at the proper distance
Diagram
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• It holds occular and objectives are lenses.
• It also provides sufficient space for image
formation
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Nose Piece
• The Nose Piece holds the objective lenses
and can be turned to increase the
magnification
Diagram
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• A base in which objectives are fixed
• Simply rotating the nosepiece can rotate
each objective into place
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Objective Lenses
• The Objective Lenses increase
magnification (usually from 10x to 100x)
Diagram
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• Second lens system of a microscope
• Mounted on nosepiece and can be rotated into
the place
• There are usually three objective lenses on a
microscope.
• Objective lenses are generally equipped with
microscope having low power, high power and
oil immersion lens and magnification of 10x, 45x
and 100x respectively
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• Function: to make real image
• Working distance: it is the distance
between objective and object under
observation
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Stage Clips
• These 2 clips hold the slide/specimen in
place on the stage.
Diagram
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Iris Diaphragm
• The Diaphragm controls the amount of
light on the slide/specimen
Turn to let more light in or to
make dimmer.
Diagram
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• It is equipped with condenser
• Control intensity of light condenser and
therefore controls the amount of light
intensity.
• Lever is equipped with it to adjust the light
intensity.
• Blue colour filter is also equipped below
the condenser
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Condenser
• It is third lens system of microscope
• It is located below the stage
• It is responsible for focusing the light on
the specimen
• There are several different types of
condensers depending upon the type of
microscope to be employed
• Abbe’s condenser is most commonly used
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Light Source (Illuminator)
• Projects light upwards through the
diaphragm, the specimen and the lenses
• Some have lights, others have mirrors
where you must move the mirror to reflect
light
Diagram
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illumination
• Abbe and Nelson
– In this system light source, such as sun light
through a window, or an open lamp flame is
placed before the microscope mirror.
– Any structure or irregularity of the source is
seen directly in the field of view.
– It creates a problem to some extent during
examination of the specimen
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• Koelhler’s type:
– It is second ,method of illumination
– Prepared by Dr. August Koehler.
– It eliminated field of view
– The koehler form of illumination is mostly
used today
– Here parallel rays of light generated usually
by a tungsten filament lamp are used to
illuminated the specimen
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Ocular Lens/Eyepiece
• Magnifies the specimen image
Diagram
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• It is the first lens system of microscope.
• It is present at top of the microscope
• In the microscope, the occular is capable
of 10x magnifications
• Eyepieces of 5x, 15x and 20x
magnification potential are also available.
• One eyepiece can be replaced by another
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• Function: to make virtual image of
specimen
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Arm
• Used to support the microscope when
carried. Holds the body tube, nose piece
and objective lenses
Diagram
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Stage
• Supports the slide/specimen
Diagram
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• It is the platform on which the specimen to
be viewed is placed.
• Some stages have clips to hold the glass
slide in place.
• Others have a mechanical stage, which
make it possible to move the slide across
the stage in both horizontal and vertical
directions
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Coarse Adjustment Knob
• Moves the stage up and down (quickly) for
focusing your image
Diagram
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• They are used to move the body
tube/stage relative to the objectives and
occular, making it possible to focus the
image
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Fine Adjustment Knob
• This knob moves the stage SLIGHTLY to
sharpen the image
Diagram
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Base
• Supports and stabilizes the microscope
Diagram
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Types of Microscopes:
1. Compound Light Microscope (what we
use most often)
2. Stereoscopes – also known as dissecting
scopes
3. Electron Microscopes
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Parts of the Microscope
Arm
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Parts of the Microscope
Light Source
Diaphragm
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Parts of the Microscope
Stage
Stage Clips
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Parts of the Microscope
Revolving
Nosepiece
Objective
Lenses
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Parts of the Microscope
Ocular Lens
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Parts of the Microscope
Coarse adjustment knob
Used only when low power objective is used!!
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Parts of the Microscope
Fine adjustment knob
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Carrying a Microscope
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Steps to Use:
1. Rotate the low power objective into place and make sure the
stage is all the way down.
2. Place slide on stage making sure object to be viewed is
centered over the hole in the stage. Use the stage clips to
hold the slide in place.
3. Turn light on.
4. Focus first with the coarse adjustment knob. Once in focus on
low power, turn the nosepiece until the next higher lens is in
place.
5. Use FINE adjustment knob ONLY and focus the object.
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Remember:
1. If you are seeing perfectly round, clear circles then you
just may be looking at air bubbles. Check your slide and
try again.
2. Microscopes must always be properly put away.
3. Slides and cover-slips should be washed, dried, and
returned to their proper place.
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Important Vocabulary :
magnification \mag-ne-fe-'ka-shen\ n 1.
apparent enlargement of an object 2.
the ratio of image size to actual size
A magnification of "100x" means
that the image is 100 times bigger
than the actual object.
resolution \rez-e-loo-shen\ n 1. clarity,
sharpness 2. the ability of a
microscope to show two very close
points separately
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Highest Typical Resolution
Optical Microscope ~200 nm
Electron Microscope ~0.1 nm
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Factor affecting magnification
• Optical tube length
• Focal length of objective
• Magnifying power of eye piece and
objective
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Formula to determine magnification
• Magnification of microscope
= Objective Magnification x eye piece magnification
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Magnification
ocular power = 10x
low power objective = 20x
high power objective = 50x
a) What is the highest magnification you
could get using this microscope ?
500x
Ocular x high power = 10 x 50 = 500. (We
can only use 2 lenses at a time, not all
three.)
b) If the diameter of the low power field is 2
mm, what is the diameter of the high
power field of view in mm ?
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.8 mm
The ratio of low to high power is 20/50. So at high
power you will see 2/5 of the low power field of
view (2 mm). 2/5 x 2 = 4/5 = .8 mm
c) in micrometers ? 800 micrometers
To convert mm to micrometers, move the decimal
3 places to the right (multiply by 1000). .8 mm x
1000 = 800 micrometers
d) If 10 cells can fit end to end in the low power field
of view, how many of those cells would you see
under high power ? 4 cells.
We can answer this question the same way we go
about "b" above. At high power we would see 2/5
of the low field. 2/5 x 10 cells = 4 cells would be
seen under high power.
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Numerical Aperture
• It is a ratio of diameter of lens to its focal
length
• Formula:
Numerical aperture(NA) = ηsinθ
η = Refractive Index
θ = Half angel of aperture
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Numerical aperture
• BAC is cone of light
• θ is the half the angle of cone light formed at objective aperture
• Theoretical limit of BAC is 180°
(2θ = 180° )
• So θ = 90 °
• NA of dry lens cannot be greater that 1, since the refractive index of air is 1 and value of sin θ = 1
θ
θ
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Resolution (Resolving power)
• It is the abililty to reveal closely adjacent
points as separate and distinct
• Formula:d = 0.5 x λ
NA
• Where
λ = Wavelength of light
NA = Numerical Aperture
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• d= become small as resolution increase
• Hence resolution is inversely proportional to the wavelength and directly proportional to the numerical aperture.
• Maximum resolution = lowest wavelength light
• Compound microscope with blue filter below condenser help to resolve the image.
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Example
• Green light wavelength =550nm
• Objective with NA = 1.4 then what will be the resolution?
d = 0.5 x λ
NA
d = 0.5 x 550
1.4
d = 196 nm
• Microscope can reveal two closely associated points by 196 nm.
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Immersion oil and its use in
compound Microscope
• Observation by compound microscope,
using the 100x objective needs special oil.
• It is also known as immersion oil in
microscopy
• Immersion oil is cedar wood oil obtained
from gymnospermic juniperous vergiana
• It is colourless liquid and has refractive
index 1.55 (same as glass)
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Advantage
• Use of oil avoids diffraction of rays.
• If air is present between specimen and
objective, some light is lost due to
diffraction of ray. Thus the image observed
is fuzzy and the finer detail may lost.
• Thus oil help to get sharper image.
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• For Maximum resolution NA value must be high.
• The value of θ cannot exceed 90°.
• Hence by increasing refractive index NA value can be
increased
• Refractive index is function of the bending of light
from air though glass and back again.
• It can be made possible by filling medium which has
refractive index larger than refractive index of air
• Refractive index of oil is larger than air
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OPTICAL MICROSCOPES
Image construction for a simple biconvex lens
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Rayleigh criterion for resolution
www.microscopy.fsu.edu ; www.imb-jena.de
See more interactive tutorials at www.microscopy.fsu.edu
Numerical Aperature Resolution Rayleigh Criterion
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ComparisonBright-
FieldDark-
Field• Full
aperture
is
illuminated
• A central
obstruction blocks
the central cone.
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www.microscopy.fsu.edu
Dark-Field
Optical Microscopy•A central obstruction
blocks the central cone.
•The sample is only
illuminated by the
marginal rays.
•These marginal rays must
be at angles too large for
the objective lens to
collect.
•Only light scattered by the
object is collected by the
lens.
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www.microscopy.fsu.edu
Dark-Field
Optical
Microscopy
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THE ELECTRON MICROSCOPE
The wavelength of the electron can be tuned by changing the accelerating voltage.
de Broglie : λ = h/mv
λ: wavelength associated with the particle
h: Plank’s constant 6.63×10-34 Js;
mv: momentum of the particle
me= 9.1×10-31 kg; e = 1.6×10-19 coulomb
P.E eV = ½mv2 λ = h/(2meV) = 12.3/V (for V in KV, λ in Å)
V of 60 kV, λ = 0.05 Å Δx ~ 2.5 Å
Microscopes using electrons as illuminating radiation
TEM & SEM
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Components of the TEM
1. Electron Gun: Filament, Anode/Cathode
2. Condenser lens system and its apertures
3. Specimen chamber
4. Objective lens and apertures
5. Projective lens system and apertures
6. Correctional facilities (Chromatic, Spherical, Astigmatism)
7. Desk consol with CRTs and camera
Transformers: 20-100 kV; Vacuum pumps: 10-6 – 10-10 Torr
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Schematic of E Gun & EM lens
Magnification: 10,000 – 100,000; Resolution: 1 - 0.2 nm
www.udel.edu
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TEM IMAGES
www.udel.edu ; www.nano-lab. com ; www.thermo.com