Microscope is an optical instrument which enable us to see the object which are too small to be seen by naked eye.

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Ganesh Kumar Singh
Department of Microbiology
Nobel Medical College Teaching Hospital

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Definition:- Microscope is an optical instrument which enable us to see the
object which are too small to be seen by naked eye.
Types
Light Electron
#employs light (visible/U.V.) #employs electron
as source of illumination. beam instead of light
e.g. Simple microscope e.g. TEM
Compound microscope SEM
Fluorescent microscope STEM
Dark field microscope
Phase contrast microscope
Interference microscope
Polarized microscope
Inverted microscope

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History of microscope:
Invention of glass lens
• long before (in the hazy unrecorded past), someone picked up a piece of
transparent crystal (convex), looked through it, and discovered that it made
things look larger.
• also found that such a crystal would focus the sun's rays and set fire to a
piece of paper or cloth-- Magnifiers and "burning glasses" or "magnifying
glasses“
• are mentioned in the writings of Seneca and Pliny the Elder, Roman
philosophers during the first century A. D., but apparently they were not used
much until the invention of spectacles, toward the end of the 13th century.
• They were named lenses because they are shaped like the seeds of a lentil.

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• In about 1000AD – The first vision aid was invented (inventor unknown)
called a reading stone. It was a glass sphere that magnified when laid on top
of reading materials.
• In about 1284 AD - Italian, Salvino D'Armate is credited with inventing the
first wearable eye glasses.
• In 1590AD – Two Dutch eye glass makers, Zaccharias Janssen and son Hans
Janssen experimented with multiple lenses placed in a tube. The Janssens
observed that viewed objects in front of the tube appeared greatly enlarged,
creating both the forerunner of the compound microscope and the telescope.
• In 1609, Galileo, father of modern physics and astronomy, heard of these
early experiments, worked out the principles of lenses, and made a much
better instrument with a focusing device.
A time line covering the history of microscope

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• In 1665AD – English physicist, Robert Hooke devised
compound microscope & looked at a sliver of cork through a
microscope lens and noticed some "pores" or "cells" in it
• He also observed organisms as diverse as insects, sponges,
bryozoans, foraminifer, and bird feathers.
• Micrographia was an accurate and detailed record of his
observations, illustrated with magnificent drawings.

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• In 1674 AD – Antony van Leewenhoek, built a simple microscope with only
one lens to examine blood, yeast, insects and many other tiny objects.
• He was fond of grinding the lenses and observe with them.
• He was inspired to take up microscopy by having seen a copy of Robert
Hooke’s illustrated book Micrographia.
• Leeuwenhoek is known to have made over 500 "microscopes,“-- fewer than
ten have survived to the present day .
• In 1674, he began writing letters to the Royal Society of London, describing
what he saw with his microscopes -- his first letter contained some
observations on the stings of bees.
• On September 17, 1683, Leeuwenhoek wrote to the Royal Society about his
observations on the plaque b/n his own teeth, his own wife and daughter, and
on two old men who had never cleaned their teeth in their lives. Looking at
these samples with his microscope, Leeuwenhoek reported: “animalcules”,
very prettily a-moving.

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A: rod shaped (bacillus) bacterium
B: short motile rod (dotted line from C-D---path
E: spherical coccus
F: long filamentous bacterium
G: spirochete

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Light and its properties:-
• Amplitude:-strength of energy or brightness of light.
when light travels through any medium, amplitude decrease.
• Wavelength:- distance b/n two successive crest/trough
measured in oA, , nm
• Frequency:- number of wave per second.

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• Retardation & refraction:-
medium through which light passes will slow down the speed in proportion to
the density of medium.
light entering a medium at 900 retard in speed, but direction unchanged
if enters at any other angle, a deviation occur in addition to retardation
therefore a curved lens will exhibit both retardation & refraction

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Air
Glass
Air
i
r
ratio of the Sine values of i and r gives the
figure called R.I.
Air--------------1.0 Cedar wood oil-------1.5
Water-----------1.3 Canada balsam-------1.5
Glass------------1.5 Paraffin oil-----------

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Defects of lenses:-
# Spherical aberration:-
• Is due to the fact that lens is a curved surfaces.
• Therefore the angle at which light rays enters & leave the surface of a lens
varies with each part of the lens.
• Those passing through the periphery----refracted to a greater angle than
those passing through centre----there is no position where the light will be
in focus----image will be hazy.
* Correction:-using central area of a lens

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# Chromatic aberration:-
• Is based on the fact that white light is composed of the different colors each
vibrating as a different wavelength.
• When such light passes through a lens, is refracted to varying extent and do not
combine at the same focus-----hazy image fringed with the color spectrum.
----
* Correction:-using monochromatic light

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Simple microscope:-
• Is simply a “simple magnifying glass”
• Is a convex lens of short focal lens.
• The object should be placed in such a way that it lies at the distance less
than the focal length of the lens.
# for normal eye, the image is formed at a distance of 25cm from the eye–
called “near point.”
F
f
Fig. Simple microscope- image formation

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# Compound microscope: that is, microscope using more than one
lens
# Components:-
* optical component
* mechanical component
* illuminating component.

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Optical components:
# The objective:
- consists of system of lenses (5-15 in no.)
- screwed to nosepiece.
- designated by magnifying power as: X5( red),
X10(yellow), X40(blue), X100(white)
- engraved to it is 100/ 1.25
160/ 0.17
# form a high quality magnified real image

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• ability of an objective to resolve details depends on its N.A. & not
on its magnifying power.
Numerical aperture (N.A.) :- is the angle of aperture which determines the
light gathering powers of a lens
expressed as :
n= R.I.of the medium b/n object & the objective.
U= half of the angle formed by two most divergent rays starting from
the center of the object & entering the objective to reach the eye.
N.A.= n Sine U
F
E G
A
B
DC
BAD=U

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• since U can’t be > 900 ,
N.A. of dry objective can’t be more than 1.0 as
R.I. Of air =1.0 & Sine 90º =1
if immersion oil is interposed
N.A.=1.5 as R.I. Of oil =1.5
in practice, highest N.A. of dry lens =0.95 &
of oil immersion =1.4

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Types
Huygenian Ramsden
The two lenses are made of the same kind
of glass, e.g.,spectale crown. The lenses
are of equal focal length and their
separation is equal to the focal length.
Focal plane
Field lens
eye lens
The lens are separated by a distance equal to half the sum
of their focal lengths. The focal plane of the eyepiece is
between the two lenses

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# Mechanical components:
The body tube:
 attached to limb
 bears oculars at upper end & objective screwed to nose piece at lower end
 size =160mm
 three main types are available:
monocular, binocular or combined photo-binocular
 latter has prism allowing 100% light to eyes or to camera
or
 beam-splitting prism : 20% to eyes & 80% to camera
 provision in binocular for adjusting interpupillary distance.

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The nosepiece:
• carrier for objectives
• fitted at lower end of body tube
• rotates on central pillar
• designated as double, triple, quadruple
• its depth affect the tube length.
• is generally 18mm deep, actual length of the body tube only 142mm.

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• The stage:
- is a platform on which the object to be examined
are placed.
- may be plain or mechanical.
- a standard type of mechanical stage take a slide
of 3 X 1 inch & move over an area of approx.
3.5 X 1.25inch.
- mechanical type is also fitted with a vernier scale
for recording the position of the slide in each
direction.
simple type
mechanical type

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A warm stage:-
• Are several types:
• Some consists of a thin, flat metal box filled with hot water or through
which warm water circulate & have an aperture in the centre by which
light passes to the preparation.
• Improved form are electrically heated & have a automatic temperature
control.
•Uses:-keeps the preparation at body temperature & enables to see the
motility of the organism better.

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• Illuminating components:
A) The sub-stage:
• is below the stage & usually attached to it.
• moved up & down by a helical screw or rack & pinion.
• Sib-stage consists of:-
1. The condenser:- focus light from source into plane of
objective
Types of condenser
Abbé condenser:- are composed of two lenses neither
of which is corrected for spherical or chromatic
aberration.
sub-stage

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Aplanatic condenser:- contains a third or
middle lens which corrects for spherical
aberration but not for colors.
- give good result when used with
monochromatic light.
Achromatic condenser:- is corrected for
both spherical & chromatic aberration.
- give best result as focus the light to the
plane of objective

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Illuminating components cont.
2. The iris diaphragm:-controls the amount of light
reaching object.
if it is closed too much object become too contra sty,
Where as if left wide open, image suffer from glare –
either case poor resolution.
# correct setting is when N.A. of condenser = that of
objective in use.

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3.The filter carrier:-
-is just below the iris diaphragm.
-is ring shaped, designed to carry colored glass filter
(e.g. a blue “daylight” filter)
-filter reduce excessive red or yellow components of light.
B) The mirror:
- is fitted to tail piece below the condenser at about
4inches below the stage.
- is plano-concave.
- reflects rays of light to sub-stage condenser.
# modern microscope, however use electric bulb instead.
The condenser has an
adjustable iris ,
controlled by knob A,
and a removable filter
carrier (B).

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Image formation:-
Parallel rays of light entering a curved lens are brought to a single point ,”focal point” or ”principal focus”
F
C
f
• in addition to principal focus, a lens also have other pair of
point, one on either side, called “conjugate foci “.
• when an object is placed at one, a clear image is formed at
other.

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• The conjugate foci vary in position and as the object is moved nearer the lens,
the image is formed further away, at a greater magnification & inverted.
• This is” real image” & is that formed by the objective.
object
objective
image

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image formed by objective

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• If the image is placed yet nearer the lens, within the principal focus, the
image is formed on the same side of the object, is enlarged.
• This is the “virtual image” and is that formed by the eyepiece
image
object
eye-piece

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• Magnification: is the degree of enlargement
• an objective forms a “real, inverted &enlarged image” which is further enlarged
by the eye piece.
 total magnification = magnification of an objective x magnification of an eyepiece
Calculation:
Mechanical tube length
Total magnification = X eyepiece magnification
focal length of objective

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 with MLT of 160mm, total magnification of a microscope using:-
Objective
magnification
Eyepiece
magnification
Total
magnification
10X(f=16mm) 10X 100X
40X(f=4mm) 10X 400X
100X(f=2mm) 10X 1000X
in some model of binocular microscope, the design of binocular heads MLT
magnification usually by a factor x1.5

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• It is perfectly possible to design an optical system which will give
enormous magnification, e.g. 100 of 1000 of times, but after a
certain point details & sharpness begin to be lost, instead picture
(image) is broken down to a series of black & white dots.
Magnification of this type is called empty magnification ----- not
useful for microbiologist

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# Resolution: is the ability of a lens to define detail
: is dependent on:
a. wave length of light used ()
b. R.I. of medium b/ n objective & object(n)
c. angular aperture of the objective lens()
The relationship is as follows:
1.2 
R =
2 n Sine 
0.60 
R =
N.A * n Sine  =N.A.

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Dark-ground microscope
• Occasion arise when it is preferable or essential to examine unstained
preparations.
• Such specimens and their components have R.I. close to that of the medium in
which suspended difficult to see by bright field due to lack of contrast
• Dark-ground microscopy overcomes these problems by preventing direct light
from entering the objective
• The only light gathered is that refracted or diffracted specimen appear
bright on dark background

37. 37Fig. dark-ground condenser

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 Dark ground condenser may be for either dry, low power
objective or for oil immersion
 Objective must have a low N.A. than that of condenser
 Condenser focus a hollow cone of light at an acute angle.
 This angle is so acute that if oil is not used in b/ n the
condenser & slide, the light rays are reflected back into the
condenser
 Oil must also be used b/ n objective & object to ensure that
max. reflected light from the object enters the objective.

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 Bright-field microscope can be converted for dark-ground
work by using simple patch stop.
 Is made of black paper,placed on top of condenser lens or
suspended in filter holder.

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Setting up the dark ground microscope:-
1. Make thin preparation. Take care not to have air
bubbles.
2. Adjust the light through the condenser so that it is
evenly distributed
3. Rack the condenser down.
4. Place a drop of immersion oil on the top of the lens
of the condenser & on the lower side of the slide.
5. Slowly rack up the condenser till the two surfaces
of the oil meet without forming the air bubbles.
6. Focus with the low power objective.
7. Place a drop of immersion oil on the cover slip
8. Focus with oil immersion objective and observe.
Uses:-
• Initial examination of suspensions of cells such as yeast, bacteria, blood cells
• Examination of lightly stained prepared slides ,determination of motility in cultures

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Phase contrast microscope
• Prof. Zernicke ---- Noble prize(1935)
• Unstained & living biological specimens have little contrast
with their surrounding medium, even though small differences
of R.I. Exist in their structure.
• To see them clearly involves:
1. Closing down the iris diaphragm of the condenser which
reduces its N.A. or
2. Using dark-ground illumination.
# often fails to reveal internal structure

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Optical principle:
screen
Increased light(constructive interference)

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screen
no light,
out of phase
Destructive interference
1/2
Out of phase

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* 1/4 out of phase
Light = diff. in amplitude

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Components of phase contrast microscope:
• Lamp:- a high intensity illumination source e.g. Hg lamp
• Annulus:-
- Different sized required for each objective according to it N.A.
- These may be inserted separately, but a rotary changer carrying a set of
- annuli, mounted below condenser is more convenient.
- Are disks of glass rendered opaque, but with a narrow ring of clear glass

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• Objective:-
-are ordinary objective at the back of which is inserted the phase plate
made of glass.
• Phase plate:-
-of glass-----trough etched in it---- depth such that the light after passing
through it ---- phase difference of ¼ of  compared with the rest of
plate.

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• Auxiliary telescope:-is used in place of an eyepiece for
examining the back focal plane of the objective to ensure
that the objective phase-plate and the condenser are
properly aligned

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• Setting up the phase contrast microscope:-
• Focus the objective on the specimen.
• Rotate the appropriate annulus for the objective in position under the
condenser.
• Without disturbing the focus remove the eyepiece and replace it with
auxiliary telescope.
• Adjust the auxiliary telescope to bring the image of the phase plate into
sharp focus.
• If the image of the light annulus does not coincide with the grey ring of the
phase plate, it is adjusted with the centering screws.
• Replace the auxiliary telescope by the eyepiece and observe a phase
contrast image.
• Uses:-Phase contrast is preferable when the specimen is colorless or the
details so fine that color does not show up well. e.g. Cilia and flagella

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Fluorescence microscopy
• Originally developed by Dr Coons & co-workers
Whenever a physical body absorbs energy, this energy can’t just disappear, but
must reappear again in some other form. This could be in the form of heat,
chemical energy, or could be emitted in the form of light as “luminescence”.
When emission is stimulate by electrical means--------- electroluminescence.
When emission is stimulate by chemical reaction--------- chemiluminescence.
When emission is in biological system---------------------- bioluminescence.
When emission is due to previous absorption of light--------- photoluminescence.

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• In photoluminescence there is always a laps of time b/ n absorption &
emission.
If this pd. is >10-4 of a sec.----------- phosphorescence.
If this pd. is <10-4 of a sec.-----------fluorescence.
u. v. light
visible light

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Dyes for the fluorescent staining:-
1. Auramine “O”
2. Acridine orange
3. Berberine sulphate
4. FITC
5. Primulin
6. Rhodamine
7. Thioflavin S
8. Trypaflavin
9. Morin

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Types of fluorescence microscope:
1. Transmission light fluorescent
2. Incident light (epifluorescence) type.

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Transmission light fluorescent

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Incident light (epifluorescence) type.
barrier filter
beam splitter
eye

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Filters:-
# Exciter filter:-
• Early used were “dyed-in-the-mass” glass filter & were designated as UG/BG along
with a number. e.g.
Schott BG12 –transmits light of 325-500nm.
Schott UG1 –transmits light of 275-400nm.
• Modern exciter filters are designated by letter: G= dyed-in-the-glass; BP=band pass,
followed by numbers which indicates  of max. transmission. e.g.
• G405= have max. transmission of 405nm
• BP405/6 =have max. transmission of 405/6nm
• .
# Barrier filters:- used to protect the eye from U.V. light.
• By using different filters, with varying absorption & transmission non-specific
background fluorescence bay be extinguished, resulting better contrast.e.g.
SchottOG4 & 5 (Zeiss47 & 50) absorbs blue auto fluorescence of the specimen.
The no. of old filters referred to  at which they transmits light. e.g. 47 ---470nm & above.
• Now, filters are identified by letters no. as: LP= long pass, e.g.
• LP495 transmits light above 495nm with 50% transmission at 495.
• Leitz use prefix “K’
• The barrier filters must be chosen so that its transmission lies completely outside that of
the exciter filter, but include the fluorescence emission.

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Filter combinations
Fluorochrome Exciter filter Barrier filter
Acridine orange BP 450-490 LP 520
Auramine BP 450-490 LP 520
FITC BP 485/20 BP 520-560
Thioflavin T BP 450-490 LP 520

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• Application of fluorescence microscopy:-
• Examination of the flourochrome stained specimen.e.g.
• Malaria parasite, trypanosomes, microfilaria, intracellular gonococci,
meningococci, AFB etc
• Immunofluorescence.

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Micrometry:-the measurements of object using calibrated eyepiece scale
(micrometer) is called micrometry.
• Requirements:-
1. Eyepiece micrometer to fit into a 10X eyepiece or special measuring 10X
eyepiece. A suitable eyepiece micrometer is one that is divided in to 50
divisions.
2. Stage graticule to calibrate the eyepiece scale. A suitable scale for the stage
graticule is one that measure 2mm in length with each large division
measuring 0.1mm (100µm) & each small division measuring 0.01mm (10µm)

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Calibrating an eyepiece micrometer using stage graticule
• Unscrew the upper lens of a 10X eyepiece & insert Eyepiece micrometer disc
with engraved side facing down. Alternatively, use a measuring eyepiece.
• Place the stage graticule slide on the stage & with required objective in place,
bring the scale in to focus both the eyepiece & calibration scale should be
focused clearly.
• Adjust the field until the “zero” of the eyepiece scale aligns exactly with “zero”
of calibration scale.
• Look along the scale & note where a division of the eyepiece scale aligns exactly
with division of calibration scale.
• Measure the distance b/n the “zero” point & where the alignments occurs.
• Count the no. of division of the eyepiece scale covered b/n the “zero” & where
the alignments occurs.
• Calculate the measurements of one division of eyepiece scale in µm.
• Make tables giving the measurements for 1 to 50 eyepiece division.
Measuring an object:-with the eyepiece micrometer scale & the object sharply
focused, position the object along the scale & count the divisions covered. Refer
to the previously prepared table to obtain the measurements of the object

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# Care of the microscope
• EVERYTHING on a good quality microscope is expensive, so be careful.
• Hold a microscope firmly by the stand, only. Never grab it by the eyepiece
holder, for example.
• Hold the plug (not the cable) when unplugging the illuminator.
• Since bulbs are expensive, and have a limited life, turn the illuminator off
when you are done.
• Always make sure the stage and lenses are clean before putting away the
microscope.
• NEVER use a paper towel, your shirt, or any material other than good
quality lens tissue or a cotton swab (must be 100% natural cotton) to clean
an optical surface. Be gentle! You may use an appropriate lens cleaner or
distilled water to help remove dried material. Organic solvents may
separate or damage the lens elements or coatings.
• Cover the instrument with a dust jacket when not in use.
• Focus smoothly; don't try to speed through the focusing process or force
anything. For example if you encounter increased resistance when
focusing then you've probably reached a limit and you are going in the
wrong direction

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