with a microscope at the - biodip · early microscope attributed to jansen (about 1595) bacteria...
TRANSCRIPT
…you look at an image of the specimen.
With amicroscope
you don’t lookat the
specimen…….
Brad Amos & Stefanie Reichelt
Light rays carry information!!!!!
Violet
Indigo
Blue
Green
Yellow
Orange
Red
400nm
425 nm
470 nm
550 nm
600 nm
665 nm
700 nm
From: http://ds9.ssl.berkeley.edu/LWS_GEMS/2/em.htm
Daylight
Incandescent lamp
Mercury lamp
Wavelength (nm)
LightLightsourcessources
forforfluorescencefluorescence
Wavelength (nm)
Spectrum of a Mercury Lamp
Spectrum of a Xenon Lamp
(Modified from: http://www.cairn-research.co.uk)
ONCE UPON A TIME………..
(Ibn al-Haytham)
Alhazen was the first to show how the image isformed on the retina in the human eye, using the
camera obscura as his model. However, as far asthe camera obscura was concerned, he explained"Et nos non inventimus ita” we did not invent this.
www.islamicspain.tv/.../Physics-and-Optics.htmImage taken from: http://micro.magnet.fsu.edu/optics/timeline/people/alhazen.html
430nm lightstimulate blue light
sensitive cones.You see blue!
The relative intensity of the stimuli of each type of cones determinesthe color!!!!
With 550nm lightyou see green
At 630nmyou see red
light
Your eyes seeACHROMATIC
light…WHITE LIGHT
When the threetypes of cones arestimulated at the
same time….
PROPERTIES OF LIGHT
• Diffraction
• Refraction
• Reflection
BASICS ON….
• Absorption
H O W L I G H T M A K E T H I N G S V I S I B L EH O W L I G H T M A K E T H I N G S V I S I B L E
H O W L I G H T M A K E T H I N G S V I S I B L EH O W L I G H T M A K E T H I N G S V I S I B L E
V i s i b l e
U l t r a v i o l e t
I n f r a r e d
Air
Water
Glass
Air
Refracted rays passing through threedifferent media
DIFRACTION
XVII Century. Zaccharias & Hans Jansen 1590
Galileo Galilei 1609
Christiaan Huygens 1621
Images taken from: http://micro.magnet.fsu.edu/optics/timeline/people/alhazen.htmlhttp://www.biol.unlp.edu.ar/historiabiologiacelular.htm
XV Century. Leonardo Da Vinci …The necessity to use lenses to facilitate vision and too see small things….
13th Century13th Century
Reading stonesReading stones
Early Microscope Attributed to Jansen(about 1595)
Bacteria
Human sperm
Antony van Leeuwenhoek1632 - 1723
www.biol.unlp.edu.ar
www.gewina.nl
Robert Hooke 1635 - 1703Micrographia 1665
O b j e c t i v e l e n s
L a m p
P o w e r
s u p p l y
C o n d e n s e r
l e n s e s
F o c u s i n g
m e c h a n i s m
M e c h a n i c a l
s t a g e
E y e p i e c e
Modified from:www.its.caltech.edu
I N V E R T E DI N V E R T E D
U P R I G H TU P R I G H T
Carl Zeiss1816 - 1888
Ernst Abbe1840 - 1905
Otto Schott(1851 - 1935)
F’ FO2F’ 2F
F’ F
FF’
A l e n s h a s t w o f o c a l p l a n e sA l e n s h a s t w o f o c a l p l a n e s
F’ F2F’ 2F
The lenses of the microscopeThe important lenses of the microscope are positive or
converging lenses,
- thicker in the middle than at their edges
From Peter Evennett
The job of an ideal lens• To accept as many rays as possible from each point in an object
• To reassemble all the rays from each point at corresponding points in theimage…
• In such a way that the distance travelled by all the rays from each objectpoint to its corresponding image point is the same- so that they all arrive ‘in phase’.
This is unfortunately not possible with a single-element lens
because of several aberrations- spherical, chromatic and others
O b j e c t B l u e i m a g e
i n f o c u s
G r e e n i m a g e
i n f o c u s
R e d i m a g e
i n f o c u s
G r e e n i m a g e
i n f o c u s
G r e e n i m a g e
i n f o c u s
The objective lens
The most important lens of the microscope
Is the microscopeThe other parts support its function
and adapt the image to the receiving device
micro.magnet.fsu.edu/.../anatomy/objectives.html
1
2
11
8
6
9
10
1
2
3
4
5
10
96
8
7
Eyepieces
Objectives
Revolver
Focusing KnobCondenser
Lamp house (Mercury lamp)
Lamp house (Halogen lamp)
Condenser adjusting knob
Field Diaphragm
Condenser Diaphragm
4 53
Filter wheel11
7
1
2
3
4
5
10
96
8
7
Eyepieces
Objectives
Revolver
Focusing Knob
Condenser
Lamp house (Mercury lamp)
Lamp house (Halogen lamp)
Condenser adjusting knobField Diaphragm
Condenser DiaphragmFilter wheel
11
1
4
5
11
9
10
7
6
2
3
8
Microscope Illumination
Two basic methods of illumination:
Source-focused or ‘Critical’ Illumination:Light-source imaged on to specimen
Köhler Illumination:Light-source imaged in the aperture ofthe condenser
T r a n s m i t t e d - l i g h t . B r i g h t - fi e l d
C o n d e n sC o n d e n s
e re r
O b j e c t iO b j e c t i
v ev e
E y e p i eE y e p i e
c ec e
T r a n s m i t t e d - l i g h t . B r i g h t - fi e l d
C o n d e n s e rC o n d e n s e r
d i a p h r a g md i a p h r a g m
C o n d e n sC o n d e n s
e re r
O b j e c t iO b j e c t i
v ev e
E y e p i eE y e p i e
c ec e
“ S e t o f p l a n e s 1 ”
T r a n s m i t t e d - l i g h t . B r i g h t - fi e l d
C o n d e n s e rC o n d e n s e r
d i a p h r a g md i a p h r a g m
C o n d e n sC o n d e n s
e re r
O b j e c t iO b j e c t i
v ev e
E y e p i eE y e p i e
c ec e
“ S e t o f p l a n e s 2 ”
C R I T I C A L I L L U M I N A T I O NC R I T I C A L I L L U M I N A T I O N
August Köhler1866 - 1948
PublishedA new system of illumination for
photomicrographic purposes(in German) in 1893.
Köhler illumination
Köhler alignment
Microscope alignment
Did you......Köhler??
F’ F
FF’
C o n d e n sC o n d e n s
e re r
O b j e c t iO b j e c t i
v ev e
E y e p i eE y e p i e
c ec e
L i g h tL i g h t
s o u r c es o u r c e
L i g h tL i g h t
s o u r c es o u r c e
Light up the specimen uniformly
Set of “aperture”Conjugated planes
+ Set of “field” Conjugated planes
= 2 Sets of conjugated Planes
2 Conjugated set of Planes
Specimen +
Diaphragm image
Primary image of the specimen +
Diaphragm image
Diaphragm
Final image of the specimen +
Diaphragm image
Lamp Filament
Condenser aperture+
Lamp Filament image
Condenser aperture image+
Lamp Filament image
Condenser aperture image+
Lamp Filament image
Field Set of planesAperture Set of planes
CR
IT
IC
AL
I
LL
UM
IN
AT
IO
NC
RI
TI
CA
L
IL
LU
MI
NA
TI
ON
KÖ
HL
ER
I
LL
UM
IN
AT
IO
NK
ÖH
LE
R
IL
LU
MI
NA
TI
ON
C o n d e n s e rC o n d e n s e r
d i a p h r a g md i a p h r a g m
C o n d e n s e rC o n d e n s e r
S p e c i m e nS p e c i m e n
p l a n ep l a n e
O b j e c t i v eO b j e c t i v e
E y e p i e c eE y e p i e c e
S o u r c eS o u r c e
i m a g e d o ni m a g e d o n
r e t i n ar e t i n a
I M A G I N G S E TI M A G I N G S E T
I L L U M I N A T I N GI L L U M I N A T I N G S E TS E T
I l l u m i n a t e dI l l u m i n a t e d
fi e l dfi e l d
d i a p h r a g md i a p h r a g m
S p e c i m e nS p e c i m e n
p l a n ep l a n e
C o n d e n s e rC o n d e n s e r
d i a p h r a g md i a p h r a g m
B a c k f o c a l p l a n eB a c k f o c a l p l a n e
o f t h e o b j e c t i v eo f t h e o b j e c t i v e
M i c r o s c o p eM i c r o s c o p e
e x i t p u p i le x i t p u p i l
(( e y e p o i n te y e p o i n t ))
P r i m a r yP r i m a r y
i m a g e p l a n ei m a g e p l a n eR e t i n aR e t i n a
I l l u m i n a t e dI l l u m i n a t e d
fi e l dfi e l d
d i a p h r a g md i a p h r a g m
S p e c i m e nS p e c i m e n
p l a n ep l a n e
P r i m a r yP r i m a r y
i m a g e p l a n ei m a g e p l a n e
C o n d e n s e rC o n d e n s e r
a p e r t u r ea p e r t u r e
B a c k f o c a l p l a n eB a c k f o c a l p l a n e
o f t h e o b j e c t i v eo f t h e o b j e c t i v e
M i c r o s c o p eM i c r o s c o p e
e x i t p u p i le x i t p u p i l
(( e y e p o i n te y e p o i n t ))
R e t i n aR e t i n a
C o n d e n s e rC o n d e n s e r O b j e c t i v eO b j e c t i v e
E y e p i e c eE y e p i e c eL i g h t S o u r c eL i g h t S o u r c e
( F i l a m e n t )( F i l a m e n t )
L i g h t S o u r c eL i g h t S o u r c e
( F i l a m e n t )( F i l a m e n t )
C o l l e c t o rC o l l e c t o r
l e n s l e n s
L i g h t S o u r c eL i g h t S o u r c e
( F i l a m e n t )( F i l a m e n t )
L i g h t S o u r c eL i g h t S o u r c e
( F i l a m e n t )( F i l a m e n t )
L M F . P h D M i c r o s c o p y c o u r s e 2 0 0 8
• Light up the specimen uniformly
– over a controllable area
• Illuminate the objective aperture uniformly
– over a controllable angle
What are we trying to do whenilluminating a microscopical
specimen??
From Peter Evennett
U P R I G H T M I C R O S C O P EU P R I G H T M I C R O S C O P E I N V E R T E D M I C R O S C O P EI N V E R T E D M I C R O S C O P E
E y e
C C D
~ 6 0 % ~ 8 0 % ~ 9 5 % ~ 1 0 0 %
Condenser Aperture too large
Image hazy and ‘washed out’From Peter Evennett
Condenser Aperture correct
Image contrast optimalFrom Peter Evennett
Condenser Aperture too small
Image contrast too high; artefacts presentFrom Peter Evennett
Condenser Aperture too small
From Peter Evennett
Condenseraperturetoo small
Condenseraperturecorrect
Note this object
This is what it‘should’ look like!
From Peter Evennett
From Peter Evennett
Köhler Illumination provides
Control of Area illuminated by theIlluminated Field Diaphragm,
which is adjusted according to magnification.
Control of Angle of illumination by theIlluminating Aperture Diaphragm
(the condenser aperture),which is adjusted according to objective aperture.