…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.