imaging and Kohler illumination, objectives and eyepieces Humberto - - PowerPoint PPT Presentation

Geometrical Optics II: paraxial theory, microscope imaging and Kohler illumination, objectives and eyepieces Humberto Cabrera

Venezuelan Institute for Scientific Research International Centre for Theoretical Physics

Preparatory School to the Winter College on Optics: Advanced Optical Techniques for Bio-imaging

Imaging

Imaging

Finite objetive lens

Infinite objetives work with tube lens to produce an image

Projecting the image to your eyes

Imaging with a camera at the intermediate imaging plane

Imaging with a camera at the eyepiece imaging plane

Illuminating the specimen

Kohler illumination

Critical illumination

Kohler illumination versus critical illumination why kohler illumination?

Ligh Light sou

• urce im

image pla lanes:

• La

Lamp fila filament

• Con

Condenser dia iaphragm

• Ba

Back foc

• cal

l pla lane e of

• f th

the e ob

• bje

jectiv ive

• The

e eyep epoint Spec ecimen im image pla lanes:

• Field

ield dia iaphragm

• Spec

ecimen

• In

Intermedia iate im image pla lane (th (the e eyep epie iece dia iaphragm)

• The

e eye e reti tina or

• r camera sen

ensor

Conjugates planes

In Infin init ite ob

• bjetive

Fin init ite ob

• bjetive

Conjugate planes are simultaneously in focus and appear superimposed when viewing through the microscope

Objective lenses and optical aberrations

Ther ere ar are e man any clas classes of

• f ob
• bjectiv

ive le lense ses Common ob

• bjectiv

ive op

• ptical correction factors

 Aberration correction  Tran ansmis ission  Resolvin ing power

Aberration correction

• Achromats
• Axial - red and blue (656 nm-486 nm)
• Spherical - Green - 546 nm
• Fluorites (Semi-Apo)
• Axia

ial l -2 2 to

• 4

4 col

• lors
• Sp

Spherical - 2 2 to 4 4 colors

• Apochromats
• Axia

ial l - 4 4 to 5 5 colors - vio viole let, blu lue, green an and red

All available in “Plan” versions

Numerical aperture NA= n.sin (α)

α: : the the ha half lf op

• penin

ing an angl gle of

• f the

the

• b
• bjectiv

ive or

• r an

angl gle of

• f the

the con

• ne of
• f

ill illum umin ination n: n: the the ref refractiv ive in inde dex of

• f the

the im immers rsion mediu edium us used be between the the ob

• bjectiv

ive an and d the the ob

• bject
• Air

ir n=1 =1.0

• Water n=1.

1.33

• Oil

il an and glas lass n=1 =1.5

Numerical aperture

Why immersion media increases NA?

Maximum NA=1.49 in oil and NA=1.27 in water

How immersion medium affects the true N.A. and consequently resolution

With immersion oil (3)

n=1.518

• No Total Reflection
• Objective aperture fully usable
• N.A.max = 1.45 > Actual angle a2 :

3 a1 a2 2 1

a2 a1

No immersion (dry)

• Max. Value for a = 90° (sin = 1)
• Attainable: sina = 0.95 (a = 72°)
• Actual angle a1:
• n

NA 73 518 . 1 45 . 1 arcsine arcsine

2

   a 1) Objective 2) Cover Slip, on slide 3) Immersion Oil

No oil Oil

• n

NA 39 52 . 1 95 . arcsine arcsine

1

   a

Beampath

Snell’s Law:

n1 sin b1 = n2 sin b2

Chromatic aberrations

Chromatic aberrations correction

Chromatic aberrations correction

Spherical aberrations

Spherical aberrations correction

Objective Magnification

Example le: Objective = = 60 60 x Eyepiece = = 10 10 x In Intermedia iate Factor = = 1 1 x Overall ll M = = 600 600 x Mag agnification of

• f th

the Mic icroscope

• M Micr

icroscope = = M Objective X X M M Eyepiece X X M In Intermediate Factor M = = Mag agnification

Features of an objective

Thanks