II From “Beam Paths” to the “Microscope” October 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging

Understanding Beam Paths  From Pin Hole Camera to the Lens  The three “Thin Lens Laws”  Image Ratio – not the same as Magnification  “Magnification”  The Concept of “Infinity”  The “simple” microscope (Leeuwenhoek, Magnifier, Eyepiece)  The “compound” microscope – Upright / Inverted  Illumination – Transmitted / Reflected  Stereo Microscopes – Greenough / Telescope Types

Geometric Optics – Creating an Image The Pinhole Camera

Geometric Optics – Creating an Image Making the aperture larger…

Geometric Optics – Creating an Image Adding a lens…

How does a lens “bend” light? Infinite number of prisms with different angles

Drawing Beam Paths Thin Lens Laws (1,2,3)

1) Draw ray through center of lens n1n1 n2n2 (small error is ignored if glass is very thin) Exact path

2) Rays that enter the lens parallel to the optical axis cross over at Back Focal Point (Back) Focal Point

2b) Rays that enter the lens from infinity, cross over at Back Focal Plane (Back) Focal Plane

3) Light rays that enter the lens from the focal point exit parallel to the optical axis. (Front) Focal Point f focal distance

 3b) Light rays that enter the lens from a point along the focal plane exit parallel (  ). Size translates to angle !

Predicting the behavior of imaging systems (principle ray technique) ff Object Back Focal Pt Front Focal Pt

1) Draw in central ray Object

1) Draw in central ray 2) In parallel; out via back focal point

1) Draw in central ray 2) In parallel; out via back focal point 3) In via front focal point; out parallel

1) Draw in central ray 2) In parallel; out via back focal point 3) In via front focal point; out parallel Intersection defines image Image B A

Our eye is a great imaging system. Its lens provides variable focal lengths to bring objects in focus at the retina

Objects appear to the eye at different magnifications, depending on their distance from the eye. Accommodation (lens) makes it possible. M B ~ 2x M A A B Magnification – unaided Eye

Conventional Viewing Distance 250 mm 1x ?

“Magnification” 1x f = 250 mm 1x 250 mm “Infinity Optics” ?

Higher Magnifications via Single Lens f = 250 mm 1x Example: f=50mm 5x Magnifying Glass (Loupe)

The Leeuwenhoek microscope

Intermediate ImageEyepoint (Exit Pupil) The Eyepiece (Ocular) is a “Simple” Microscope If you need a magnifier, remove eyepiece, turn upside down and move close to eye; subject will be about 25mm away from lens

Objective Eyepiece The Compound Microscope Specimen Intermediate Image Eye (Retina)

Tube lens Objective Eyepiece The Compound Microscope Specimen Intermediate Image Eye (Retina)  -corrected Infinity Space

Advantage of Infinity Correction Infinity System Specimen off-center Finite System Specimen off-center  ax  lat. Intermediate image is 1) “in registration” 2) fully corrected Objective

The  -corrected Compound Microscope Objective Eyepiece Tube Lens

Cross-section through an ∞ corrected Microscope Intermediate image (fully corrected) Infinity Space Objective Tube Lens Eyepiece

Upright microscope. Inverted microscope The basic light microscope types

Illuminating the Specimen Transmitted Light The sample must be transparent !

Upright microscope. Inverted microscope

Eg. Fluorescence, Opaque Samples Illuminating the Specimen Reflected (Incident) Light

Upright microscope. Inverted microscope

Upright microscope. Inverted microscope Mixed Illumination

Which Microscope types typically use these types of illumination? External Sources, e.g.  Fiber Optics  Reflectors  Ring Lights  Various Combinations Source ?

“Couldn’t one build a microscope for both eyes, and thereby generate spatial images?” Question addressed to Ernst Abbe in 1896 by Horatio S. Greenough

1896: Drawing by Horatio S. Greenough 1897 – the first Stereo Microscope in the world, built by Zeiss according to the “Greenough” principle

Greenough Type

What happens if we take the objective away from the microscope? Tube lens (Zeiss: f=164.5mm) Objective Eyepiece Tube f 250mm f M  We have created a “Telescope” ∞ ∞

Greenough TypeTelescope Type Introduced first by Zeiss

Comparison Greenough Type: 2 separate beam paths going through centers of lenses Excellent correction Economical Telescope Type: One common objective for both beam paths Flexibility to interchange tubes and objectives Possibility to add intermediate tubes e.g. for Fluorescence, Co- observation, Drawing

Questions? Short break? Next: Dissecting an infinity-corrected microscope