Ophthalmic Optical Instruments I Telescopes and Microscopes C. I. OPHTMALMOMETER, CHICAGO ILLINOIS ca 1899

TELESCOPES

Astronomical (Keplerian) Telescope Image is Inverted objective eyepiece Fe Fo fe fo M = - fe fo Virtual image at 25 cm

Astronomical Telescope objective eyepiece Fe Fo fe fo Virtual image at infinity D

Galilean Telescope plus lens fp fp negative lens fp fp’s coincide final telescope parallel rays

Viewing Through a Galilean Telescope parallel rays emmetropic eye object iImage UPRIGHT OBJECT APPEARS UPRIGHT D GTT 04

MICROSCOPES

ANGULAR MAGNIFICATION Apparent size of object depends on angle it subtends at eye.

ANGULAR MAGNIFICATION On average, an object cannot be closer than 25 cm from the eye to be seen clearly. Average distance of most distinct vision

cm (cm)

BASIC MICROSCOPE magnifier real image magnification

MICROSCOPE MAGNIFICATION 25 M = 2 f Im Im 25 M = = M X 1 Ob Ob f total

n a a D NA = sin n OBJECTIVES w.d. EXAMPLE a = 14 n = 1.00 (air) Numerical Aperture (NA) Light gathering ability Resolution a n a NA = sin n EXAMPLE a D = 14 n = 1.00 (air) w.d. NA = 1.00 x sin(14 ) NA = 0.24

OBJECTIVES N.A. Examples

parallel rays from eyepiece EYEPIECES (OCULARS) Huygens Ramsden parallel rays from eyepiece Real image Real image converging rays from objective D

REAL MICROSCOPE

EXPERIMENT 4 Basic Microscope iris diaphragm real image on card onion skin f Produce real image of onion skin on card. Mark distance of real image on base.

EXPERIMENT 4--CONTINUED View real image with magnifier (“eyepiece”) real image plane f Adjust iris diaphragm. How does image change? Im 25 What is the total magnification? = M X Ob f total

Slit-lamp Biomicroscope

The slit-lamp biomicroscope begins with a microscope…. Eyepiece Objective Specimen

….change specimen, objective & eyepiece ….turned on its side ….change specimen, objective & eyepiece Huygens eyepiece objective subject image plane …….fundamental slit-lamp biomicroscope

Galilean telescope to change mag Build in magnification change without changing working distance working distance fobj Galilean telescope to change mag no image in image plane

Galilean telescope to change mag Build in magnification change without changing working distance working distance fobj Galilean telescope to change mag no image in image plane D

astronomical telescope …..add lens to form image in eyepiece image plane astronomical telescope D

Porro* prism 2 right-angle prisms 1800 image rotation reduce length of telescope displace image horizontally Porro -Abbe *Ignazio Porro. 1801 – 1875. Italian optical instrument maker

Slit-lamp with folded optical path

D

binocular slit-lamp viewing system

Operating Microscope

Operating microscope optics are very similar to those of the slit-lamp.

binocular astronomical telescopes Change magnification without changing working distance magnification change: Galilean telescopes prism objective lens

Specular Microscope specular == “mirror-like” Endothelial cells posterior surfaces flat & adjacent to aqueous Difference in index of refraction gives specular reflection Useful in seeing corneal endothelial cells

Specular Microscope D halogen lamp condenser slit dipping cone lens M objective Ramsden eyepiece film or CCD D

endothelium { slit image specular reflections and stray light

Typical image of endothelium from specular microscope

Confocal Principle Red cell in thick sample imaged by lens Blue cell, nearer to surface, imaged at different point Pinhole in image plane passes all light from blue cell Pinhole blocks most of light from red cell Based on Webb, RH, Rep Prog Phys 59:427

Confocal Principle Point source CONFOCAL with blue cell & pinhole selectively illuminates blue cell Confocal point source gives less light to red cell, and most is blocked by pinhole Beam splitter makes confocal microscope epitaxial Based on Webb, RH, Rep Prog Phys 59:427

Confocal Optical Systems: Pinhole allows light from small volume in sample. Other stray light blocked. Confocal point source confines light to small volume in sample. Rejects stray light Allows Z-axis “sectioning”

Confocal systems can improve imaging Standard specular microscope Confocal specular microscope

Koester’s confocal microscope

M M Koester’s Confocal scanning microscope CCD camera lamp confocal slit slit rotating mirror Koester’s Confocal scanning microscope objective specimen

Other Scanning Methods and Confocal Microscopes Tandem Scanning Confocal Microscope Scanned (laser) Spot Confocal Microscope Scanned Slit Confocal Microscope

Tandem Scanning Confocal Microscope q r Uses Nipkow disk Paul Nipkow (1860 -1940) Studied with Helmholtz Invented disk in 1883 Used for telegraphing pictures Later used in 1st television r = a + bq Sets of holes in plate Holes on Archimedes spirals D

Petran Tandem Confocal Scanning Microscope Very light inefficient Petran Tandem Confocal Scanning Microscope

TCSM for imaging cornea

X-Y mirror confocal laser scanning

Confocal slit scanning

3-D reconstruction from confocal images d fiber from subepithelial plexus Guthoff et al. Cornea 24:608

Commercial Confocal Scanning Corneal Microscopes Heidelberg HRT II*-Rostock attachment Nidek Confoscan Topcon SP-2000p *also scans retina