Basic Illuminating Light Paths and Proper Microscope

AlignmentE. D. Salmon

University of North Carolina at Chapel Hill

Today

• Answers to last lecture’s questions

• Light Paths for Trans illumination

• Koehler Illumination

• Action of Field and Condenser Diaphragms

• Conjugate Planes in Properly Aligned Microscope

• Major components of Research Microscope

Homework

1.

A beam of light in glass hits a surface at an angle. At what angle does the light just become total internally reflected if the glass has a refractive Index of 1.52 and the interface has a refractive index of :

a. Airb. Waterc. Immersion oil

In each case, what is the numerical aperture (NA) of the beam relative to the normal to the interface?

Snell' Law: n1 sin(q1) = n2sin (q2); q2 = 90 degrees, sin (90) = 1; n1sin(q1) = n2*1; q1 = sin-1(n1/n2)

n(glass) = 1.52n2 angle

air 1 41.1381water 1.33 61.04289oil 1.515 85.34851

q1 90

Homework 2: What is an easy way to measure the approximate focal length of

a lens

f

LAMP

LAMP

Homework 3: What is The Ocular Focal Length for the Following

Magnifications?• 5X _________

• 10X _________

• 20X _________

• 25X _________

5 51

10 25.5

15 17

20 12.7525 10.2

foc =255/Mag (mm)

Homework 4: For Finite Focal Length Objective and OTL = 160 mm, what is focal length for the following Objective

Magnifications• 4X _________

• 10X ________

• 20X ________

• 40X ________

• 60X ________

• 100X _______

Mag. f = 160/Mag4 40

10 1620 840 460 2.6

100 1.6

(mm)

A Lamp Collector Lens and Microscope Condenser Lens are Used to Concentrate Light

on the Specimen

Two Schemes for Specimen Illumination Using a Condenser

a. Critical Illumination with tungsten filament sources.

b. Kohler illumination with tungsten filament source.

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Critical Illumination

a. Critical Illumination with tungsten filament sources.

b. Kohler illumination with tungsten filament source.

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Light source out-of-focus at condenser aperture and in-focus at specimen.Produces bright, but un-even illumination of specimen.

Koehler Illumination

Light source in-focus at condenser aperture and out-of-focus at specimen.Produces bright, and even illumination of specimen.

a. Critical Illumination with tungsten filament sources.

b. Kohler illumination with tungsten filament source.

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

Condensor apertureor front focal plane

Specimenplane

Lamp filament

Collector Condenser

The Field Diaphragm Controls the Area Illuminated

The field diaphragm is focused onto the specimen by moving the condenser back-and-forthThe lamp image is focused at the condenser aperture (diaphragm) by moving the Collector lens Back-and –forth.

Lamp CL FD CD Cond SP OBJ OBA

FD' CD'

focus

Field and Condenser Diaphragms in Koehler Illumination

The Condenser Diaphragm Controls the Illumination NA

An image of the Condenser Diaphragm is in-focus in the Objective Back Focal Plan (Aperture). As the condenser diaphragm is opened, the illumination NA increases without changing the area of specimenIlluminated (area controlled by Field Diaphragm).

Condenser and Objective Apertures

obm

Qcond

CD Cond SP OBJ OB FFP BFP

Summary of Koehler Illumination

• The lamp filament and collector lens must both be centered on the same optical axis in the lamp housing.

• The collector lens is used to project an image of the lamp centered and in-focus at the condenser diaphragm.

• The condenser lens is used to project an image of the field diaphragm centered and in-focus on the specimen.

Summary of Koehler Illumination (cont.)

• A telescope is used to view the objective back back focal plane (aperture) in order to:

a) Adjust the opening of the condenser diaphragm so that the diameter of its image is equal to or slightly less than the diameter of the objective aperture;

b) Adjust the focus and x-y position of the lamp image so it is centered and in-focus at the objective back aperture

Collector Lens in Lamp Housing is Translated Along Optical Axis to Bring Lamp Image into Focus at Condenser

Front focal Plane (Diaphragm)

Condenser focus

Condenser is Translated Along Optical Axis to Bring Field Diaphragm into

Focus

Condenser focus

CondenserX-Y TranslationScrewsAre Used toCenter Image of Field-Diaphragm

IMAGING LIGHT PATHS ILLUMINATING LIGHT PATHS

Eyeor

Camera

Eyepiece

IntermediateImage plane

Objective Lens

Specimen

Condenser Lens

CondenserDiaphragm.

FieldDiaphragm

Collector Lens

Lamp

Objective Back FocalPlane

Tube Lens

MICROSCOPE ALIGNMENT FORTRANSMITTED LIGHT KOEHLER ILLUMINATIONImage Planes for

The Field DiaphragmAnd Light Source (Condenser Diaphragm)Alternate AlongOptical Path of a ProperlyAligned Microscope

The Light Path is Extended or Re-Directed Using Projection Lenses,

Mirrors and Prisms and Beam-Switches

Objective Lens

Specimen

Objective BackFocal Plane

Eye

Eyepiece

Tube Lens

IntermediateImage Plane

Emission Filter

Filter Cube

Dichromatic Mirror

Excitation Filter

LampArc

Arc Image

Condenser Diaphragm

FieldDiaphragm

Arc image

MICROSCOPE ALIGNMENT FOR EPI-FLUORESCENCE KOEHLER ILLUMINATION

Collector Lens

Homework Problem 5

 The light source is a 3-mm square tungsten filament. The design

of the illumination system requires that (1) the filament be 300 mm away from the condenser diaphragm, (2) the image of the filament must be in focus at the condenser diaphragm and (3) the filament must be 15-mm square to fill the condenser aperture with light. Assuming the lamp collector lens is an ideal thin lens, determine the focal length, and theposition of the collector lens between the lamp filament and the condenser diaphragm.

Homework Problem 6

A field diaphragm or iris is placed in front of the collector lens as shown for the Koehler illumination system. The field iris is used to control the illuminated area of the specimen. The condenser lens is translated back and forth along the central axis until an image of the field diaphragm is in sharp focus on the specimen. When the opening of the field diaphragm is 20 mm, the image on the specimen must be 2 mm in diameter. In addition, the field diaphragm is placed 160 mm away from the condenser lens. What is the focal length of the condenser needed to meet these requirements?

Homework Problem 7

Indicate “In-focus” or “out-of-focus”for:Field Diaphragm Light Source

at:Field Diaphragm _____________ ____________Condenser Diaphragm _____________ ____________Specimen _____________ ____________Objective BFP _____________ ____________Ocular FFP _____________ ____________Ocular BFP (RamdensDisk) _____________ ____________Retina (or camera detector) _____________ ____________

Homework 8

Work through the Microscope Illumination Section under Microscope Anatomy at: http://micro.magnet.fsu.edu/primer/index.html

Camera

Camera AdapterBinocular

Eyepiece

Beam Switch

Filter Cube ChangerSlot for Analyzer

Slot for DIC Prism

Objective Nosepiece

Objective

Stage

Condenser: Diaphragm&Turret Centering Focus

Field Diaphragm

Coarse/Fine Specimen Focus

Filtersand Diffuser

Lamp: Focus, Centering

Mirror:Focus andCentering

Mirror:Focus andCentering

Focus, Centering

Trans-Lamp Housing

Epi-Lamp HousingEpi-Field Diaphragm

Epi-CondenserDiaphragm

ShutterFilters& Centering

Slot for Polarizer

Upright Microscope Stand

Body Tube

MICROSCOPE COMPONENTS

Magnification Changer

Homework Problem 9:

IdentifyMajor ComponentsAndTheir LocationsAnd FunctionsWithinModern Research LightMicroscope(See Salmon And Canman,2000, CurrentProtocols in CellBiology, 4.1)

Condenser NA Relative to Objective NA

ob

cond

NUMERICAL APERTURE (NA) OF OBJECTIVE LIGHT COLLECTION AND CONDENSER ILLUMINATION

CondenserDiaphragm

Objective Back Focal Plane

NAob = nsinob

NAcond = nsincond

A Teloscope is Often Used to Observe the Image of the Lamp and Condenser Diaphragm at

the Objective Back focal Plane

-Remove Ocular and insert Teloscope to view objective back aperture(back focal plane) -In some microscopes, a focusable “Bertrand” lens is inserted between the ocular and objective to produce a teloscope while the ocular

I'

Image

at

I

O

F'oc

Telescope TelescopeObjective Eye Piece

FeiFobj