Basic Illuminating Light Paths and Proper Microscope Alignment E. 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. Air b. Water c. Immersion oil In each case, what is the numerical aperture (NA) of the beam relative to the normal to the interface? q1 90

Homework 2: What is an easy way to measure the approximate focal length of a lens f LAMP

Homework 3: What is The Ocular Focal Length for the Following Magnifications? 5X _________ 10X _________ 20X _________ 25X _________ 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 _______ (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

Critical Illumination 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.

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

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 specimen Illuminated (area controlled by Field Diaphragm).

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 Condenser X-Y Translation Screws Are Used to Center Image of Field- Diaphragm

Image Planes for The Field Diaphragm And Light Source (Condenser Diaphragm) Alternate Along Optical Path of a Properly Aligned Microscope

The Light Path is Extended or Re- Directed Using Projection Lenses, Mirrors and Prisms and Beam-Switches

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 the position 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:

Homework Problem 9: Identify Major Components And Their Locations And Functions Within Modern Research Light Microscope (See Salmon And Canman, 2000, Current Protocols in Cell Biology, 4.1)

Condenser NA Relative to Objective NA

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