Transcript Image Forming and Illuminating Systems of the Bright field Microscope

Geometrical Optics and Basic
Imaging Light Paths of the Bright
Field Microscope
E. D. Salmon
University of North Carolina at
Chapel Hill
Major Imaging Functions of the
Microscope
• Magnification: Needed to overcome resolution
limitations produced by finite size of recording
sensors- rods and cones in eye, silver grains in
film; pixels in CCDs
• Resolution: Limited by lens aberrations and finite
wavelength of light
• Contrast: How to make resolvable structural detail
visible-absorbing stains, phase contrast, DIC, Pol.,
immunofluorescence, fluorescent analogs, GFPfusion proteins, other fluorescent molecular probes
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Some Visible Spectrum Light Sources
The eye is most sensitive
to green light!
Primer on Geometrical Optics
• Light moves in straight lines through homogeneous media
at velocity: v = c/n(l)
• Example values for n(546nm):
Air
1.0
Water
1.3333
Cytoplasm
1.38
Glycerol
1.46
Crown Glass
1.52
Immersion Oil
1.515
Protein
1.51-1.53
Flint Glass
1.62
Reflection
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Reflection and Refraction at Transparent
Surface
Snell’s Law of Refraction
n1sin(Qi) = n2sin(Qr)
Total Internal Reflection Can Occur
Snell’s Law of Refraction
n1sin(Qi) = n2sin(Qr)
Total Internal Reflection is Used to ReDirect Light
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Light Guide
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?
Refraction is Usually Greater at Shorter
Wavelengths
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Refraction at Curved Lens Surfaces:
Action of Convex or Concave Lenses
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F’ is Primary Front Focal Point of Lens
Basic Action of Converging Lenses
Light parallel to the optical axis comes
into focus at a point F’, the back focal
point, located one focal length, f, from
principle plane (PPL) of the lens.
Parallel light at angle, Q, to the optical
axis comes into focus at a point, F”,
located in the back focal plane and at
a distance a = fsin(Q) from the focal
point, F’.
Homework: What is an easy way to
measure the approximate focal length of
a lens
Basic Action of Converging Lenses (cont.)
Light emanating from the front focal
point, F’, located a distance, f, on the
optical axis from the PPL will emerge
Parallel to the optical axis.
Light emanating from a point in the
front focal plane, FFP, at distance a
from the optical axis, will emerge as a
parallel beam of light at angle, Q, to
the optical axis, where a = fsin(Q).
Example: Flashlight
Real-Image Formation
As Object Moves
Closer to Lens
Three Light Rays Can Define Real
Image Formation
M = I/O =i/o
1/i +1/o = 1/f
Image Formation in the Human Eye
Resolution Limitations of the Human Eye
Limits to Accommodation
Unresolved
Resolved
Resolution Test
Ocular is a Single Lens Magnifier
Magnification (angular) = 250 mm/f
Homework: What is The Ocular Focal
Length for the Following
Magnifications?
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5X _________
10X _________
20X _________
25X _________
The Objective Forms a Real Image At the
Ocular Front Focal Plane: The Primary or
Intermediate Image Plane (IIP)
Conventional Optics
Objective with finite Focal Length
(Optical Tube Length, OTL, Typically 160 mm)
Mob = OTL/fob
Total Magnification = Mob x Moc = OTL/fob x 250mm/foc
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Homework: For Finite Focal Length
Objective and OTL = 160 mm, what is
focal length for the following Objective
Magnifications
4X _________
10X ________
20X ________
40X ________
60X ________
100X _______
How to
Insert
Filters
Above
Objective
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Inducing
Image
Aberration
Mob = ffocusing/fob
Zeiss introduced infinity corrected
objective for biomedical scopes in late
1980’s, Nikon, Leica and Olympus
followed by Mid-1990’s
Also, field of view in ocular enlarged
from 18 mm to 24-25 mm.
Numerical Aperture (NA) of Collection
(a) or Illumination (b)
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How to Measure Objective NA
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Homework for Thursday: Go to
http://micro.magnet.fsu.edu/primer/index.html
and work through:
• Light and color
• Anatomy of Microscope:
-Introduction
-concept of Magnification
-Microscope Optical components:
-Geometrical construction of Ray Diagrams
-Perfect Lens Characteristics
-Perfect 2 Lens Characteristics