Light Microscopy Lecture, LSU Spring 2010

8/9/2019 Light Microscopy Lecture, LSU Spring 2010

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Course Expectations

A final exam 30%

Research project involving at least 3

techniques (written paper and

presentation) 40%

Lab exercises (notebook) 10%

A final practical exam 20%


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Examples of Techniques

Fluorescent localizations Modified contrast microscopy (phase, DIC,

Hoffman, Polarized, Rheinberg)

Confocal/deconvolution Laser microdissection techniques

Video microscopy

Digital photography Film photography


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Discovery is based on the ability to

observe phenomena that everyone else

has overlooked Microscopy is the interpretive use of the

microscope

Importance of Observation


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History

Lenses have been used for well over2000 years - burning spheres

Simple hand lenses available for over1000 years (magnifiers)

1st useable compound microscope wasproduced in 1590 Janssen

Use elegantly demonstrated by R. Hooke

in 1670s Popularized by Leeuwenhoek 1673

Initiated race for shorter focal lengths


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Improving a 325 year old instrumentImproving a 325 year old instrument

Major Improvements in 1800s

1827, Amici; produced achromatic lenses,

coverslips and immersion media optics

1860s to 1890s Ernest Abbe, improved condensers, immersion oils

Otto Schott, formulated glass melts-color correction;

First apochromatic lenses /c Karl Zeiss

Professor Kohler, illumination systems


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Advent of new Techniques

Phase Microscopy Zernicke, 30s Darkfield contrast

Polarizing microscopy

Rheinberg iillumination

Differential Interference Contrast

Nipkow disk confocals 1970s

Laser scanning confocal microscopy 1980s

Two photon microscopy 1998-2000 Quest for a more powerful microscope


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Defining Microscope Power

Function of the course

Relates ultimately to resolution (not magnification)

Dependent on specimen properties Related to functions of a diffraction limited

system


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The Modern Microscope


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Understanding Light

Electromagnetic radiations, 4 properties Wave (sine wave oscillations ofE and B fields)

Particle (quanta of energy)

Ray (depicting light paths as straight line) Vector (amplitude and angle referenced to a

point)

Most of light phenomena are associated with

wave properties - for our purposes


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Light

Qualities

Depicted as E

vectors

Having coaxial planes of

propagation


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Basic properties of waves

Propagation

Velocity

Interference

Constructive

Destructive


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Depicting a Wave Remember these are electro magnetic- waves


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The Electromagnetic Spectrum


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Properties of light perceived by the Eye

Color Wavelength dependent

Brightness intensity of light is

proportional to amplitude of waves I E A2

Contrast light intensity of object must be

different from background C = I/Ib


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Basic wave properties


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Basic Definitions

wavelength distance between two successive in

phase points v velocity (speed of light in medium, phase

velocity)

n refractive index (ratio of v in vacuum vs medium) R - frequency (1/)


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Interactions of Waves with Matter

Absorption - interference with litepropagation

Transmission just passing thru

Refraction Get Bent

Reflection Mirror, Mirror

Diffraction Get Bent Again

Polarization No, not a cold snap

Fluorescence/phosphorescence Shine On


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Constructive/destructive


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Velocity Changes in Media


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Substance n

Vacuum 1.000000

Air 1.000277

Water 1.33

Ethanol 1.36

Rock salt 1.54

Carbon disulfide 1.63

Optical glasses 1.45-1.95

Diamond 2.42

Example Refractive Indices


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Refraction Snells Law

n1

n2

i

r

Sin I n2

=Sin r n1

May also determine by knowing the velocities of light in the two different media


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Demo of Snells Law


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Reflection

normalIncident ray Reflected ray

i r

Critical angle


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Image Formation with a thin lens

Diagram of simple thin lens image formation

f

f

F F


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http://micro.magnet.fsu.edu/primer/anatomy/kohler.html

Light Microscopy Lecture, LSU Spring 2010

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