View
220
Download
0
Category
Preview:
Citation preview
Overview – Schedule for Basic Microscopy Training – MBL – December 2008
I. Basics Properties of Light
Basic Theories – Corpuscular, Wave (Quantum Theory omitted)
Terms
Interaction of Light with Materials
II. Geometric Optics Understanding Beam Paths
Constructing a “Microscope”
III. The modern Microscope and its Components Basic Discussion of Components – from the Light Source to the Eyepieces
Proper Setup and Alignment – Koehler Illumination
IV. Practical Aspects Glass
Airy Disk, PSF
Aberrations
Selecting an Objective
Overview
Morning
Before D
inner
II
Properties, Terms, Phenomena of LightProperties, Terms, Phenomena of Light
December 2008December 2008
Rudi Rottenfusser – Carl Zeiss MicroImaging
I. Properties of Light and Basic Theories– Corpuscular, Wave Theories (Quantum Theory not considered)
II. Characteristics of Waves; Spectrum– Amplitude– Wavelength / Spectrum– Metric Terms
III. Interaction of Waves with each other– Coherence - Incoherence– Interference (Coherence)
IV. Phenomena of Light - Interaction of Waves with Material– Diffraction – Reflection– Refraction– Critical Angle– Total Reflection – Dispersion, Cover Slips– Transmission; Absorbance; ND Filters– Polarized Light
Basics about Light and Waves
The apparent linear propagation of light was known since antiquity. The ancient Greeks believed that light consisted of a stream of corpuscles.
The first measurement of the velocity of light was carried out by the Danish astronomer Olaus Roemer in 1676. Today the velocity of light is known very accurately as 2.992926• 108 m/sec ( 300,000 km/sec 187,500 miles/sec).
Any satisfactory theory of light must explain its origin and disappearance and its changes in speed and direction while it passes through various media. Partial answers to these questions were proposed in the 17th century by Newton, who based them on the assumptions of a corpuscular theory.
Corpuscular Theory
(Excerpt from Encyclopedia Britannica)
The English scientist Robert Hooke and the Dutch astronomer, mathematician, and physicist Christiaan Huygens, propose a “wave” theory in place of the corpuscular theory.
In the early 19th century, the British physicist and physician Thomas Young distinguishes between the two theories by demonstrating interference. The French physicist Augustin Jean Fresnel decisively favors the wave theory.
In 1872 Ernst Abbe formulates his theory of microscopic imaging, defining what’s known as the Abbe sine condition, which becomes the basis for
modern microscope design.
Wave Theory(Excerpt from Encyclopedia Britannica)
Amplitude
The Amplitude of a wave is half the difference in height between the crest and the trough.
The Intensity is proportional to the square of the amplitude.
II - Characteristics of Waves
11
Wavelength
II - Characteristics of Waves
Related Terms
The period of a wave is the time it takes for two crests or two troughs to travel to the same point in space.
Example: Measure the time from the peak of a water wave as it passes by a specific marker to the next peak passing by the same spot.
II - Characteristics of Waves
The frequency of a wave is the reciprocal of its period = 1/period [Hz or 1/sec]
Example: If the period of a wave is three seconds, then the frequency of the wave is 1/3 per second, or 0.33 Hz.
The velocity (or speed) at which a wave travels can be calculated from the wavelength and the period.
It is determined by dividing the distance one wave travels by the time it takes to do this.
frequencywavelengthperiod
wavelengthvelocity
II - Characteristics of Waves
The frequency remains constant while light travels through different media. It is the wavelength, which changes.
A combination of all wavelengths originating from the source
What is “White Light”?
Pl.note that wavelength relationship exceeds visible range
II - Characteristics of Waves
Named Spectral Lines
404.7 h Violet Hg
435.8 g Blue Hg
480.0 F‘ Blue Cd
486.1 F Blue H
546.1 e Green Hg
587.6 d Yellow He
589.3 D Sodium
643.8 C‘ Red Cd
656.3 C Red H
706.5 r Red He
759.4 A Potassium
(µm)
En
erg
y
Speed of light = 2,99,792458 300,000 km/sec
Energy
c
hhE 23410626176.6 Wsh
Metric
P
refi
xes
Prefix Symbol Factor Zeta Z 1021 1,000,000,000,000,000,000,000
Exa E 1018 1,000,000,000,000,000,000
Peta P 1015 1,000,000,000,000,000
Tera 1) T 1012 1,000,000,000,000
Giga 2) G 109 1,000,000,000
Mega 3) M 106 1,000,000
kilo 4) k 103 1,000
hecto 5) h 102 100
Deka D 101 10
100 1
deci 6) d 10-1 0.1
centi 7) c 10-2 0.01
milli 8) m 10-3 0.001
micro 9) µ 10-6 0.000 001
nano 10) n 10-9 0.000 000 001
Ångstrøm 13) Å 10-10 0.000 000 000 1
pico 11) p 10-12 0.000 000 000 001
femto 12) f 10-15 0.000 000 000 000 001
atto a 10-18 0.000 000 000 000 000 001
zepto z 10-21 0.000 000 000 000 000 000 001
1) TBytes = TeraBytes = 1012 Bytes (storage capacity of computers)
2) Ghz = Gigahertz = 109 Hertz = 109 1/s (frequency)
3) M= MegOhm = Million Ohm (resistance)
4) kW = kilowatt = 1000 Watt (power) ¾ HP
5) hl = hectoliter = Hundred liters (volume of barrels)
6) (dm)3 = decimeter3 = cubic decimeter = 1 liter
7) cm = centimeter (length) 3/8”
8) mV = millivolt (voltage)
9) µA = microampere (current)
10) ng = nanogram (weight)
11) pf = picofarad (capacitance)
12) fl = femtoliter (volume)
13) Ångstrøm – used primarily in Electron Microscopy
Exam
ple
s:
English/metric conversion (exact): 1” = 25.4 mm | 1/1000” = 1 mil = 25.4 µm
1 Nm = 1 Ws
N = Newton = force that’s given to a mass of
1 kg and acceleration of 1 m/s2
Ws = Watt sec or Joule = energy released in one
second by a current of one Ampere through a resistance of one Ohm
State of Polarization
II - Characteristics of Waves
Will be covered in great detail during Bob Hard’s Pol Section later on
Small phase differences between 2 waves cannot be detected by the human eye
Shifts between waves (Phase)
III – Interaction of Waves
• Coherence - What is it?
• Constructive Interference
• Destructive Interference
• Difference between Coherent and Incoherent waves
Interference
III – Interaction of Waves
d) Interference
Constructive Interference
BAC 222 2 BABABAI Intensity
Amplitude
Interference Term
Coh
ere
nt W
aves
Example:
A=5 >I = 25
B=3 >I = 9
C=5+3=8 >I = 64
III – Interaction of Waves
d) Interference
Constructive Interference
Destructive Interference
Amplitudes
Intensity
BAC 222 2 BABABAI
Interference Term
Coh
ere
nt W
aves
Example:
A=5 >I = 25
B=3 >I = 9
C=5-3=2 >I = 4
III – Interaction of Waves
Intensity 2222222222 JIHGFEDCBAI
From “msnucleus.org”
ABCDEFGHIJ
Incoherent Waves
III – Interaction of Waves
Phenomena of Light Phenomena of Light
IV – Interaction of Waves with Material
Reflection
When a beam of light strikes a surface at an angle measured from a line perpendicular to that surface, it is reflected in the opposite direction at an angle equal in size
90°
’
(angle of incidence) = ’(angle of reflection)
Normal (perpendicul
ar to interface of
different materials)
Variations
(Types of Reflection)
Specular Diffuse Retro
- the bending of light as it passes from one material to another
n1
1
2
n2
Normal (perpendicular to interface of different materials)
Snell’s Law:
n1 sin 1 = n2 sin 2
Refraction
2
1
1sin
2sin
n
nβ
Medium Refractive Index
Vacuum 1
Air 1.0003
Water 1.33
Glycerin 1.46
Immersion Oil 1.518
Glass 1.56 – 1.46
Diamond 2.42
Quartz 1.544e / 1.553o
medium
sm
medium
vacuum
velocityvelocity
velocityn
81099792.2
Refractive indexRefractive index n
Velocity in medium [km/s]
299792.458
299703
225408
205337
197492
192175 - 205337
123881
194166 / 193041
medium
skm
medium nv
299792
n1
1
2
n2 n1
Light beam through a plane-parallel glass plate
4
3
2
1
?
n1
1
2
n2 n1
Light beam through a plane-parallel glass plate
1
90o 90o
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction (Marching Band Analogy)
Refraction in a Prism
Going from dense to less dense medium
n2
n1
“Critical Angle”
.crit
n2
n1
2
11
.sin
n
ncrit
Snell’s Law: n1 sin 1 = n2 sin 2
2
1sinn
ncritical
sin 1 = 1
2
11
.sin
n
ncrit
n1 = water (tissue) = 1.35
n2 = crown glass = 1.52
n1 = air = 1.00
n2 = crown glass = 1.52
Example 1
n1 = air = 1.00
n2 = diamond = 2.42
Example 2 Example 3
sin = 0.658
Critical angle = 41o
sin = 0.888
Critical angle = 62.6o
sin = 0.413
Critical angle = 24.4o
Total Reflection
n2
n1
'
The separation of white light into spectral colors as a result of different amounts of refraction by different wavelengths of light.
Dispersion
nF‘ = refractive index at the blue Cadmium line (480nm)
nC‘ = refractive index at the red Cadmium line (644nm)
'' CF nnn
Dispersion in a plane-parallel glass plate
(e.g. slide, cover slip, window of a vessel)
Which expression is commonly used for “unwanted” dispersion?Chromatic Aberration…
“White” Light
Dispersion of different materials:
Material nblue (486nm) nyellow (589nm) nred (656nm) Dispersion
(~)
Crown Glass 1.524 1.517 1.515 0.009
Flint Glass 1.639 1.627 1.622 0.017
Water 1.337 1.333 1.331 0.006
Cargille Oil 1.530 1.520 1.516 0.014
Use 0.170 mm thick cover slips !
Types and Thickness Ranges
No.0 ......... 0.08 - 0.12 mm
No.1 ......... 0.13 - 0.17 mm
No.1.5....... 0.16 - 0.19 mm
No.2 ......... 0.19 - 0.23 mm
No.3 ......... 0.28 - 0.32 mm
No.4 ......... 0.38 - 0.42 mm
No.5 ......... 0.50 - 0.60 mm
Choose the right cover glass!
No.1.5....... 0.16 - 0.19 mm
Absorbance (Extinction) A
λI
IT
0
1
dCA TA
1log
= Absorption Coefficient
C = Concentration
d = Path Length
I0 I1
Transmission T
d
AT
101
(Lambert-Beer Law)
Neutral Density (ND) Filters
Optical Density Transmission (%) f-stop Optical Density Transmission (%) f-stop
0 (no filter) 100.000 0 1.7 2.000
0.1 80.000 1/3 1.8 1.600 6
0.2 63.000 2/3 1.9 1.250
0.3 *50.000 1 2 1.000
0.4 40.000 1+ 1/3 2.1 0.800 7
0.5 32.000 1+ 2/3 2.2 0.630
0.6 *25.000 2 2.3 0.500
0.7 20.000 2.4 0.400 8
0.8 16.000 2.5 0.320
0.9 *12.500 3 2.6 0.250
1 10.000 2.7 0.200 9
1.1 8.000 2.8 0.160
1.2 *6.300 4 2.9 0.125
1.3 5.000 3 0.100 10
1.4 4.000 3.1 0.080
1.5 *3.200 5 3.2 0.063
1.6 2.500 3.3 0.050 11
*standard Zeiss filters
Refracted visible light / Dispersion
Primary reflected light
Illumination
Secondary reflected
light
Secondary Refracted + Stray light
Scatter and decrease in intensity with increasing pathlength
Multiple Phenomena in Optical Systems
Filter
Polarized Light
Birefringent Material
Polarizer
Analyzer
Polarized Light
Birefringent Material
Polarizer
Analyzer
Polarizer
Analyzer
Polarized Light
Birefringent Material
Polarizer
AnalyzerPolarizer
Analyzer
Polarized Light
Birefringent Material
Polarizer
Analyzer
Polarized Light
Birefringent Material
Polarizer
Analyzer
• The numerical difference between the maximum and minimum refractive indices of anisotropic substances. nγ - nα.
• Birefringence may be qualitatively expressed as • low (0 - 0.010), • moderate (0.010 – 0.050)• high (>0.050) • extreme (>0.2)
• Birefringence may be determined by use of compensators, or estimated through use of a Michel-Lévy Interference Color Chart.
Birefringence
dn Path Optical
dnndndn boBackgroundObject Difference Path Optical
The Michel Lévy Color Chart
3rd
OrderRed
1st OrderRed
1st OrderRed
2nd OrderRed
2nd
OrderRed
3rd
OrderRed
•An excellent introduction to this chart is provided at McCrone’s website http://www.modernmicroscopy.com/main.asp?article=15
LOW
< 0.010Moderate
0.010 – 0.050
High
> 0.050
Some Types of Birefringence
• Intrinsic or crystalline (Quartz, Calcite, Myosin Filaments, Chromosomes, Keratin, Cellulose Fibers)
• Form or Textural (Plasma membranes, Actin filaments, microtubules)
• Edge (resulting from diffraction at edges of objects embedded in a medium of different refractive Index)
• Strain (resulting from mechanical stress e.g. glass, plastic sheets)
• Circular –also known as- Optical Rotation (sugars, amino acids, proteins)
The wave exhibits electric (E) and magnetic (B) fields whose amplitudes oscillate as a sine function over dimensions of space or time. The amplitudes of the electric and magnetic components at a particular instant or location are described as vectors that vibrate in two planes perpendicular to each other and perpendicular to the direction of propagation. At any given time or distance the E and B vectors are equal in phase. For convenience it is common to show only the electric field vector (E vector) of a wave in graphs and diagrams.
Light as an electromagnetic wave
E
y
xz
Ey
Ex
E E
Polarized Light
Polarized Light and Birefringence
Polarized Light and Birefringence
Interface with birefringent Material
n = higher refractive index > slower waven = lower refractive index > faster wave
Linearpolarizer
¼ wave plate
Unpolarizedlight linearlypolarized
Circularlypolarized
How to create circularly polarized light
xz
Ex
E
Circularly Polarized Light
1
1
2
2
3
3
4
4
5
5
E
y
xz
Ey E
x
E E
Sénarmont Compensator*
¼ wave plate, located before analyzer, is oriented with its birefringence parallel to the polarizer or analyzer. Therefore, there will be no effect on the polarized beam.
Birefringence produced by specimen (occurring at 45˚), will be converted by ¼ wave plate into circular polarized light which can pass through the analyzer.
By rotating the analyzer, it is possible to introduce “bias” birefringence because it will not be parallel to ¼ wave plate any more. * 1st described by de Sénarmont in 1840
Recommended