Light Microscopy and

Digital Imaging

Levi A. Gheber
Department of Biotechnology Engineering
Ben-Gurion University of the Negev
Course Outline
Electromagnetic waves
Color and light
Sources of visible light
Introductory Optics
Basics of Optical Microscopy
Specialized Microscopy Techniques
Digital Imaging in Optical Microscopy
(?)Light Sources for Optical Microscopy
Electromagnetic waves
Electromagnetic radiation
Duality of light
Reflection
Refraction
Diffraction
Interference
Polarization
Birefringence
Electromagnetic radiation
Electromagnetic wave
Amplitude
n=c/l
Wavelength
Frequency

E = hn = hc/l
Electromagnetic radiation states
Electromagnetic radiation states (cont.)
monochromatic light consists of waves all having the same
wavelength and frequency (same color)
polychromatic light usually appears as white due to contributions from
the mixture of all or most wavelengths in the spectrum
non-polarized: the electric field vectors vibrate in all planes lying
perpendicular to the direction of propagation
plane-polarized: all of the electric vectors vibrating in a single plane
perpendicular to the direction of propagation.
non-coherent light displays a variety of phase relationships among the
wavelengths present in the spectrum
coherent light is composed of wavelengths that are in phase with each
other
collimated: light waves that have coaxial, relatively non-diverging
paths as they travel through space
divergent or non-collimated light spreads to varying degrees while
traveling through space
Duality of Light
 Duality of light

Duality of light (cont.)

 Duality of light

Duality of light (cont.)

E = mc2 = hn
Reflection of light
Refraction of light
n (Refractive Index) = c/v

Snell's Law
n1 sin(q1) = n2 sin(q2)

Material Refractive Index

Air 1.0003

Water 1.333

Glycerin 1.473

Immersion Oil 1.515

Glass (Crown) 1.520

Glass (Flint) 1.656

Zircon 1.920

Diamond 2.417

Lead Sulfide 3.910

 Refraction of light

Refraction of light (cont.)

Relative Index of Refraction = sin(q1)/sin(q2) = nr = n2/n1

 Refraction of light

Dispersion of light

Blue Yellow Red

Dispersion = n = (n(D)-1)/(n(F)-n(C))
Material (486.1 nm) (589.3 nm) (656.3 nm)
F line (H) D line (sodium) C line (H)

Crown Glass 1.524 1.517 1.515 57.4

Flint Glass 1.639 1.627 1.622 37.9

Water 1.337 1.333 1.331

55.5
Cargille Oil 1.530 1.520 1.516
37.1

Carbon Disulfide 1.652 1.628 1.618

18.5
 Refraction of light

Critical Angle of Reflection

sin(qc) = n(1)/n(2)
Diffraction of light

sin(q) = ml/d
 More details on Fraunhofer diffraction

d=1, L=0.5 d=5, L=0.5 d=25, L=0.5

1.2 1.2 1.2

1 1 1

0.8 0.8 0.8

0.6 0.6
0.6

0.4 0.4
0.4

0.2 0.2
0.2
0 0
0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
0 5 10 15 20 25 30 35 -0.2 -0.2
 Diffraction of light

Airy Patterns

d = 1.22 l(f/D)

NA (Numerical Aperture) = n sin (q)

r = 1.22 l/(2NA)
Interference of light
 Interference of light

Youngs double slit experiment

Polarization of light
 Polarization of light

Brewsters angle

n = sin(qi)/sin(qr) = sin(qi)/sin(q90-i) = tan(qi)

 Polarization of light

Nicol Polarizing Prism

 Polarization of light

Cross Polarizers

Malus Law

I = I(o) cos2q
 Polarization of light

Polarization of Scattered Light

Rayleigh scattering

blue skies
white clouds
red sunsets
atmospheric
polarization
 Polarization of light

Elliptical and Circular Polarization

 Polarization of light

Applications
 Birefringence

O-ray: E Optical Axis

E-ray: E || Optical Axis
 Birefringence

Optical Path Difference

Birefringence (B) = |ne - no|

Optical Path Difference D = (n1 - n2) t (Thickness)

Retardation (G) = Thickness (t) x Birefringence (B)

G = t |ne - no|
 Birefringence

Birefringence between cross-polarizers

Color and Light
Color temperature
Primary colors
Light Filters
Color Temperature Daylight Sources
Color Temperature
(K)

Skylight 12000 to 18000

Overcast Sky 7000

Noon Sun/Clear Summer Sky 5000 to 7000

Noon Sun/Clear Winter Sky 5500 to 6000

Photographic Daylight 5500

Noon Sunlight
4900 to 5800
(Date Dependent)

Average Noon Sunlight

5400
(Northern Hemisphere)

Sunlight at 30-Degree Altitude 4500

Sunlight at 20-Degree Altitude 4000

Sunlight at 10-Degree Altitude 3500

Sunrise and Sunset 3000

Color Temperature
Artificial Sources
(K)

White LED 6500 to 9500

Electronic Flash 5500 to 6500

Xenon Burner 6000

White Flame Carbon Arc 5000

Warm White Fluorescent Tubes 4000

Aluminum-Filled Flash Bulbs

3800
(M2, 5, & 25)

500-Watt 3400 K Photoflood 3400

12 Volt/100 Watt
3200
Tungsten-Halogen @ 9 Volts

12 Volt/50 Watt
3200
Tungsten-Halogen @ 9 Volts

100-Watt Household Lamp 2900

40-Watt Household Lamp 2650

Gaslight 2000 to 2200

Candlelight
2900
(British Standard)
 Color Temperature

Color conversion and Balancing filters

Planks black body radiation
 Color Temperature

White balance calibration in digital imaging

Primary colors

W R G B
 Primary Colors

Primary colors (cont.)

 Light Filters

OD (Optical Density) = log(Absorbance)

A (Absorbance) or Opacity = 1/T (Transmittance)

OD = log(1/T) = -log(T)
 Light Filters

Absorption filters

Color Compensating, Conversion, and Balancing Filters

 Light Filters

Interference Filters
 Light Filters

Neutral Density Filters

Neutral Density Filter Specifications

Transmission
Designation Density
(Percentage)

ND-80 0.1 80

ND-70 0.15 70

ND-60 0.2 60

ND-50 0.3 50

ND-40 0.4 40

ND-30 0.5 30

ND-25 0.6 25

ND-20 0.7 20

ND-16 0.8 16

ND-13 0.9 13

ND-10 1.0 10

ND-1 2.0 1

ND-0.1 3.0 0.1

ND-0.01 4.0 0.01

Sources of Visible Light
Wavelength Range
Perceived Color
(nanometers)

340-400 Near Ultraviolet (UV; Invisible)

400-430 Violet

430-500 Blue

500-570 Green

570-620 Yellow to Orange

620-670 Bright Red

670-750 Dark Red

Over 750 Near Infrared (IR; Invisible)

 Sources of Visible Light

Incandescent light sources

 Sources of Visible Light

Fluorescent light sources

 Sources of Visible Light

Arc Discharge Lamps

 Sources of Visible Light

White LED
 Sources of Visible Light

LASER
Introductory Optics
Lenses and geometrical optics
Mirrors
Prisms and beamsplitters
Lenses and geometrical optics
How Lenses work
Ray construction
 Simple thin lens
1/a + 1/b = 1/f

1/a + 1/b = 1/f b=af/(a-f)

Magnification (M) = (Image Height)/(Object Height) = b/a = f/(a-f)

Wave diagram representation
 Lenses and Geometrical Optics

Aberrations
On-Axis
Chromatic
Spherical
Off-Axis
Coma
Astigmatism
Geometrical
Field Curvature
Barrel
Pincushion
 Lenses and Geometrical Optics

Chromatic Aberration

Dispersion! Chromatic difference of magnification

 Lenses and Geometrical Optics

Spherical Aberration
 Lenses and Geometrical Optics

Coma Aberration
 Lenses and Geometrical Optics

Astigmatism
 Lenses and Geometrical Optics

Field curvature
 Lenses and Geometrical Optics

Geometric Aberrations
Summary
Mirrors
 Mirrors

Hyperbolic and Elliptical Mirrors

 Mirrors

Spherical Mirrors

1/d0 + 1/d1 = 1/f

 Mirrors

Reflectance of metallic coatings

 Mirrors

Dielectric mirrors

Applications
Dichroic mirrors
Hot mirrors (IR reflecting)
Cold mirrors
 Mirrors

Aberration in Mirrors
 Prisms and beamsplitters

Prisms and Beamsplitters

 Prisms and beamsplitters

Tunnel Diagrams

Apparent thickness = d/n

 Prisms and beamsplitters

Other prisms
 Prisms and beamsplitters

Polarizing Prisms

O-ray: E Optical Axis

E-ray: E || Optical Axis

ne >no

ne >no
Birefringent Indices of refraction

Tourmaline 1.669 1.638

Calcite 1.6584 1.4864
Quartz 1.5443 1. 5534
Sodium Nitrate 1.5854 1. 3369
Ice 1.309 1.313
Rutile (TiO2 ) 2.616 2.903

Birefringence (B) = |ne - no|

 Prisms and beamsplitters

Refracting (Dispersing) Prisms

Glass Refractive Abbe

Formula Index Number

Fused Quartz 1.4585 67.8

BK 7 1.5168 64.17

Light Barium Crown 1.5411 59.9

Light Flint 1.5725 42.5

Dense Flint Glass 1.620 36.37

Extra Dense Flint Glass 1.6725 32.20

Very Dense Flint Glass 1.728 28.41

 Prisms and beamsplitters

Beamsplitters
Basics of Optical Microscopy
Magnification Concept
Microscope optical components
Stereo Microscopy
Magnification
Magnification
 Concept of Magnification

Compound microscope
Microscope Components
Microscope Component Attributes

Light Source, Collector Lens, Field

Illuminator Diaphragm, Heat Filters, Light Balancing
Filters, Diffuser, Neutral Density Filters

Condenser Iris, Darkfield Stop, Aperture

Light Mask, Phase Annulus, Polarizer, Off-Center
Conditioner Slit Aperture, Nomarski Prism, Fluorescence
Excitation Filter

Numerical Aperture, Focal Length,

Condenser Aberrations, Light Transmission, Immersion
Media, Working Distance

Slide Thickness, Cover Glass Thickness,

Immersion Media, Absorption, Transmission,
Specimen
Diffraction, Fluorescence, Retardation,
Birefringence

Magnification, Numerical Aperture, Focal

Length, Immersion Media, Aberrations, Light
Objective
Transmission, Optical Transfer Function,
Working Distance

Compensator, Analyzer, Nomarski Prism,

Objective Iris, Phase Plate, SSEE Filter,
Image Filter Modulator Plate, Light Transmission,
Wavelength Selection, Fluorescence Barrier
Filter

Magnification, Aberrations, Field Size, Eye

Eyepiece
Point

Human Eye, Photographic Emulsion,

Detector Photomultiplier, Photodiode Array, Video
Camera
Microscope components
Overview
Microscope Illumination
Objectives
Eyepieces
Condensers
Stages
Perfect Lens Revisited

d = f sin(a)

object space image space

Conjugate Planes
1/a + 1/b = 1/f
b=af/(a-f)

M = h(2)/h(1) = b/a=f/(a-f)
Illuminator (lamp)
Condenser
Objective
 Eyepiece (projection mode)

Intermediate image between f and 2f!

Real image!
Eyepiece (virtual image)
Fixed tube length
Infinity corrected
Conjugate image planes
Cutaway diagram Olympus BX51
Cutaway Diagram Nikon Eclipse E200
Cutaway Diagram Olympus BH2
Microscope Illumination
Afocal (non-focused) Illumination
does not form an image of the light source at some point in the optical pathway

Critical (Nelsonian) Illumination

Focused image of light source in the plane of the sample

Khler Illumination
Critical Illumination
Khler Illumination
Illumination Systems
Condenser Illuminating Cones
Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the
condenser and reaching the specimen from all azimuths

NA=1.2 NA=0.60 NA=0.30 NA=0.15

Condenser NA
Planachromat objective (NA = 0.75)

Cond NA = 0.81 0.54 0.18

Area of illumination
The illuminated area of the specimen plane must be at least as large as the
field of view for any given objective
For Eyepiece with FN 18
Objective Designation Numerical Aperture Field of View Diameter

Planachromat 1x 0.04 18.0

D = (FN) / M
Planachromat 2x 0.06 9.0
Field (of view) Number = the diameter of the viewfield
Planachromat 4x 0.10 4.50
expressed in millimeters and measured at the
Planachromat 5x 0.15 3.60
intermediate image plane
Planachromat 10x 0.25 1.80

Planachromat 20x 0.40 0.90

Planachromat 40x 0.65 0.45

Planapochromat 50x 0.90 0.36

Planapochromat 60x 0.95 0.30

Planapochromat 100x 1.40 0.18

 Khler Illumination Summary
Conjugate planes in the path of the illuminating light rays :

The lamp filament.

The condenser aperture diaphragm (at the front focal plane of the condenser).
The back focal plane of the objective.
The eye point (also called the Ramsden disk) of the eyepiece

Conjugate planes in the image-forming light path:

The field diaphragm.
The focused specimen.
The intermediate image plane (i.e., the plane of the fixed diaphragm of the eyepiece).
The retina of the eye or the film plane of the camera.

Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the
condenser and reaching the specimen from all azimuths. Has large influence on working NA of the
system and resolution.

The field diaphragm in the base of the microscope controls only the width of the bundle of light rays
reaching the condenser--it does not affect the optical resolution, numerical aperture, or the intensity of
illumination.

Illumination intensity should only be controlled through the use of neutral density filters placed into
the light path or by reducing voltage to the lamp (although the latter is not usually recommended,
especially for photomicrography) (color temperature!!).
Practical adjustment of Illumination

60% - 90%

Phase telescope or Bertrand lens

Start with 10X and repeat for

any change of objective!!
Reflected light illumination
 Image Brightness
Image Brightness (NA/M)2

F(trans) = 104 NA2/M2

F(epi) = 104 (NA2/M)2
Numerical
Correction Magnification F(trans) F(epi)
Aperture

Plan Achromat 10x 0.25 6.25 0.39

Plan Fluorite 10x 0.30 9.00 0.81

Plan Apo 10x 0.45 20.2 4.10

Plan Achromat 20x 0.40 4.00 0.64

Plan Fluorite 20x 0.50 6.25 1.56

Plan Apo 20x 0.75 14.0 7.90

Plan Achromat 40x 0.65 2.64 1.11

Plan Fluorite 40x 0.75 3.52 1.98

Plan Apo 40x (oil) 1.30 11.0 18.0

Plan Fluorite 60x 0.85 2.01 1.45

Image Brightness (Fluorescence) NA4/M2 Plan Apo 60x (oil) 1.40 5.4 10.6

Plan Apo 100x (oil) 1.40 1.96 3.84

Plan Apo 100x (oil) 1.45 2.10 4.42

Plan Apo 100x (oil) 1.65 2.72 7.41

Reflection of lenses
 Brightness of Illumination
The size of the opening in the field diaphragm affects only
the diameter of the illuminated field and not its brightness
Arc Size
Current Luminous Flux Mean Luminous
Lamp (H x W)
(Amperes) (Lumens) Density (cd/mm2)
(Millimeters)

Mercury Arc
5 2200 1700 0.25 x 0.25
(100 Watt)
Xenon Arc
5.4 850 400 0.25 x 0.50
(75 Watt)
Xenon Arc
30 9000 3500 0.30 x 0.30
(500 Watt)
Tungsten-
8 2800 45 4.2 x 2.3
Halogen
Objectives
Parameters of interest

Manufacturer (Zeiss, Olympus, Nikon)

Linear Magnification (20X, 40X, 100X)
Optical Correction (Achro, Apo, Plan)
Numerical Aperture (0.45, 0.9, 1.3, Iris.)
Mechanical tube length (, 160, 180)
Cover glass thickness (0.17, corr)
Working distance (L, LWD)
Specialized optical properties (Ph, DIC)
Screw threads (RMS, M25, M32)
Immersion medium (Oil, Water)
Color codes
Parfocal distance
Objective Components
Common Corrections

Objective Spherical Chromatic Field

Type Aberration Aberration Curvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes

 Apochromats

Taylor-Cook Triplet Zeiss Tessar Orthoscopic Doublet Zeiss Orthometer

Field curvature correction
Coverslip Correction Collar
 Objective Specifications by Magnification
Plan Fluorite

4x 0.13 17.10

10x 0.30 16.00

20x 0.50 2.10

Magnification Numerical Aperture Working Distance (mm)

Achromat 40x 0.75 0.72

4x 0.10 30.00 40x (oil) 1.30 0.2

10x 0.25 6.10 60x 0.85 0.3

20x 0.40 2.10 100x (dry) 0.90 0.30

40x 0.65 0.65 100x (oil) 1.30 0.20

60x 0.80 0.30 100x (oil with iris) 0.5-1.3 0.20

100x (oil) 1.25 0.18 Plan Apochromat

Plan Achromat 2x 0.10 8.50

0.5x 0.02 7.00 4x 0.20 15.70

1x 0.04 3.20 10x 0.45 4.00

WD
2x 0.06 7.50 20x 0.75 1.00

4x 0.10 30.00 40x 0.95 0.14

10x 0.25 10.50 40x (oil) 1.00 0.16

20x 0.40 1.30 60x 0.95 0.15

40x 0.65 0.57 60x (oil) 1.40 0.21

50x (oil) 0.90 0.40 60x

1.20 0.22
(water immersion)
100x (oil) 1.25 0.17
100x (oil) 1.40 0.13
40x 0.65 0.48
100x (NCG oil) 1.40 0.17
100x 0.90 0.26
NCG = No Cover Glass
 l
R
2 NA
Depth of field l 1
2 NA Z
= sin a
nl 1
2 NA Z
= NA

nl
Z
NA2
dtot = ln/NA2

l/2NA Magnification Numerical Aperture

Depth of Field Image Depth
(mm) (mm)

a
Z 4x 0.10 15.5 0.13

10x 0.25 8.5 0.80

20x 0.40 5.8 3.8

40x 0.65 1.0 12.8

60x 0.85 0.40 29.8

100x 0.95 0.19 80.0

Depth of field vs. NA
Specification and identification
 Specification abbreviations
Abbreviation Type

Achro, Achromat Achromatic aberration correction

Fluor, Fl, Fluar, Neofluar, Fluotar Fluorite aberration correction

Apo Apochromatic aberration correction

Phase, PHACO, PC Phase Contrast
Plan, Pl, Achroplan, Plano Flat Field optical correction
Ph 1, 2, 3, etc. Phase Condenser Annulus 1, 2, 3, etc.
Extended Field
EF, Acroplan
(field of view less than Plan)
DL, DLL, DM, BM Phase Contrast: Dark Low, Dark Low Low, Dark medium, Bright Medium
N, NPL Normal field of view plan
PL, PLL Phase Contrast: Positive Low, Positive Low Low
Plan Apo Apochromatic and Flat Field correction
Phase Contrast: Positive Medium, Positive High Contrast (Regions with higher
PM, PH
refractive index appear darker.)
UPLAN Olympus Universal Plan (Brightfield, Darkfield, DIC, and Polarized Light)
Phase Contrast: Negative Low, Negative Medium, Negative High Contrast
LU Nikon Luminous Universal (Brightfield, Darkfield, DIC, and Polarized Light)
NL, NM, NH (Regions with higher
refractive index appear lighter.)
L, LL, LD, LWD Long Working Distance
Strain-Free, Low Birefringence,
ELWD Extra-Long Working Distance P, Po, Pol, SF
for Polarized Light
SLWD Super-Long Working Distance
UV transmitting
U, UV, Universal (down to approximately 340 nm)
ULWD Ultra-Long Working Distance
for UV-excited epifluorescence
Corr, W/Corr, CR Correction Collar
UIS Universal Infinity System (Olympus)
Adjustable numerical aperture
I, Iris, W/Iris M Metallographic (no coverslip)
(with iris diaphragm)
NC, NCG No Coverslip
Oil, Oel Oil Immersion
EPI Oblique or Epi illumination
Water, WI, Wasser Water Immersion
TL Transmitted Light
HI Homogeneous Immersion
BBD, HD, B/D Bright or Dark Field (Hell, Dunkel)
Gly Glycerin Immersion
D Darkfield
Differential or
DIC, NIC
Nomarski Interference Contrast
H For use with a heating stage
Chrome-Free,
CF, CFI U, UT For use with a universal stage
Chrome-Free Infinity-Corrected (Nikon)
Interferometry, Noncontact,
ICS Infinity Color-Corrected System (Zeiss) DI, MI, TI
Multiple Beam (Tolanski)
Royal Microscopical Society
RMS
objective thread size

Metric 25-mm objective thread;

M25, M32
Metric 32-mm objective thread
Numerical aperture and working distance
Optical Correction*
Numerical Working Distance
and
Aperture (Millimeters)
Magnification

ACH 10x 0.25 6.10

ACH 20x 0.40 3.00

ACH 40x 0.65 0.45

ACH, Achromat
ACH 60x 0.80 0.23
PL, Plan Achromat
ACH 100x (Oil) 1.25 0.13
PL FL, Plan Fluorite
PL 4x 0.10 22.0
PL APO, Plan Apochromat
PL 10x 0.25 10.5

PL 20x 0.40 1.20

PL 40x 0.65 0.56

PL 100x (Oil) 1.25 0.15

PL FL 4x 0.13 17.0

PL FL 10x 0.30 10.00

PL FL 20x 0.50 1.60

PL FL 40x 0.75 0.51

PL FL 100x (Oil) 1.30 0.10

PL APO 1.25x 0.04 5.1

PL APO 2x 0.06 6.20

PL APO 4x 0.16 13.00

PL APO 10x 0.40 3.10

WD
PL APO 20x 0.70 0.65

PL APO 40x 0.85 0.20

PL APO 60x (Oil) 1.40 1.10

PL APO 100x (Oil) 1.40 0.10

Objective Color Codes
Magnification Color Code

1/2x No Color Assigned

1x Black

1.25x Black

1.5x Black

2x Brown (or Orange)

2.5x Brown (or Orange)

4x Red

5x Red

10x Yellow

16x Green

20x Green

25x Turquoise

32x Turquoise

40x Light Blue

50x Light Blue

60x Cobalt Blue

63x Cobalt Blue

100x White

150x White

250x White

Immersion Media Color Code

Oil Black

Glycerol Orange

Water White

Special Red
Objectives for Specialized Applications
 Resolution and numerical aperture
Objective Type
Wavelength (nanometers) Resolution (micrometers)

Plan Achromat Plan Fluorite Plan Apochromat 360 .19

400 .21
Resolution Resolution Resolution
Magnification N.A N.A N.A
(m) (m) (m)
450 .24
4x 0.10 2.75 0.13 2.12 0.20 1.375
500 .26
10x 0.25 1.10 0.30 0.92 0.45 0.61
550 .29
20x 0.40 0.69 0.50 0.55 0.75 0.37

600 .32
40x 0.65 0.42 0.75 0.37 0.95 0.29

650 .34
60x 0.75 0.37 0.85 0.32 0.95 0.29

100x 1.25 0.22 1.30 0.21 1.40 0.20 700 .37

Numerical Aperture and Airy disc size
 Objective selection
Brightness (Fluorescence) NA4/M2
R = 0.61 l /NA
6 6

dtot = ln/NA2
5 40 X 5

4 Depth of field (mm) 4

100 X

Viewfield (mm)
Brightness

3 3

2 2

20 X
1 1
10 X
0 0

0.2 0.4 0.6 0.8 1.0

Resolution (mm)
Image Formation
Conoscopic analysis of image formation

NAb < NAc < NAd

Immersion medium and resolution
Immersion media
 Standard immersion oils
Normal Light Microscopy - The most common immersion oil meeting two viscosity requirements (Types
A and B): 150 and 1250 centistokes. These oils can be blended to produce intermediate viscosities.
High Viscosity Oil (Type NVH) - Having a viscosity of 21,000 centistokes, this oil is popular for inverted,
horizontal, and inclined applications and also for very long working distance objectives and samples that
utilize wide condenser gaps.
Fluorescence Microscopy (Type DF) - An oil that yields the highest resolution with fluorescence
microscopy. This oil produces a background color that is pale green.
Fluorescence Microscopy (Type FF) - Improved oil that has essentially no background fluorescence
and is crystal clear and non-hydroscopic. This oil is often termed "universal" oil because it can be
substituted with confidence for virtually any other immersion oil for fluorescence applications.

Type A Type B Type NVH

Property

nF (Hydrogen F-line) 1.5236 1.5236 1.5227

nC (Hydrogen C-line) 1.5115 1.5115 1.5118
nD (Sodium D-line) 1.5150 1.5150 1.5150
ne (extraordinary) 1.5180 1.5180 1.5176
Mean Dispersion (nF - 0.0121 0.0121 0.0109
nC )
Abbe Number (VD) 42.6 42.6 47.2
Temperature Coefficient 0.00033 0.00033 0.00031
(-dn/dt)
Fluorescence very low low low
(ultraviolet)

Viscosity (cSt or mm2/s) 150 (low) 1250 (high) 2100

(v. high)

Cargille Immersion Oils

Common immersion media

Material Refractive Index

Air 1.0003
Water 1.333
Glycerin 1.4695
Paraffin oil 1.480
Cedarwood oil 1.515
Synthetic oil 1.515
Anisole 1.5178
Bromonaphthalene 1.6585
Methylene iodide 1.740
Water immersion objectives
Working distance and parfocal length

Common Objective Working Distances

Numerical Working
Manufacturer Correction Magnification
Aperture Distance

Nikon PlanApo 10x 0.45 4.0 mm

Nikon PlanFluor 20x 0.75 0.35 mm

PlanFluor
Nikon 40x 1.30 0.20 mm
(oil)

PlanApo
Nikon 60x 1.40 0.21 mm
(oil)

PlanApo
Nikon 100x 1.40 0.13 mm
(oil)

Long Working Distance Objectives

Numerical Working
Designation Magnification
Aperture Distance

ELWD 20x 0.40 11.0 mm

ELWD 50x 0.55 8.7 mm

ELWD 100x 0.80 2.0 mm

SLWD 10x 0.21 20.3 mm

SLWD 20x 0.35 20.5 mm

SLWD 50x 0.45 13.8 mm

SLWD 100x 0.73 4.7 mm

Mechanical tube length
Infinity Optical Systems
Tube Lens
Parfocal Distance
Manufacturer Focal Length Thread Type
(Millimeters)
(Millimeters)

Leica 200 45 M25

Nikon 200 60 M25

Olympus 180 45 RMS

Zeiss 160 45 RMS

M=fT/fobj
Eyepieces
Corrections
 Abbreviations
Super Viewfield
Magnifi
Eyepiece Type Finder Eyepieces Widefield Wide Field Eyepieces Diameter
Eyepiece cation
(mm)

35 CROSSW 1/2x 42.4

Descriptive PSWH PWH SWH WH WH
SWH H
Abbreviation 10x 10x 10x H 15x 10x H
10x 10x H
1x 21.2
Field Number 26.5 22 26.5 26.5 22 14 22
2x 10.6
Diopter
-8~+2 -8~+2 -8~+2 -8~+2 -8~+2 -8~+2
Adjustment 4x 5.3

3"x4 3"x4 diopter

35mm diopter diopter 10x 2.12
" " correctio
Remarks photo correctio correctio
photo photo n
mask n n
mask mask crossline 20x 1.06

Diameter of 40x 0.53

Micrometer --- --- --- --- --- 24 24
Reticle
50x 0.42

60x 0.35

100x 0.21

150x 0.14

250x 0.085
Useful (and empty) magnification

Useful Magnification (total) = 500 to 1000 NA (Objective)

Objective Eyepieces

(NA) 10x 12.5x 15x 20x 25x

2.5x
--- --- --- x x
(0.08)

4x
--- --- x x x
(0.12)

10x
--- x x x x
(0.35)

25x
x x x x ---
(0.55)

40x
x x x --- ---
(0.70)

60x
x x x --- ---
(0.95)

100x
x x --- --- ---
(1.42)
Measurement accessories
Projection eyepieces
 Field Number
field number is the diameter of the view field in millimeters measured at the intermediate image plane

Field Size = Field Number (fn) Objective Magnification (Mo)

Abbe Condenser
Achromatic and Aplanatic condensers
Achromat/Aplanat
Condensers
Condenser
Aberrations Corrected
Type

Spherical Chromatic

Abbe --- ---

Aplanatic x ---

Achromatic --- x

Aplanatic-
x x
achromatic
Condenser types

2 lens elements 3-4 lens elements (2 colors)

5 lens elements 8 lens elements

Swing-Out Top Lens
Applications

Condenser Phase
Brightfield Darkfield DIC Polarizing
Type Contrast

Achromat/

Aplanat
[10x~100x]
N.A. 1.3

Achromat

Swing-out
[4x~100x]
N.A. 0.90

Low-Power
N.A. 0.20 [1x~10x]

Phase

Contrast Abbe [up to N.A.
[10x~100x]
N.A. 1.25 0.65]

Phase

Contrast
[up to N.A.
Achromat [4x~100x]
0.70]
N.A. 0.85

DIC Universal

Achromat/ [up to N.A. [20x, 40x,
[10x, 100x]
Aplanat 0.70] 100x]

Darkfield, dry
N.A. 0.80~0.95 [4x~40x]

Darkfield, oil
N.A. 1.20~1.43 [4x~100x]

Stain-Free
Achromat

Swing-Out [4x~100x]
N.A. 0.90
Stages
Simple stage
Circular stage
Inverted microscope stages
High Precision stages
Micromanipulators
 Stereo Microscopy

(Keystone) (Perspective)
Cons and Pros
Greenough:
High NA
Easy correction of aberrations
Keystone (tilted objectives)
CMO
Infinity Space
Perspective distortion
Nikon SMZ-1500
Zoom Configuration
 Stereo microscope objective specs
Working
Objective Numerical
Color Code Distance
Magnification Aperture
(Millimeters)
Numerical Aperture and Equivalent f-Number Values
ED Plan 0.5x Red 0.045 155

ED Plan 0.75x Yellow 0.68 117

Numerical Aperture f-Number
ED Plan 1x White 0.09 84
0.023 21.7
ED Plan 1.5x Green 0.14 50.5
0.029 17.2
ED Plan 2x Blue 0.18 40
0.052 9.6
Plan Apo 0.5x N/A 0.066 136
0.085 5.9
Plan Apo 1x N/A 0.13 54
0.104 4.8
Plan Apo 1.6x N/A 0.21 24
0.118 4.2

0.128 3.9

0.131 3.8

Depth of Field in Stereomicroscope Objectives

f-Number (f) = 1 / (2 x NA) and NA = 1 / (2 x f)
Depth of Field
Zoom Numerical (Micrometers)
Objective
Factor Aperture
10x 15x 20x 30x

0.75 0.023 1,348 1,072 934 796

1 0.029 820 655 573 491

2 0.052 239 193 170 147

4 0.085 80 66 59 52
HR Plan
Apo 1x
6 0.104 48 41 37 33

8 0.118 35 30 27 25

10 0.128 28 24 22 21

11.25 0.131 26 21 21 19
Specialized Microscopy Techniques
Contrast Enhancing Techniques
Fluorescence Microscopy
Basics of Confocal Microscopy
Advanced Fluorescent microscopic
techniques
Near-field Scanning Optical Microscopy
Combinations
Contrast in optical microscopy

Percent Contrast (C) = ((I(s) - I(b)) x 100)/I(b)

Contrast enhancing

Brightfield Phase DIC

Amplitude and Phase specimens
Optical Path Difference

Optical Path Difference (OPD) = D = (t ns - t nm) = t (ns - nm)

Phase shift: d = D(2p/l)

 Optical gradients

tan f = tan a (ns - nm)

OG (Optical Gradient) = F = a(ns - nm)
f = a (ns - nm) (in radians, small angles)
Coherence preservation
 Contrast Enhancing Techniques
Specimen Imaging
Type Technique Reflected Light

Transmitted Light
Specular (Reflecting) Surface
Brightfield Illumination
Transparent Specimens Thin Films, Mirrors
Phase Contrast Phase Contrast, DIC
Phase Objects Polished Metallurgical Samples
Differential Interference Contrast (DIC) Darkfield Illumination
Bacteria, Spermatozoa, Integrated Circuits
Hoffman Modulation Contrast
Cells in Glass Containers,
Oblique Illumination
Protozoa, Mites, Fibers, etc.

Rheinberg Illumination Diffuse (Non-Reflecting) Surface

Light Scattering Objects Darkfield Illumination Thin and Thick Films Brightfield Illumination
Diatoms, Fibers, Hairs, Phase Contrast and DIC Rocks and Minerals Phase Contrast, DIC
Fresh Water Microorganisms, Hairs, Fibers, and Bone Darkfield Illumination
Radiolarians, etc. Insects

Light Refracting Specimens

Phase Contrast Amplitude Surface Features
Colloidal Suspensions
Dispersion Staining Dyed Fibers
powders and minerals Brightfield Illumination
DIC Diffuse Metallic Specimens
Liquids Darkfield Illumination
Composite Materials
Polymers
Amplitude Specimens
Stained Tissue
Naturally Colored Specimens Brightfield Illumination
Hair and Fibers
Insects and Marine Algae Birefringent Specimens
Mineral Thin Sections
Hairs and Fibers
Fluorescent Specimens Polarized Illumination
Bones and Feathers
Cells in Tissue Culture Single Crystals
Fluorescence Illumination
Fluorochrome-Stained Sections Oriented Films
Smears and Spreads

Birefringent Specimens
Mineral Thin Sections Fluorescent Specimens
Liquid Crystals Mounted Cells
Polarized Illumination Fluorescence Illumination
Melted and Recrystallized Chemicals Fluorochrome-Stained Sections
Hairs and Fibers Smears and Spreads
Bones and Feathers
Contrast enhancing
Dark field
Hoffman modulation contrast
Oblique (anaxial) illumination
DIC
Phase contrast
 Darkfield Microscopy

Works nicely with magnifications up to 40x, N.A. up to 0.65

Darkfield contrast

Brightfield Darkfield + red filter

Light stops

Numerical
Magnification Stop Size (mm)
Aperture

1X 0.03 25-30

2x 0.05 8-11

4X 0.10 8-14

10x 0.25 16-18

20X 0.40 18-20

20x 0.65 20-22

40X 0.65 22-24

Darkfield microscopy at high
magnifications

Dry darkfield (NA ~0.7) Paraboloid (immersion oil)

No light without oil: TIR
Advanced darkfield condensers
High NA Darkfield condensers

Hollow Cone Objective Maximum Number of Reflecting

Condenser Type Optical Corrections
Numerical Aperture Numerical Aperture Surfaces

Paraboloid 1.00-1.40 0.85 1 Parabolic Achromatic

1 Spherical
Cardioid 1.20-1.30 1.05 Achromatic/Aplanatic
1 Cardioidal

1 Cardioidal
Bicentric 1.20-1.30 1.05 Aplanatic
1 Spherical

Bispheric 1.20-1.30 1.05 2 Spherical Aplanatic

1 Aspheric
Cassegrain 1.40-1.50 1.30 Aplanatic
1 Spherical

Spot Ring
1.40-1.50 1.30 2 Spherical Aplanatic
(Bicentric)

Nelson 1 Aspheric
1.30-1.45 1.20 Aplanatic
Cassegrain 1 Spherical
Reflected Dark field microscopy
Reflected DF components
Rheinberg illumination
Rheinberg Illumination Filters
Differential Interference Contrast
Wollaston / Nomarsky modification
DIC Microscope
DIC in depth
Wavefronts in DIC
Bias retardation in DIC
Hoffman Modulation Contrast
Slit / Modulator arrangements

Resolution: l/NA Resolution: l/2 NA

Phase gradients
Oblique Illumination
Achieving oblique illumination
Asymmetry with oblique illumination
Oblique illumination in transmission
and reflection
Phase Microscope
Phase Microscopy
Phase contrast components
Positive (dark)
Negative (bright)
Interaction of light with phase
specimens
P=S+D

F = 90 + f/2

Particle= result
Surrounding = no interaction
(background)
Diffracted = through sample
Phase microscope train
Positive and negative phase contrast
 Positive and negative phase contrast

Pos

Neg
Halo and shade-off
Apodized Phase contrast
Apodized phase plates
 Phase objectives specifications
DL (Dark-Light) - DL objectives produce a dark image outline on a light gray background. These
objectives are designed to furnish the strongest dark contrast in specimens having major
differences in refractive indices. The DL phase contrast objective is the most popular style for
examination of cells and other semi-transparent living material and is especially suited for
photomicrography and digital imaging.

DLL (Dark Lower Contrast) - Similar to the DL objective, the DLL series allows better images in
brightfield and is often used as a "universal" objective in microscope systems that utilize multiple
illumination modes such as fluorescence, DIC, brightfield, and darkfield.

ADL (Apodized Dark-Light) - Recently introduced by Nikon, the apodized phase contrast ADL
objectives contain a secondary neutral density ring on either side of the phase ring. Addition of
the secondary rings assists in reducing unwanted "halo" effects often associated with imaging in
phase contrast microscopy.

DM (Dark-Medium) - DM objectives produce a dark image outline on a medium gray

background. These objectives are designed to be used for high image contrast with specimens
having small phase differences, such as fine fibers, granules, and particles.

BM (Bright-Medium) - Often referred to as negative phase contrast, BM objectives produce a

bright image outline on a medium gray background. BM objectives are ideal for visual
examination of bacterial flagella, fibrin bundles, minute globules, and blood cell counting.
Phase objectives abbreviations
Phase,
PHACO, Phase Contrast
PC
Ph 1, 2, 3,
Phase Condenser Annulus 1, 2, 3, etc.
etc.
DL, DLL, Phase Contrast: Dark Low, Dark Low Low, Dark
DM, BM medium, Bright Medium
PL, PLL Phase Contrast: Positive Low, Positive Low Low
Phase Contrast: Positive Medium, Positive High
PM, PH Contrast (Regions with higher
refractive index appear darker.)
Phase Contrast: Negative Low, Negative Medium,
NL, NM, Negative High Contrast
NH (Regions with higher
refractive index appear lighter.)
Comparison of phase and DIC
Aperture
Azimuth effects

DIC DIC Phase

Halos
Depth of field and optical sectioning
Birefringent samples
Stained specimens
Comparison
Characteristic Phase Contrast DIC

Image Brightness
1.3 Percent 0.36 - 2.3 Percent
(Brightfield = 100 Percent)

Epi-Fluorescence Light Loss

28 Percent 73 Percent
(Brightfield = 0 Percent)

Condenser
Lateral Resolution Annulus Superior
Restricted

Axial Resolution
Poor Superior
(Depth Discrimination)

10 Percent of
Illuminating Aperture Variable
Objective NA

Phase Shift
< l/100 < l/100
Detection Limit

Utility at High Phase Shifts Not Useful Useful

Azimuthal Effects No Yes

Halos and Shade-Off Yes No

Stained Specimens Not Useful Useful

Birefringent Specimens Useful Not Useful

Birefringent Specimen
Yes No
Containers

Brightfield Image
Slight None
Deterioration

Cost Moderate High

Fluorescence microscopy
Fluorescence the more complete story
Theoretical Absorption and emission
Mirror image and Stokes Shift
Extinction coefficient, Quantum Yield,
Fluorescence lifetime
(molar) Extinction coefficient (e) is a direct measure
of the ability of a fluorophore to absorb light

Quantum yield: ratio of photons emitted to the number of

photons absorbed
Fluorescence lifetime (t):
t

I (t ) = I 0 e t

F = photons emitted/photons absorbed = kf/(kf + knr) = tf/to

Quantum yield data

Excitation Emission
Quantum
Compound Solvent Wavelength Wavelength
Yield
(nm) (nm)

Acridine
Ethanol 493 535 0.46
Orange
Benzene Ethanol 248 300-350 0.04

Chlorophyll-A Ethanol 440 685 0.23

Eosin Water 521 544 0.16

Fluorescein Water 437 515 0.92
Rhodamine-B Ethanol 555 627 0.97
Quenching and Photobleaching

Quenching: competing processes that induce non-radiative relaxation

Photobleaching: photon induced covalent modification

Antifade Reagents

Antifade Reagent Comments

The most effective reagent for FITC. Also
effective for Rhodamine. Should be
adjusted to 0.1% p-phenylenediamine in
p-phenylene-
glycerol/PBS for use. Reagent blackens
diamine
when subjected to light exposure so it
should be stored in a dark place. Skin
contact is extremely dangerous.
DABCO Highly effective for FITC. Although its
(1,4-diazabi- effect is slightly lower than p-
cyclo-2,2,2- phenylenediamine, it is more resistant to
octane) light and features a higher level of safety.
The most effective reagent for
Rhodamine, also effective for FITC.
n-propylgallate
Should be adjusted to 1% propylgallate in
glycerol/PBS for use.
Used to observe chromosome and DNA
specimens stained with propidium iodide,
2-mercapto-
acridine orange, or Chromomysin A3.
ethylamine
Should be adjusted to 0.1mM 2-
mercaptotheylamine in Tris-EDTA
The fluorescence microscope
Epi-fluorescence arrangement
Fluorescence illuminator
Lamps and filter cubes
Inverted fluorescence microscope
 Light sources

Lamp Type XBO 150W/1 XBO 75W/2 HBO 200W/2 HBO 100W/2 HBO 50W/3

Current DC DC DC DC DC

Rate Power
150 75 200 100 50
(Watts)

Luminous
Flux 3000 950 10000 2200 1300
(Lumens)

Light
Intensity 300 100 1000 260 150
(Candella)

Average
Brightness 15000 40000 40000 170000 90000
(cd/sqcm)

Arc Size
(W x H in 0.50 x 2.20 0.25 x 0.50 0.60 x 2.20 0.25 x 0.25 0.20 x 1.35
Millimeters)

Life (Hours) 1200 400 400 200 200

Laser sources
Filter cubes
Introduction to confocal microscopy
LSCM
Nipkow rotating Disk confocal
microscope
Why confocal?
Optical sectioning
3D rendering from confocal stacks
Advanced Fluorescence Microscopy
Techniques
Multi-photon Microscopy
Total Internal Reflection Fluorescence
Microscopy
Multi-photon excitation microscopy
Geometrical Description
Comparison with confocal
TIRFM
n(1) sinq(1) = n(2) sinq(2)

n(1) sinq(c) = n(2)

q(c) = sin-1n(2)/n(1)

I(z) = I(o)e-z/d

Penetration depth: d = l(o)/4p (n(1)2sin2q(1) - n(2)2)-1/2

TIRFM
TIRFM on Cells
Epifluorescence
TIRFM
Inverted Microscope Configurations
Inverted and upright configurations
The setup at NIBN
TIR through the objective
Total Reflection Maximum
n(1) n(2) Angle A(1) Angle A(2)
(NA) (NA)

NA Immersion oil Cells 65.63 67.53

1.4 1.515 1.38 (1.38) (1.40)

Immersion
NA Cells 50.83 67.97
liquid
1.65 1.38 (1.38) (1.65)
1.78
Dynamics of MHC-I - GFP
 Individual Clusters
C17
C16
3.0
3.4
2.8
3.2
2.6
3.0
2.4
2.8 68
2.2
2.6
30

I/I0
2.0
2.4 14.9
I/I0

2.2 103.7 1.8

2.0 1.6

1.8
1.4
1.6
1.2
1.4
-20 0 20 40 60 80 100 120 140 160 180 -20 0 20 40 60 80 100 120 140 160 180

Time (s) Time (s)

 More clusters
C29
C26
3.5
4.0

3.5 46
3.0
51

3.0
2.5

I/I0
I/I0

2.5
30
34
2.0
2.0

1.5
1.5

-20 0 20 40 60 80 100 120 140 160 180 -20 0 20 40 60 80 100 120 140 160 180

Time (s) Time (s)

Distribution of lifetimes

40 individual clusters

20

15
% of clusters

10

0
0 20 40 60 80 100 120
Time constant (s)
Combinations: Fluorescence and DIC
Combinations: Fluorescence and Phase
Digital Imaging in Optical Microscopy
Concepts in digital imaging technology
Basic Properties of Digital Images
Basic concepts in digital image processing
Deconvolution in optical microscopy
Concepts in digital imaging
technology
CCD
Binning
Blooming
Dynamic range
Anatomy of CCD
CCD Clocking
Microlens Arrays
Binning
Blooming
Dynamic Range
Dynamic Range = 20 x Log(Nsat/Nnoise)
Bit Depth (A/D and images)
Quantum efficiency
 Resolution requirements
Objective Resolution Projected Required Pixel
(Numerical Limit Size on CCD Size
Aperture) (Microns) (Microns) (Microns)
Manufacturer
4x (0.20) 1.5 5.8 2.9 Pixel Size Array Size
and Format
(Microns) (Millimeters)
Model
10x (0.45) 0.64 6.4 3.2
Kodak
20x (0.75) 0.39 7.7 3.9 1732 x 1172 13 x 13 22.5 x 15.2
KAF-2001CE
40x (0.85) 0.34 13.6 6.8 Kodak
2016 x 1512 9x9 18.1 x 13.6
KAF-3000CE
40x (1.30) 0.22 8.9 4.5
Kodak
60x (0.95) 0.31 18.3 9.2 2144 x 1432 6.8 x 6.8 14.6 x 9.7
KAF-3040CE
60x (1.40) 0.21 12.4 6.2 Kodak
3052 x 2016 9x9 27.5 x 18.1
KAF-6302CE
100x (0.90) 0.32 32.0 16.0
Kodak
100x (1.25) 0.23 23.0 11.5 2048 x 2048 7.4 x 7.4 15.16 x 15.16
KAI-4000
100x (1.40) 0.21 21.0 10.5 Sony
1392 x 1040 4.65 x 4.65 7.6 x 6.2
ICX205AK

SITe
2048 x 4096 15 x 15 30.72 x 30.72
ST-002A

Marconi
4608 x 2048 13.5 x 13.5 27.6 x 62.2
CCD 42-90

Marconi
1028 x 1033 13 x 13 13.3 x 13.3
CCD 48-20

Philips
3072 x 2048 12 x 12 36.8 x 24.6
FTF3020-C

Philips
1024 x 1024 7.5 x 7.5 7.68 x 7.68
FT18
Intensifiers

Fast, ~8bit dynamic

range, low S/N (relative
to cooled, slow scan)
Higher spatial resolution than
intensified, lower gain and
sensitivity.
Sequential Color CCD
Basic Properties of Digital Images
Spatial Resolution
Color Models
RGB
CYM
HSI
Image Histograms
Graphic file formats

Pixel Grayscale Bitmap JPEG TIFF

Dimensions (8-Bit) (24-Bit) (24-Bit) (24-Bit)

16 x 16 2k 2k 2k 2k

64 x 64 6k 13k 5k 13k

128 x 128 18k 49k 12k 49k

256 x 256 66k 193k 22k 193k

320 x 240 77k 226k 24k 226k

512 x 512 258k 769k 52k 770k

640 x 480 302k 901k 56k 902k

800 x 600 470k 1,407k 75k 1,408k

1024 x 768 770k 2,305k 104k 2,306k

1280 x 1024 1,282k 3,841k 147k 3,842k

1600 x 1200 1,877k 5,626k 161k 5,627k

2250 x 1800 3,960k 11,869k 276k 11,867k

3200 x 2560 8,002k 24,001k 458k 24,002k

3840 x 3072 11,522k 34,561k 611k 34,562k

Digital Image Processing
Look-up Table Operations
Flat-Field Correction
Background correction

B(x, y) = c0 + c1x + c2y + c3x2 + c4y2 + c5xy

Histogram Operations

Histogram Stretching

Output(x, y) = (Input(x, y) - B) / (W - B)

Histogram Equalization
Spatial Convolution Kernels (Masks)
Smoothing and Sharpening
Kernel size
Median Filtering
 Derivative

000
0 -1 1 111
0 -1 1 -1 -1 -1
0 -1 1
Edge detection (Sobel)

-1 0 1 121
-2 0 2 000
-1 0 1 -1 -2 -1

x y
Unsharp Mask Filtering

Gaussian
Original Result
smoothed
 Fourier Series

a0 np x np x f(x) is defined in the

f ( x) = an cos bn sin
2 n =1 L L interval c x c 2L

1 c2 L np x
L c
an = f ( x) cos dx
L
1 c2 L np x
bn = f ( x) sin dx
L c L

c2 L
a0 = f ( x)dx D.C. offset
c
c c+2L
 n=1
n=2
1.0 n=3 1.0

n=1
0.5 0.5
n=2
cos (n p x /L)

n=3

sin (n p x /L)
0.0 0.0

-0.5 -0.5

-1.0 -1.0

0 2 4 6 8 10
0 2 4 6 8 10
x
x
2L=10
2L = 10

an bn
1.4

4
1.2
0.5
3
1.0

2
0.8 0.0
0 1 2 3
f(x)

1
an

bn
0.6
n
0 -0.5
0.4

-1
0.2
-1.0
-2
0 5 10 15 20 25 30 0.0
0 1 2 3
X
n
 sin (a ) = sin a cos cos a sin
np x np x np x
sin = A cos B sin
L L L

1.0

A2 B 2 0.5

np x
0.0
0 5 10 15 20 25 30
A sin X
L -0.5

np x -1.0
B cos
L

Power Spectrum 4.0

3.5
3.0
2.5
2.0
1.5
1.0
2.0 0.5
0.0
-0.5 0 5 10 15 20 25 30
-1.0 X
1.5 -1.5
2

2
a n +b n

1.0
2

0.5
0
0 5 10 15 20 25 30
X
0.0
0 1 2 3

n -2
Fourier transforms
Filtering in frequency domain
Low Pass (gaussian)
High pass
Band pass
 Filtering

LP HP BP BS
Band stop example
Deconvolution
Blur

Noise (random, poisson or

gauss, => well defined)
Scatter (passage through the
sample, heterogeneity of
refractive index = > random
(thickness))
Glare (in the optical train,
minimized by advanced optics)
Blur (out-of-focal-plane light =
> diffraction limit =>
deconvolution)
 Point Spread Function (PSF)

"It is impossible to bring out detail not present in the primary image by increasing the
power of the eyepiece, for each element of the primary image is a small diffraction
pattern, and the actual image, as seen by the eyepiece, is only the ensemble of the
magnified images of these patterns".
PSF
Fourier Integral Theorem

f ( x) = A( ) cos x B( ) sin x d
0

A( ) =
1
p f ( x) cos xdx


B( ) =
1
p f ( x) sin xdx

Fourier Transform

F f ( x ) F ( ) = f ( x ) e i x dx


F -1
F ( ) = f ( x ) = F ( ) ei x d

Point spread function
Deconvolution algorithms
Deblurring (nearest neighbors)
Inverse filter Algorithms
Restoration algorithms (iterative)
Blind deconvolution

A original
B deblurring
C inverse filter
D - iterative