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Slide 1 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Lecture 2
The Principles of MicroscopyBMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine
J.Paul Robinson, Ph.D.Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text.
UPDATED January, 2000
Slide 2 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Review• Microscope Basics, Magnification, Optical systems
Properties of Light• Refraction• A Lens• Refractive Index• Numerical Aperture• Resolution• Aberrations• Fluorescence
Slide 3 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Refraction & Dispersion
Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.
dispersion
Short wavelengths are “bent” more than long wavelengths
refraction
Slide 4 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Reflection and Refraction• Snell’s Law: The angle of
reflection (Ør) is equal to the angle of incidence (Øi) regardless of the surface material
• The angle of the transmitted beam (Øt) is dependent upon the composition of the material
t
i
r
Incident Beam
Reflected Beam
Transmitted(refracted)Beam
n1 sin Øi = n2 sin Øt
The velocity of light in a material of refractive index n is c/n
Slide 5 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Properties of thin Lensesf
1
p+
1
q=
1
f
f
p q
Resolution (R) = 0.61 xNA
Magnification = q
p(lateral)(Rayleigh criterion)
Slide 6 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Microscope Components• Ocular • Objectives• Condenser • Numerical Aperture• Refractive Index• Aberrations• Optical Filters
Slide 7 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Ocular - Eyepiece• Essentially a projection lens (5x to 15x magnification) Note: there is usually
an adjustment call the inter-pupillary distance on eyepieces for personal focusing
• Huygenian– Projects the image onto the retina of the eye– your eye should not be right on the lens, but
back from it (eyecups create this space)
• Compensating– designed to work with specific apochromatic or flat field objectives - it is
color compensated and cannot be mixed with other objectives (or microscopes)
• Photo-adapter– designed to project the image on the film in the camera - usually a longer
distance and lower magnification from 0.5x to 5x
Slide 8 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Condenser• Has several purposes
– must focus the light onto the specimen
– fill the entire numerical aperture of the objective (i.e. it must match the NA of the objective)
• Most microscopes will have what is termed an “Abbe” condenser (not corrected for aberrations)
• Note if you exceed 1.0 NA objective, you probably will need to use oil on the condenser as well (except in inverted scopes)
Slide 9 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Microscope Objectives
Slide 10 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Objectives
PLAN-APO-40X 1.30 N.A. 160/0.22
Flat field Apochromat Magnification Numerical Tube CoverglassFactor Aperture Length Thickness
∞ - Infinity corrected
Slide 11 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Objectives
Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion):
d = λ/2 N.A.
Thus high NUMERICAL APERTURE is critical for high magnification
This defines a “resel” or “resolution element”
d = 0,61λ/N.A.
Slide 12 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Numerical Aperture
• The wider the angle the lens is capable of receiving light at, the greater its resolving power
• The higher the NA, the shorter the working distance
Slide 13 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Numerical Aperture• Resolving power is directly related to numerical
aperture.
• The higher the NA the greater the resolution
• Resolving power:The ability of an objective to resolve two distinct lines
very close together
NA = n sin μ
– (n=the lowest refractive index between the object and first objective element) (hopefully 1)
– μ is 1/2 the angular aperture of the objective
Slide 14 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Numerical Aperture• For a narrow light beam (i.e. closed illumination aperture diaphragm) the finest
resolution is (at the brightest point of the visible spectrum i.e. 530 nm)…(closed condenser).
NA
2 x NA
.000532 x 1.00= 0.265 μm
.000531.00 = 0.53 μm
• With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..
=
=
http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html
Slide 15 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Object Resolution• Example:
40 x 1.3 N.A. objective at 530 nm light
2 x NA
.000532 x 1.3 = 0.20 μm=
40 x 0.65 N.A. objective at 530 nm light
2 x NA
.000532 x .65 = 0.405 μm=
Slide 16 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Microscope Objectives
SpecimenCoverslip
Oil
MicroscopeObjective
Stage
60x 1.4 NAPlanApo
Slide 17 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Refractive Index
Objective
n=1.52
n = 1.52
n = 1.52
Specimen
Coverslip
Oil
n=1.33
n = 1.52
n = 1.0
n = 1.5
Water
n=1.52
Air
Slide 18 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
• Monochromatic Aberrations– Spherical aberration
– Coma
– Astigmatism
– Flatness of field
– Distortion
• Chromatic Aberrations– Longitudinal aberration
– Lateral aberration
Sources of Aberrations
Slide 19 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Monochromatic Aberration– Spherical aberration
Generated by nonspherical wavefronts produced by the objective, and increased tube length, or inserted objects such as coverslips, immersion oil, etc. Essentially, it is desirable only to use the center part of a lens to avoid this problem.
F1 F2
F1
Corrected lens
Slide 20 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Fig 12 p117
From:”Handbook of Biological Confocal Microscopy”J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
The figure is not reproduced in this presentation becausewe do not have permission to place this figure onto a public site.
Monochromatic Aberrations– Coma
Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems.
Fig From:Handbook of Biological Confocal MicroscopyJ.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
Note: For class useFigure is under box
Slide 21 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Fig 13 p118
From:”Handbook of Biological Confocal Microscopy”J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
The figure is not reproduced in this presentation becausewe do not have permission to place this figure onto a public site.
Monochromatic Aberrations–Astigmatism
Fig 13 p118
If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated. From:Handbook of Biological Confocal Microscopy
J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed.Note: For class useFigure is under box
Slide 22 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
• Monochromatic Aberrations– Flatness of Field– Distortion
Lenses are spherical and since points of a flat image are focused onto a spherical dish, the central and peripheral zones will not be in focus. Complex Achromat and PLANAPOCHROMAT lenses partially solve this problem but at reduced transmission.
DISTORTION occurs for objects components out of axis. Most objectives correct to reduce distortion to less than 2% of the radial distance from the axis.
Slide 23 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Chromatic aberrations
Chromatic aberration of a single lens causes different wavelengths
of light to have differing focal lengths
Slide 24 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Chromatic aberrations
Diffractive optical element with complementary dispersion
properties to that of glass can be used to correct for color aberration
Slide 25 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Chromatic aberrations
For an achromatic doublet, visible wavelengths have approximately
the same focal length
Slide 26 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Useful Factoids
The intensity of light collected The intensity of light collected decreasesdecreases as the square of the magnificationas the square of the magnification
The intensity of light The intensity of light increasesincreases as the as the square of the numerical aperturesquare of the numerical aperture
Thus when possible, use Thus when possible, use low magnificationlow magnification and and high NAhigh NA objectives objectives
Slide 27 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Fluorescence Microscopes• Cannot view fluorescence emission in a single optical plane
• Generally use light sources of
much lower flux than confocal systems
• Are cheaper than confocal systems
• Give high quality photographic images
(actual photographs) whereas confocal
systems are restricted to small resolution images
Slide 28 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Fluorescent Microscope
Dichroic Filter
Objective
Arc Lamp
Emission Filter
Excitation Diaphragm
Ocular
Excitation Filter
EPI-Illumination
Slide 29 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Interference in Thin Films
• Small amounts of incident light are reflected at the interface between two material of different RI
• Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths
• Optical filters can thus be created that “interfere” with the normal transmission of light
Slide 30 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Interference and Diffraction: Gratings
• Diffraction essentially describes a departure from theoretical geometric optics
• Thus a sharp objet casts an alternating shadow of light and dark “patterns” because of interference
• Diffraction is the component that limits resolution
Slide 31 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Polarization and Phase: Interference
• Electric and magnetic fields are vectors - i.e. they have both magnitude and direction
• The inverse of the period (wavelength) is the frequency in Hz
Wavelength (period T)
Axis of
Magnetic F
ield
Axis of Propagation
Axi
s of
Ele
ctri
c F
ield
Modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p78
Slide 32 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Interference
ConstructiveInterference
DestructiveInterference
A
B
C
D
A+B
C+D
Am
plitude
0o 90o 180o 270o 360o Wavelength
Figure modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p79
Here we have a phase difference of 180o (2 radians) so the waves cancel each other out
The frequency does not change, but the amplitude is doubled
Slide 33 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Construction of Filters
Dielectric filtercomponents
Single Opticalfilter
“glue”
Slide 34 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Anti-Reflection Coatings
Optical Filter
MultipleElements
Coatings are often magnesium fluoride
Dielectric filtercomponents
Slide 35 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Standard Band Pass Filters
Transmitted LightWhite Light Source
630 nm BandPass Filter
620 -640 nm Light
Slide 36 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Standard Long Pass Filters
Transmitted LightLight Source520 nm Long Pass Filter
>520 nm Light
Transmitted LightLight Source575 nm Short Pass Filter
<575 nm Light
Standard Short Pass Filters
Slide 37 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Optical Filters
Dichroic Filter/Mirror at 45 deg
Reflected light
Transmitted LightLight Source
510 LP dichroic Mirror
Slide 38 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Filter Properties Light Transmission
%T
Wavelength
100
0
50
Notch
Ban
dp
ass
Slide 39 t:/classes/BMS524/524lect2.ppt© 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Summary Lecture 2
• Parts of the microscope (ocular, condenser)
• Objectives
• Numerical Aperture (NA)
• Refractive Index/refraction (RI)
• Aberrations
• Fluorescence microscope
• Properties of optical filters