slide 1 t:/classes/bms524/524lect2.ppt © 1995-2000 j.paul robinson - purdue university cytometry...

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


Review• Microscope Basics, Magnification, Optical systems

Properties of Light• Refraction• A Lens• Refractive Index• Numerical Aperture• Resolution• Aberrations• Fluorescence


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


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


Properties of thin Lensesf

1

p+

1

q=

1

f

f

p q

Resolution (R) = 0.61 xNA

Magnification = q

p(lateral)(Rayleigh criterion)


Microscope Components• Ocular • Objectives• Condenser • Numerical Aperture• Refractive Index• Aberrations• Optical Filters


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


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)


Microscope Objectives


Objectives

PLAN-APO-40X 1.30 N.A. 160/0.22

Flat field Apochromat Magnification Numerical Tube CoverglassFactor Aperture Length Thickness

∞ - Infinity corrected


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.


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


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


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


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=


Microscope Objectives

SpecimenCoverslip

Oil

MicroscopeObjective

Stage

60x 1.4 NAPlanApo


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


• Monochromatic Aberrations– Spherical aberration

– Coma

– Astigmatism

– Flatness of field

– Distortion

• Chromatic Aberrations– Longitudinal aberration

– Lateral aberration

Sources of Aberrations


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


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


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


• 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.


Chromatic aberrations

Chromatic aberration of a single lens causes different wavelengths

of light to have differing focal lengths



Diffractive optical element with complementary dispersion

properties to that of glass can be used to correct for color aberration



For an achromatic doublet, visible wavelengths have approximately

the same focal length


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


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


Fluorescent Microscope

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination


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


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


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


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


Construction of Filters

Dielectric filtercomponents

Single Opticalfilter

“glue”


Anti-Reflection Coatings

Optical Filter

MultipleElements

Coatings are often magnesium fluoride

Dielectric filtercomponents


Standard Band Pass Filters

Transmitted LightWhite Light Source

630 nm BandPass Filter

620 -640 nm Light


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


Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted LightLight Source

510 LP dichroic Mirror


Filter Properties Light Transmission

%T

Wavelength

100

0

50

Notch

Ban

dp

ass


Summary Lecture 2

• Parts of the microscope (ocular, condenser)

• Objectives

• Numerical Aperture (NA)

• Refractive Index/refraction (RI)

• Aberrations

• Fluorescence microscope

• Properties of optical filters

slide 1 t:/classes/bms524/524lect2.ppt © 1995-2000 j.paul robinson - purdue university cytometry...

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