BMS 631 - Lecture 4
J.Paul Robinson
Professor of Immunopharmacology & Biomedical Engineering
Purdue University
Optical Systems
optical geometry; light sources, laser illumination,
& other useful means; optics and shaping the incoming beam;
forward angle light scatter - what it is, why it is useful.
Side angle (90 degree) light scatter, what does it measure?
References:
Shapiro 3rd ed. 93-115
WWW.CYTO.PURDUE.EDU
©1990-2002 J.Paul Robinson, Purdue University
Page 1
Review
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•
•
•
•
•
Scatter - Rayleigh Scatter - directly proportional to property of the scattering molecule
called molecular polarizability (ie dipole formation), inversely proportional to the fourth power
of the wavelength of the incident light (blue light has highest scatter - thus blue sky!)
Scatter - Raman Scatter - (p 93 3rd ed) molecules undergo vibrational transitions at the
same time as scatter occurs- if is transition to higher level is known as Stoke's Raman
emission. Normally 1/1000th intensity of Rayleigh Scatter, but is significantly increased
when using lasers for excitation.. Raman emission of water at 488 nm excitation is around
570-590 nm.
Polarizations - E vectors - larger changes in E vectors not incident light plane; Mie
scattering - increased scatter in the forward angle for larger particles (1/4 wavelength to
tens of wavelength). (p89, 3rd ed)
Incident light, reflected light, transmitted light, refractive index - note the angle of incidence =
angle of reflection regardless of the material of surface. tt transmission angle depends upon
the composition of material according to Snell's law of refraction n1 sin Ti =n2 sin Tt
n1, n2 are the refractive indices respectively through which the incident beam passes (air = 1
essentially)
Brewster's Angle, chromatic aberration, filters, interference, band pass, dichroic,
absorption, laser blocker.
Fluorescence lifetime, polarization, fluidity, anisotrophy, resonance energy transfer,
quenching, bleaching (p82 3rd ed)
©1990-2002 J.Paul Robinson, Purdue University
Page 2
Light Propagation &
Vergence
• Considering a point source emission of light, rays
emanate over 4pi steradians
• If the ray of light travels through a length L of a
medium of RI n, the optical path length S=Ln (thus S
represents the distance light woul dhave traveled in a
vacuum in the same time it took to travel the distance
L in the medium (RI n).
• Rays diverge (because the come from a point source
• Vergence is measured in diopters
Shapiro p 93
©1990-2002 J.Paul Robinson, Purdue University
Page 3
Image Formation
• Object plane - (originating image)
• Image plane - inverted real image
• A real image is formed whenever rays
emanating from a single point in the object
plane again converge to a single point
Shapiro p 94
©1990-2002 J.Paul Robinson, Purdue University
Page 4
Properties of thin Lenses
f
f
p
q
1
p
Resolution (R) = 0.61 x
(lateral)
(Rayleigh criterion)
+
1
q
l
NA
©1990-2002 J.Paul Robinson, Purdue University
=
1
f
q
Magnification =
p
Page 5
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
Shapiro p 96
©1990-2002 J.Paul Robinson, Purdue University
Page 6
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 m
– (n=the lowest refractive index between the object and
first objective element) (hopefully 1)
– m is 1/2 the angular aperture of the objective
©1990-2002 J.Paul Robinson, Purdue University
Page 7
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).
l
.00053
1.00 = 0.53 mm
=
NA
• With a cone of light filling the entire aperture the
theoretical resolution is…(fully open condenser)..
l
2 x NA
=
.00053
2 x 1.00 = 0.265 mm
©1990-2002 J.Paul Robinson, Purdue University
Page 8
Depth of Field and
Resolution
• Depth of field is designated as the longitudinal
distance for the formation of a sharp image is
obtained at a fixed point in the image plane
• Limits of resolution are diffraction limited - the
diffraction image is a point is a bright central spot
surrounded by what is called the Airy disk
(alternating light and dark rings)
• at wavelength l, the radius of the Airy disk is 0.61
l Thus to resolve two points they need to be at
least this distance apart (radius of the Airy disk)
thus the resolution is defined as 0.61 l /NA
Shapiro p 97
©1990-2002 J.Paul Robinson, Purdue University
Page 9
Object Resolution
• Example:
40 x 1.3 N.A. objective at 530 nm light
l
2 x NA
.00053
2 x 1.3 = 0.20 mm
=
40 x 0.65 N.A. objective at 530 nm light
l
2 x NA
=
.00053
2 x .65 = 0.405 mm
©1990-2002 J.Paul Robinson, Purdue University
Page 10
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
©1990-2002 J.Paul Robinson, Purdue University
Shapiro p 101
Page 11
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
©1990-2002 J.Paul Robinson, Purdue University
Page 12
Refraction
He sees the
fish here….
But it is really here!!
©1990-2002 J.Paul Robinson, Purdue University
Page 13
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
©1990-2002 J.Paul Robinson, Purdue University
Page 14
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced
depending upon the color absorbed. Thus the selective
absorption of white light produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent
medium to another with different optical density. A ray from
less to more dense medium is bent perpendicular to the
surface, with greater deviation for shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated
at sharp edges - the smaller the aperture the lower the
definition
• Dispersion
– Separation of light into its constituent wavelengths when
entering a transparent medium - the change of refractive index
with wavelength, such as the spectrum produced by a prism or a
rainbow
©1990-2002 J.Paul Robinson, Purdue University
Page 15
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
©1990-2002 J.Paul Robinson, Purdue University
blue
blue
Page 16
Light absorption
Control
Absorption
No blue/green light
red filter
©1990-2002 J.Paul Robinson, Purdue University
Page 17
Light absorption
white light
blue light
©1990-2002 J.Paul Robinson, Purdue University
red light
green light
Page 18
The light spectrum
Wavelength = Frequency
Blue light
488 nm
short wavelength
high frequency
high energy (2
times the red)
Photon as a
wave packet
of energy
Red light
650 nm
long wavelength
low frequency
low energy
©1990-2002 J.Paul Robinson, Purdue University
Page 19
Technical Aspects of Flow
Cytometry
• Illumination Sources
Lamps
Xenon
Mercury
Lasers
Argon Ion (Ar)
Krypton (Kr)
Helium Neon (He-Ne)
Helium Cadmium (He-Cd)
YAG
©1990-2002 J.Paul Robinson, Purdue University
Page 20
Elite Cytometer with 4 Lasers
353 nm
325 nm
488 nm
633 nm
UV\Beam Splitter
He-Cd Laser 325/441
395 longPass
Argon Laser 353/488 nm
(High speed sorting)
633 Beam Splitter
He-Ne Laser 633 nm
Argon Laser 488 nm
Mirror
Optical bench
Height Translators
©1990-2002 J.Paul Robinson, Purdue University
Page 21
Elite Cytometer with 4 Lasers
He-Cd laser
Santa clause
Air-cooled argon
laser
Water cooled
argon laser
©1990-2002 J.Paul Robinson, Purdue University
Page 22
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Laser
Bandpass
Filters
©1990-2002 J.Paul Robinson, Purdue University
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Coulter Optical System - Elite
PMT4
PMT2
PMT3
PMT1
555 - 595
575 BP
525 BP
488 BP
655 - 695
L
L
D
5
2
D
5
6
5
0
488 BK
632 BP
675 BP
PMT5
L
D
0
9
4
APC
PMT6
TM
PMT7
• The Elite optical
system uses 5 side
window PMTs and a
number of filter
slots into which any
filter can be
inserted
Purdue Cytometry Labs
©1990-2002 J.Paul Robinson, Purdue University
Page 24
Coulter Optical System - Elite
Empty PMT
slot
PMTs
Dichroic filter slot
Light Scatter
Detector
©1990-2002 J.Paul Robinson, Purdue University
Page 25
Detection Systems
Bio-Rad Bryte HS
PMTs
Light source
Fluorescence
emission
filters
Excitation dichroic
filter
Fluorescence Detectors and Optical Train
©1990-2002 J.Paul Robinson, Purdue University
Fluorescence
signal
viewing
telescope
Dsc00050.jpg
Page 26
Forward Angle
Scatter PMT
Lamp
Housing
“Red”PMT
Large Angle
Scatter PMT
Sample Inlet
“Orange” PMT
Emission
Filter
Block
Emission
FilterBlock
“Green” PMT
Slit
Slit
Retractable
Mirror
Ocular
Microscope
Objective
Microscope Excitation
Objective Filter
Block
The Bryte Optical Layout
©1990-2002 J.Paul Robinson, Purdue University
Retractable
Mirror
Ocular
Page 27
Bryte HS Optical System
Cells
Scatter Objective
Light
Cover Glass
Fluorescence
Objective
Water
Flow
Dark
Xenon Light
Field
Light
Focus
Water
Flow
Dark Spot
Immersion Oil
©1990-2002 J.Paul Robinson, Purdue University
Page 28
Summary Slide
• Light propagation and image planes
• We use optical filters to separate the
spectrum
• Each cytometer has a different optical
train
• PMTs are used for signal collectio
www.cyto.purdue.edu
©1990-2002 J.Paul Robinson, Purdue University
Page 29