Week 2
Excitation, fluorescence, optical systems, resolution
BME 695Y / BMS 634
Confocal Microscopy: Techniques and Application Module
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
& Department of Biomedical Engineering, Schools of Engineering
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology & Biomedical Engineering
Director, Purdue University Cytometry Laboratories
J
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.
One useful 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 February 2004
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 1 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Overview of lecture 2
1. Excitation Sources
2. Fluorescence
3. Raman & Raleigh Scatter
4. Photobleaching
5. CCD cameras for fluorescence
6. Fluorescent probes for biological material
7. The structure of a confocal microscope
8. Optical properties of confocal systems
9. Confocal principals
11.Resolution, gray scales and image structure
12 Sampling theory and electronic zoom
13.Reflection Imaging.
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 2 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Excitation Sources
Excitation Sources
Lamps
Xenon
Xenon/Mercury
Lasers
Argon Ion (Ar)
Krypton (Kr)
Helium Neon (He-Ne)
Helium Cadmium (He-Cd)
Krypton-Argon (Kr-Ar)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 3 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence
• Chromophores are components of
molecules which absorb light
• They are generally aromatic rings
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 4 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence
Jablonski Diagram
Singlet States
Triplet States
Vibrational energy levels
Rotational energy levels
Electronic energy levels
S2
ENERGY
T2
S1
IsC
T1
ABS
FL
I.C.
PH
IsC
S0
[Vibrational sublevels]
ABS - Absorbance
S 0.1.2 - Singlet Electronic Energy Levels
FL - Fluorescence
T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC
- Intersystem Crossing
PH - Phosphorescence
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 5 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Simplified Jablonski Diagram
S’1
S1
hvex
hvem
S0
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 6 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence
The longer the wavelength the lower the energy
The shorter the wavelength the higher the energy
eg. UV light from sun causes the sunburn
not the red visible light
Intensity
related to the probability of the event
Wavelength
the energy of the light absorbed or emitted
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 7 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence
Stokes Shift
Fluorescnece Intensity
– is the energy difference between the lowest energy
peak of absorbence and the highest energy of emission
Fluorescein
molecule
Stokes Shift is 25 nm
495 nm
520 nm
Wavelength
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 8 t:/classes/BMS602 B/Lecture 2 602_B.ppt
350
300 nm
457 488 514
400 nm
500 nm
Common Laser Lines
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 9 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Parameters
• Extinction Coefficient
– 
refers to a single wavelength (usually the absorption maximum)
• Quantum Yield
– Qf
is a measure of the integrated photon emission over the
fluorophore spectral band
• At sub-saturation excitation rates,
fluorescence intensity is proportional to the
product of  and Qf
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 10 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Excitation Saturation
• The rate of emission is dependent upon the time the molecule remains
within the excitation state (the excited state lifetime f)
• Optical saturation occurs when the rate of excitation exceeds the
reciprocal of f
• In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in
1 second requires a dwell time per pixel of 2 x 10-6 sec.
• Molecules that remain in the excitation beam for extended periods
have higher probability of interstate crossings and thus
phosphorescence
• Usually, increasing dye concentration can be the most effective means
of increasing signal when energy is not the limiting factor (ie laser
based confocal systems)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 11 t:/classes/BMS602 B/Lecture 2 602_B.ppt
How many Photons?
• Consider 1 mW of power at 488 nm focused to a Gaussian
spot whose radius at 1/e2 intensity is 0.25m via a 1.25
NA objective
• The peak intensity at the center will be 10-3W [.(0.25 x
10-4 cm)2]= 5.1 x 105 W/cm2 or 1.25 x 1024 photons/(cm2
sec-1)
• At this power, FITC would have 63% of its molecules in
an excited state and 37% in ground state at any one time
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 12 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Raman Scatter
• A molecule may undergo a vibrational transition (not an
electronic shift) at exactly the same time as scattering occurs
• This results in a photon emission of a photon differing in
energy from the energy of the incident photon by the
amount of the above energy - this is Raman scattering.
• The dominant effect in flow cytometry is the stretch of the
O-H bonds of water. At 488 nm excitation this would give
emission at 575-595 nm
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 13 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Rayleigh Scatter
• Molecules and very small particles do
not absorb, but scatter light in the
visible region (same freq as excitation)
• Rayleigh scattering is directly
proportional to the electric dipole and
inversely proportional to the 4th power
of the wavelength of the incident light
the sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 14 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Photobleaching
• Defined as the irreversible destruction of an
excited fluorophore (discussed in later lecture)
• Methods for countering photobleaching
–
–
–
–
–
Scan for shorter times
Use high magnification, high NA objective
Use wide emission filters
Reduce excitation intensity
Use “antifade” reagents (not compatible with viable
cells)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 15 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Photobleaching example
• FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC
bleaches with a quantum efficiency Qb of 3 x 10-5
• Therefore FITC would be bleaching with a rate
constant of 4.2 x 103 sec-1 so 37% of the molecules
would remain after 240 sec of irradiation.
• In a single plane, 16 scans would cause 6-50%
bleaching
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 16 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Antifade Agents
• Many quenchers act by reducing oxygen concentration to
prevent formation of singlet oxygen
• Satisfactory for fixed samples but not live cells!
• Antioxidents such as propyl gallate, hydroquinone, pphenylenediamine are used
• Reduce O2 concentration or use singlet oxygen quenchers such
as carotenoids (50 mM crocetin or etretinate in cell cultures);
ascorbate, imidazole, histidine, cysteamine, reduced
glutathione, uric acid, trolox (vitamin E analogue)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 17 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Excitation - Emission Peaks
Fluorophore
FITC
Bodipy
Tetra-M-Rho
L-Rhodamine
Texas Red
CY5
EXpeak EM peak
496
503
554
572
592
649
518
511
576
590
610
666
% Max Excitation at
488
568 647 nm
87
58
10
5
3
1
0
1
61
92
45
11
0
1
0
0
1
98
Note: You will not be able to see CY5 fluorescence
under the regular fluorescent microscope because
the wavelength is too high.
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 18 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescent Microscope
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular
Dichroic Filter
Objective
Emission Filter
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 19 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence Microscope with
Color Video (CCD)
35 mm Camera
camera
Camera viewer
ocular
filters
objectives
stage
condensor
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 20 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Cameras and emission filters
Cooled color
CCD camera
Camera
goes here
Color CCD camera does not need optical filters to collect all wavelengths but if you want
to collect each emission wavelength optimally, you need a monochrome camera with
separate emission filters shown on the right (camera is not in position in this photo).
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 21 t:/classes/BMS602 B/Lecture 2 602_B.ppt
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 22 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Types of Probes
•Proteins
•Nucleic Acids
•DNA
•Ions
•pH Sensitive Indicators
•Oxidation States
•Specific Organelles
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 23 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Probes for Proteins
Probe
Excitation
Emission
FITC
PE
APC
PerCP™
Cascade Blue
Coumerin-phalloidin
Texas Red™
488
488
630
488
360
350
610
550
540
640
525
575
650
680
450
450
630
575
575
670
Tetramethylrhodamine-amines
CY3 (indotrimethinecyanines)
CY5 (indopentamethinecyanines)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 24 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Probes for Ions
•
•
•
•
INDO-1
QUIN-2
Fluo-3
Fura -2
Ex350
Ex350
Ex488
Ex330/360
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Em405/480
Em490
Em525
Em510
Slide 25 t:/classes/BMS602 B/Lecture 2 602_B.ppt
pH Sensitive Indicators
Probe
Excitation
Emission
• SNARF-1
488
575
• BCECF
488
440/488
525/620
525
[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 26 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Probes for Oxidation States
Probe
Oxidant
• DCFH-DA
• HE
• DHR 123
(H2O2)
(O2-)
(H2O2)
DCFH-DA
HE
DHR-123
Excitation
488
488
488
Emission
525
590
525
- dichlorofluorescin diacetate
- hydroethidine
- dihydrorhodamine 123
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 27 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Specific Organelle Probes
Probe
BODIPY
NBD
DPH
TMA-DPH
Rhodamine 123
DiO
diI-Cn-(5)
diO-Cn-(3)
Site
Golgi
Golgi
Lipid
Lipid
Excitation
505
488
350
350
Mitochondria 488
Lipid
488
Lipid
550
Lipid
488
Emission
511
525
420
420
525
500
565
500
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammonium
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 28 t:/classes/BMS602 B/Lecture 2 602_B.ppt
DNA Probes
• AO
– Metachromatic dye
• concentration dependent emission
• double stranded NA - Green
• single stranded NA - Red
• AT/GC binding dyes
– AT rich: DAPI, Hoechst, quinacrine
– GC rich: antibiotics bleomycin, chromamycin A3,
mithramycin, olivomycin, rhodamine 800
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 29 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Multiple Emissions
• Many possibilities for using multiple probes
with a single excitation
• Multiple excitation lines are possible
• Combination of multiple excitation lines or
probes that have same excitation and quite
different emissions
– e.g. Calcein AM and Ethidium (ex 488)
– emissions 530 nm and 617 nm
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 30 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Energy Transfer
• Effective between 10-100 Å only
• Emission and excitation spectrum must
significantly overlap
• Donor transfers non-radiatively to the
acceptor
• PE-Texas Red™
• Carboxyfluorescein-Sulforhodamine B
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 31 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescence
Resonance Energy Transfer
Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Absorbance
Absorbance
Wavelength
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 32 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Benefits of Confocal Microscopy
•
•
•
•
•
•
•
Reduced blurring of the image from light scattering
Increased effective resolution
Improved signal to noise ratio
Clear examination of thick specimens
Z-axis scanning
Depth perception in Z-sectioned images
Magnification can be adjusted electronically
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 33 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fluorescent Microscope
Confocal Microscope
Arc Lamp
Laser
Excitation Diaphragm
Excitation Filter
Excitation Pinhole
Excitation Filter
Ocular
PMT
Objective
Objective
Emission Filter
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Emission
Filter
Emission Pinhole
Slide 34 t:/classes/BMS602 B/Lecture 2 602_B.ppt
MRC 1024 System
UV Laser
Optical Mixer
Kr-Ar Laser
Scanhead
Microscope
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 35 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Bio-Rad MRC 1024
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 36 t:/classes/BMS602 B/Lecture 2 602_B.ppt
MRC 1024 System
Light Path
PMT
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 37 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Optical Mixer - MRC 1024 UV
Fast Shutter
Argon Laser
353,361 nm
UV
Visibl
e
Filter
Wheels
UV Correction
Optics
ArgonKrypton
Laser
488, 514
nm
488,568,647 nm
Beam Expander
To Scanhead
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 38 t:/classes/BMS602 B/Lecture 2 602_B.ppt
MRC 1024 Scanhead
3
2
Emission
Filter
Wheel
From Laser
PMT
1
Galvanometers
To and from Scope
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 39 t:/classes/BMS602 B/Lecture 2 602_B.ppt
From Scanhead
To Scanhead
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 40 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Scanning Galvanometers
Point Scanning
x
y
Laser out
To
Microscope
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Laser in
Slide 41 t:/classes/BMS602 B/Lecture 2 602_B.ppt
The Scan Path of the Laser Beam
Start
767, 1023, 1279
0
0
Specimen
511, 1023
Frames/Sec
# Lines
1
2
4
8
16
512
256
128
64
32
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 42 t:/classes/BMS602 B/Lecture 2 602_B.ppt
How a Confocal Image is
Formed
Pinhole 1
Pinhole 2
Specimen
Detector
Condenser
Lens
Objective
Lens
Modified from: Handbook of Biological Confocal
Microscopy. J.B.Pawley, Plennum Press, 1989
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 43 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Fundamental Limitations of Confocal
Microscopy
From
Source
n1 photons
PIXEL
1
y
VOXEL
x
1
.
z
x,y,z
2
n2 photons
2
To Detector
From: Handbook of Biological Confocal Microscopy.
J.B.Pawley, Plennum Press, 1989
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 44 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Optical Resolution:
Gray Level & Pixelation
• Analogous to intensity range
For computer images each pixel is assigned a value. If the image is 8
bit, there are 28 or 256 levels of intensity If the image is 10 bit there
are 1024 levels, 12 bit 4096 levels etc.
• The intensity analogue of a pixel is its grey level which shows up as
brightness.
• The display will determine the possible resolution since on a TV
screen, the image can only be displayed based upon the number of
elements in the display. Of course, it is not possible to increase the
resolution of an image by attributing more “pixels” to it than were
collected in the original collection!
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 45 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Pixels
• Pixels & image structure
Hardcopy usually compromises pixel
representation. With 20/20 vision
you can distinguish dots 1 arc second
apart (300 m at 1 m) so 300 DPS on
a page is fine. So at 100 m, you
could use dots 300 mm in size and
get the same effect! Thus an image
need only be parsimonius, i.e., it only
needs to show what is necessary to
provide the expected image.
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
T
Slide 46 t:/classes/BMS602 B/Lecture 2 602_B.ppt
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 47 t:/classes/BMS602 B/Lecture 2 602_B.ppt
320x240 x 24
Zoom x 4
Magnifying with
inadequate
information. This
is known as
“empty
magnification”
because there are
insufficient data
points.
Zoom x 2
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
The final image appears to be very “boxy”
this is known as “pixilation”.
Zoom x 8
Slide 48 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Socrates?….well
perhaps not...
180x200x8
(288,000) 1X
361x400x8
Magnifying with
(1,155,200) 2x
adequate
information.
Here, the original
image was
collected with
541x600x8
many more pixels (2,596,800) 1.5x)
so the magnified
©image
1995-2004looks
J.Paul Robinson
better!- Purdue University Cytometry Laboratories
Slide 49 t:/classes/BMS602 B/Lecture 2 602_B.ppt
320x240 x 24
Originals
collected at
high resolution compared to a
low resolution
image magnified
1500x1125x24
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 50 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Sampling Theory
• The Nyquist Theorem
– Nyquest theory describes the sampling frequency (f) required to represent
the true identity of the sample.
– i.e., how many times must you sample an image to know that your sample
truly represents the image?
– In other words to capture the periodic components of frequency f in a signal
we need to sample at least 2f times
• Nyquist claimed that the rate was 2f. It has been determined that in
reality the rate is 2.3f - in essence you must sample at least 2 times
the highest frequency.
• For example in audio, to capture the 22 kHz in the digitized signal,
we need to sample at least 44.1 kHz
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 51 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Digital Zoom
1x
1024 points
2x
1024 points
4x
1024 points
Note that we have reduced the field
of view of the sample
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 52 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Reflection Imaging
Backscattered light imaging
Same wavelength as
excitation
CD-ROM pits
Increasing
mag
Advantages: no photobleaching since not
using a photo-probe (note: does not
mean no possible damage to specimen)
Problems: optical reflections from
components of microscope
Collagen
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 53 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Issues for good confocal imaging
• Axial Resolution
– Must determine the FWHM (full width half maximum) intensity values of a vertical
section of beads
• Field Flatness
– Must be able to collect a flat field image over a specimen - or z-axis information will
be inaccurate
• Chromatic Aberration
– must test across an entire field that emission is constant and not collecting radial or
tangential artifacts due to chromatic aberration in objectives
• Z-drive precision and accuracy
– must be able to reproducibily measure distance through a specimen - tenths of microns
will make a big difference over 50 microns
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 54 t:/classes/BMS602 B/Lecture 2 602_B.ppt
Conclusions
• Fluorescence is the primary energy source for confocal microscopes
• Dye molecules must be close to, but below saturation levels for optimum
emission
• Fluorescence emission is longer than the exciting wavelength
• The energy of the light increases with reduction of wavelength
• Fluorescence probes must be appropriate for the excitation source and the
sample of interest
• Correct optical filters must be used for multiple color fluorescence
emission
• Sampling rate must be appropriate for specimen(Nyquist Theorem)
© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 55 t:/classes/BMS602 B/Lecture 2 602_B.ppt