Week 5
Live Cell Imaging in Confocal Microscopy
Multiphoton Microscopy
Spectral Imaging
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
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 .
A 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 or of the WEB.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 1 t:/classes/BMS 602B/lecture 5 602_B.ppt
Lecture Summary
•
•
•
•
1. Live cell confocal microscopy
2. Live cell applications and examples
3. Multiphoton microscopy
4. Spectral Imaging
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 2 t:/classes/BMS 602B/lecture 5 602_B.ppt
Specific Organelle Probes
Probe
BODIPY
NBD
DPH
TMA-DPH
Rhodamine 123
DiO
diI-Cn-(5)
diO-Cn-(3)
Site
Excitation
Golgi
505
Golgi
488
Lipid
350
Lipid
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 4 t:/classes/BMS 602B/lecture 5 602_B.ppt
Organelle Function
•
•
•
•
Mitochondria
Endosomes
Golgi
Endoplasmic Reticulum
Purdue University Cytometry Laboratories
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3) Carbocyanine
© 1995-2004 J.Paul Robinson, Purdue University
Slide 5 t:/classes/BMS 602B/lecture 5 602_B.ppt
Calcium Related Applications
• Probe Ratioing
– Calcium Flux (Indo-1)
– pH indicators (BCECF, SNARF)
Molecule-probe
Excitation
Emission
Calcium - Indo-1
Calcium- Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Magnesium - Mag-Indo-1
Phospholipase A- Acyl Pyrene
351 nm
488 nm
363 nm
488 nm
351 nm
351 nm
405, >460 nm
525 nm
>500 nm
515 nm
405, >460 nm
405, >460 nm
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 6 t:/classes/BMS 602B/lecture 5 602_B.ppt
Probes for Ions
•
•
•
•
INDO-1
QUIN-2
Fluo-3
Fura -2
Purdue University Cytometry Laboratories
Ex350
Ex350
Ex488
Ex330/360
© 1995-2004 J.Paul Robinson, Purdue University
Em405/480
Em490
Em525
Em510
Slide 7 t:/classes/BMS 602B/lecture 5 602_B.ppt
Ionic Flux Determinations
• Calcium
• Intracellular pH
Indo-1
BCECF
How the assay works:
• Fluorescent probes such as Indo-1 are able to bind to calcium in a
ratiometric manner
• The emission wavelength
decreases as the probe binds
available calcium
600
400
200
RATIO [short/long]
800
1000
Ratio: intensity of 460nm / 405nm signals
0.8
0
Stimulation
0
36
72
108
Time (Seconds)
144
180
Flow Cytometry
Purdue University Cytometry Laboratories
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Time (seconds)
0
0
50
100
150
200
Image Analysis
© 1995-2004 J.Paul Robinson, Purdue University
Slide 8 t:/classes/BMS 602B/lecture 5 602_B.ppt
Oxidative Reactions
•
•
•
•
Superoxide
Hydrogen Peroxide
Glutathione levels
Nitric Oxide
Purdue University Cytometry Laboratories
Hydroethidine
Dichlorofluorescein
Monobromobimane
DAF-FM (4-amino-5methylamino-2',7'-difluorofluorescein)
© 1995-2004 J.Paul Robinson, Purdue University
Slide 9 t:/classes/BMS 602B/lecture 5 602_B.ppt
DCFH-DA
DCFH
DCF
2’,7’-dichlorofluorescin diacetate
O
O
CH3-C-O
O
O-C-CH3
Cl
2’,7’-dichlorofluorescin
Cl
H
COOH
O
HO
Cellular Esterases
OH
Cl
Fluorescent
Cl
H
COOH
Hydrolysis
2’,7’-dichlorofluorescein
O
HO
O
H 2O 2
Cl
Oxidation
DCFH-DA
Cl
H
COOH
Neutrophils
DCFH-DA
8
0
Monocytes
H 2O 2
counts
DCFH
60
DCF
PMA-stimulated PMN
Control
40
20
Lymphocytes
0
.
1
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
1
100
log FITC 10
Fluorescence
100
0
Slide 10 t:/classes/BMS 602B/lecture 5 602_B.ppt
Hydroethidine
HE
EB
H2N
NH2
H
N
O2-
H2N
NH2
N + Br
CH2CH3
-
CH2CH3
Phagocytic Vacuole
NADPH Oxidase
NADPH
O2
HE
O2-
NADP
SOD
O2H2O2
DCF
H2O2
DCF
OH-
Example: Neutrophil Oxidative Burst
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 11 t:/classes/BMS 602B/lecture 5 602_B.ppt
Macrovascular Endothelial Cells
in Culture
0
Purdue University Cytometry Laboratories
Time (minutes)
© 1995-2004 J.Paul Robinson, Purdue University
60
Slide 12 t:/classes/BMS 602B/lecture 5 602_B.ppt
Hydrogen peroxide measurements with DCFH-DA
1
2
3
4
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
5
525 nm
Step 7B: Export data from Excel data
base to Delta Graph
% change (DCF fluorescence)
Step 6B: Export data from measured
regions to Microsoft Excel
2000
1800
1600
1400
1200
1000
800
600
400
200
0
cell 1
cell 2
cell 3
cell 4
cell 5
0
Purdue University Cytometry Laboratories
500
1000 1500 2000 2500 3000
Time in seconds
© 1995-2004 J.Paul Robinson, Purdue University
Slide 13 t:/classes/BMS 602B/lecture 5 602_B.ppt
Superoxide measured with hydroethidine
cell 1
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
cell 3
cell 4
cell 2
Export data from measured
regions to Microsoft Excel
Export data from Excel data
base to Delta Graph
%change (DCF fluorescence)
cell 5
1800
1600
1400
1200
1000
800
600
400
cell 1
cell 2
cell 3
cell 4
200
0
cell 5
-200
200 400
600 800
1000 1200 1400 1600 1800
Time in seconds
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 14 t:/classes/BMS 602B/lecture 5 602_B.ppt
H2O2 stimulation and DCF & EB loading in Rat
Pulmonary Artery Endothelial Cells
24 treatments - 5000 cells each
200
ENDO HBSS
ENDO HBSS TNFa
ENDO L-arg
ENDO/ L-arg TNFa
ENDO/ D-arg
ENDO/ D-arg TNFa
Endo + 200uM H2O2
Endo + 200uM H2O2
Endo + 200uM H2O2
Endo / TNFa + 200uM H2O2
Endo / TNFa + 200uM H2O2
Endo / TNFa + 200uM H2O2
Confocal System Fluorescence
Measurements
.
180
160
DCF Fluorescence
Mean EB Fluorescence
Endo / L-arg + 200uM H2O2
Endo / L-arg + 200uM H2O2
Endo / L-arg + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
140
120
100
80
60
40
20
200uM
H2O2
added
200uM
H2O2
added
0
0
20
40
60
80
100
Time (minutes)
Purdue University Cytometry Laboratories
120
140
© 1995-2004 J.Paul Robinson, Purdue University
Time (seconds)
Slide 15 t:/classes/BMS 602B/lecture 5 602_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]
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 16 t:/classes/BMS 602B/lecture 5 602_B.ppt
Exotic Applications of
Confocal Microscopy
•
•
•
•
FRAP (Fluorescence Recovery After Photobleaching)
Release of “Caged” compounds
Lipid Peroxidation (Parinaric Acid)
Membrane Fluidity (DPH)
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 17 t:/classes/BMS 602B/lecture 5 602_B.ppt
“Caged” Photoactivatable Probes
Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester
undergoes photolysis upon exposure to UV light at 340-350 nm
Available Probes
•
•
•
•
•
•
•
Purdue University Cytometry Laboratories
Ca++: Nitr-5
Ca++ - buffering: Diazo-2
IP3
cAMP
cGMP
ATP
ATP--S
© 1995-2004 J.Paul Robinson, Purdue University
Slide 18 t:/classes/BMS 602B/lecture 5 602_B.ppt
Release of “Caged” Compounds
UV Beam
Culture dish
Release of “Cage”
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 19 t:/classes/BMS 602B/lecture 5 602_B.ppt
Release of Caged Compounds
UV excited
D
Fluorescence Emission at 515 nm
Control Region
Fluorescence Emission at 515 nm
CONTROL STUDY
250
C
200
150
100
50
0
0
100 200 300
400
Time (seconds) CONTROL
Purdue University Cytometry Laboratories
Release of Caged Nitric Oxide in
Attached PMN
250
200
150
100
50
0
0
20 40
60 80 100 120 140 160
Time (seconds) after UV FLASH
© 1995-2004 J.Paul Robinson, Purdue University
Slide 20 t:/classes/BMS 602B/lecture 5 602_B.ppt
Membrane Polarization
• Polarization/fluidity
Diphenylhexatriene
How the assay works: The DPH partitions into liphophilic portions of the cell and is
excited by a polarized UV light source. Polarized emissions are collected and changes
can be observed kinetically as cells are activated.
An image showing
DPH fluorescence in
cultured endothelial
cells.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 21 t:/classes/BMS 602B/lecture 5 602_B.ppt
Calcium ratios with Indo-1
1
1
2
2
3
3
460 nm
405/35 nm
Changes in the fluorescence were measured
using the Bio-Rad calcium ratioing software.
The same region in each wave length was
measured and the relative change in each region
was recorded and exported to a spread sheet for
analysis..
Export data from measured regions to
Microsoft Excel
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
0.8
Export data from Excel data
base to Delta Graph
cell 1
cell 2
cell 3
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
Purdue University Cytometry Laboratories
50
100
© 1995-2004 J.Paul Robinson, Purdue University
150
200
Slide 22 t:/classes/BMS 602B/lecture 5 602_B.ppt
FRAP
%F
Intense laser Beam
Bleaches Fluorescence
Time
Recovery of fluorescence
Zero time
Purdue University Cytometry Laboratories
10 seconds
© 1995-2004 J.Paul Robinson, Purdue University
30 seconds
Slide 23 t:/classes/BMS 602B/lecture 5 602_B.ppt
Imaging 3D ECM structures
• Mainly collagen based materials
• Usually 40-120 microns thick
• Require both transmitted and fluorescent
signals
• Often require significant image processing
to extract information
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 24 t:/classes/BMS 602B/lecture 5 602_B.ppt
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 25 t:/classes/BMS 602B/lecture 5 602_B.ppt
Thick Tissue - Bone and
Cartilage
• Very difficult to image
thick specimens
• Can use live specimens
if appropriately stained
• Special preparation
techniques
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 26 t:/classes/BMS 602B/lecture 5 602_B.ppt
Multi-Photon Microscopy
An introduction
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 27 t:/classes/BMS 602B/lecture 5 602_B.ppt
History
• Developed in 1961 by Kaiser and Garret
• A process unknown in Nature except in stars
• Can be reproduced in a laser beam whereby more than
one photon can be absorbed by a molecule in a short
time
• The energy of both photons is summed in a way similar
to that of a photon of shorter wavelength, but the
emission is almost identical to that of a single photon
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 28 t:/classes/BMS 602B/lecture 5 602_B.ppt
Energy states in 2-photon
Note that the end result is
essentially the same for 1
photon and 2 photon. The
emission is the same in
both cases.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 29 t:/classes/BMS 602B/lecture 5 602_B.ppt
Advantages of 2 Photon
 Longer observation times for live cell studies
 Increased fluorescence emission detection
 Reduced volume of photobleaching and phototoxicity. Only the focal-plane being
imaged is excited, compared to the whole sample in the case of confocal or wide-field
imaging.
 Reduced autofluorescence of samples
 Optical sections may be obtained from deeper within a tissue that can be achieved by
confocal or wide-field imaging. There are three main reasons for this: the excitation
source is not attenuated by absorption by fluorochrome above the plane of focus; the
longer excitation wavelengths used suffer less Raleigh scattering; and the fluorescence
signal is not degraded by scattering from within the sample as it is not imaged.
 All the emitted photons from multi-photon excitation can be used for imaging (in
principle) therefore no confocal blocking apertures have to be used.
 It is possible to excite UV flourophores using a lens that is not corrected for UV as
these wavelengths never have to pass through the lens.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 30 t:/classes/BMS 602B/lecture 5 602_B.ppt
2-Photon Excitation
•
•
•
•
•
•
•
•
The sample is illuminated with a wavelength of twice the wavelength of the absorption peak
of the fluorochrome being used. For example, in the case of fluorescein which has an
absorption peak around 500 nm, 1000 nm excitation could be used. Essentially no excitation
of the fluorochrome will occur at this wavelength and hence no bleaching will occur in the
bulk of the sample.
A high-powered pulsed laser is required with has a peak power of >2Kw
Power should be in pulses shorter than a picosecond (so that the mean power levels are
moderate and do not damage the specimen)
Two-photon events will occur at the point of focus give above conditions
The photon density is sufficiently high that two photons can be absorbed by the fluorochrome
essentially simultaneously.
This is equivalent to a single photon with an energy equal to the sum of the two that are
absorbed.
Thus, fluorochrome excitation will only occur at the point of focus
This eliminates unnecessary phototoxicity as there is little excitation out of the plane of focus
Image quality is excellent as there is practically no out-of-focus interference.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 31 t:/classes/BMS 602B/lecture 5 602_B.ppt
3-Photon Microscopy
Three-photon excitation can also be used in certain circumstances. In this case three photons
are absorbed simultaneously, effectively tripling the excitation energy. Using this technique,
UV excited fluorophores may be imaged with IR excitation. Because excitation levels are
dependent on the cube of the excitation power, resolution is improved compared to two
photon excitation where there is a quadratic power dependence. It is possible to select
fluorophores such that multiple labeled samples by can be imaged by combination of 2- and 3
photon excitation, using a single IR excitation source.
Advantages
 UV fluorophore excitation without UV irradiation
 Similar resolution to 2 photon excitation of UV fluorophores
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 32 t:/classes/BMS 602B/lecture 5 602_B.ppt
Limitations of 2-Photon
 Slightly lower resolution with a given fluorochrome when
compared to confocal imaging. This loss in resolution can be
eliminated by the use of a confocal aperture at the expense of a loss
in signal.
 Thermal damage can occur in a specimen if it contains
chromophores that absorb the excitation wavelengths, such as the
pigment melanin.
 Only works with fluorescence imaging.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 33 t:/classes/BMS 602B/lecture 5 602_B.ppt
Why 2-photon is very specific
• Fluorescence from the two-photon effect depends on the square of the
incident light intensity, which in turn decreases approximately as the
square of the distance from the focus.
• Because of this highly nonlinear (~fourth power) behavior, only those
dye molecules very near the focus of the beam are excited.
• The tissue above and below the plane of focus is merely subjected to
infrared light that causes neither photobleaching nor phototoxicity.
• Although the peak amplitude of the IR pulses is large, the mean power of
the beam is only a few tens of milliwatts, not enough to cause substantial
heating of the specimen.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 34 t:/classes/BMS 602B/lecture 5 602_B.ppt
Multi-Photon Fluorescence Microscopy
The experimental benefits of multi-photon excitation:
•Localized excitation provides high spatial resolution
•Inherent z-axis resolution improves sensitivity and three-dimensional optical
sectioning
•Reduced photodamage/ photobleaching
•Increased penetration depth in specimen
•Provides selective excitation of fluorophores by two and three photons
•Increased detection sensitivity of fluorophores by reducing autofluorescence
or background
•Elimination of confocal aperture
Applications for multi-photon microscopy are:
•In-vivo and in-vitro imaging
•Fluorescent Lifetime Imaging
•Optical Tomography Imaging
•Semiconductor Imaging
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 35 t:/classes/BMS 602B/lecture 5 602_B.ppt
Instrument Setup
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 36 t:/classes/BMS 602B/lecture 5 602_B.ppt
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 37 t:/classes/BMS 602B/lecture 5 602_B.ppt
Instrumentation
Typical Instrumentation for Multi-Photon TimeResolved Microscopy:
•Femtosecond, Picosecond or CW Lasers
•Near Infra-Red Optics coated for high peak power
lasers
•Special Dichroics for Multiphoton Excitation
•Laser Scanning Microscope optimized for Infra-Red
high peak power lasers
•Time-Resolved Instrumentation for Imaging
Dichroic For Two-Photon Excitation
Dichroic For Three-Photon Excitation
Wavelength (nm)
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 38 t:/classes/BMS 602B/lecture 5 602_B.ppt
Comparison Between Confocal and Two-Photon Detection
Confocal one-photon excitation imaging compared with two-photon imaging in scattering tissue. Due to the
longer wavelength, less excitation light is lost to scattering when using two-photon excitation. Ballistic and
diffusing fluorescence photons can be used in the two-photon case, but only ballistic photons can be used in
the confocal case.
In multi-photon excitation more fluorescence photons are detected from a focal point than from a confocal
method.
Ref. W. Denk, J. Biomedical Optics (1996) 1(3), 296-304. Ballistic photons are non-scattering photons.
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 39 t:/classes/BMS 602B/lecture 5 602_B.ppt
2-photon Vs single photon (confocal)
The cuvette is filled with a solution of a
dye, safranin O, which normally requires
green light for excitation. Green light (543
nm) from a continuous-wave helium-neon
laser is focused into the cuvette by the lens
at upper right. It shows the expected pattern
of a continuous cone, brightest near the
focus and attenuated to the left. The lens at
the lower left focuses an invisible 1046-nm
infrared beam from a mode-locked Nddoped yttrium lanthanum fluoride laser into
the cuvette. Because of the two-photon
absorption, excitation is confined to a tiny
bright spot in the middle of the cuvette.
From Current Protocols in Cytometry Online
Copyright © 1999 John Wiley & Sons, Inc. All rights reserved.
Purdue University Cytometry Laboratories
Photo from Brad Amos
© 1995-2004 J.Paul Robinson, Purdue University
Slide 40 t:/classes/BMS 602B/lecture 5 602_B.ppt
Comparison of One-Photon Excitation vs. Two-Photon Excitation
One-Photon and Two-Photon Excitation images were obtained by CW 5 mW
Laser at 442 nm. (Recent findings indicate that 2-photon can be obtained
with high power CW lasers) and Ti:sapphire laser at 800 nm respectively.
Two-photon excitation exhibits localized excitation, the inherent
advantage which accounts for the improved resolution available with this
method.
One- Photon Excitation
Two- Photon Excitation
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 41 t:/classes/BMS 602B/lecture 5 602_B.ppt
Lasers
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 42 t:/classes/BMS 602B/lecture 5 602_B.ppt
Dye Excitation Spectra
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 43 t:/classes/BMS 602B/lecture 5 602_B.ppt
Lasers and Probes
Pulsed laser source of 1047 nm which can excite most blue and red, and some
green emitting fluorophores.
BLUE EMITTING: AMCA, Hoechst 33342, Hoechst 33258, DAPI
GREEN EMITTING: Oregon Green 514, red-shifted GFP, JC-1, FITC, Ca
Green
ORANGE EMITTING: Calium Orange, Mitotracker Rosamine, Rhodamine
123, FM4-64
RED EMITTING: Nile Red, Calcium Crimson, TRITC, Texas Red, DiI, PPI,
CY-3
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 44 t:/classes/BMS 602B/lecture 5 602_B.ppt
Z-axis Resolution in 3-Photon and 2-Photon Excitation
Comparing the signals obtained when moving the focus from the cover glass into (a) BBO/ toluene and (b)
Rhodamine 6G / immersion oil layer. This compares the axial resolution of a three-photon and two-photon
microscope, respectively. The excitation wavelength is 900 nm. No confocal spatial filtering is used. The
steeper signal in (a) shows the higher axial resolution of three-photon excitation microscopy. The z-axis
represents the focal point in the experiment.
Ref. Stefan Hell ........ (1995)
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 45 t:/classes/BMS 602B/lecture 5 602_B.ppt
Selective Detection of Fluorophores in Multi-Photon Excitation
Ref. J. R. Lakowicz and I. Gryczynski, "Topics in Fluorescence
Spectroscopy", volume V, Plenum Press, 1997
Calcium dependent emission spectra of Indo-1 for one-, twoand three-photon excitation at 295, 590 and 885 nm,
respectively. The results suggest that the relative cross-section
for three-photon excitation of Indo-1 is less for the Ca2+ - bound
form, as compared to relative cross-section for twophoton. Hence the calcium bound or free form of Indo- 1 can
be selectively sense by two- or three-photon excitation
respectively.
http://www.microcosm.com/tutorial/tutorial.html
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 46 t:/classes/BMS 602B/lecture 5 602_B.ppt
Examples
DNA tagged fluorescence image without using an UV source. The DAPI stained
nuclei were excited with the Nd:YLF pulsed laser (1047nm) via three-photon
excitation.
(349 nm)
3-photon image of a
DAPI stained
Caenorhabitis
elegans worm
Images from: Multi-Photon Excitation Fluorescence
Microscope Coordinator, Madison, WI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 47 t:/classes/BMS 602B/lecture 5 602_B.ppt
1047 nm
488nm
Sequence of images showing a
comparison between confocal imaging
(488nm excitation) and 2-photon
imaging (1047nm excitation). The
sample is a zebra fish that is heavily
stained with safranine (the sample was
prepared by B. Amos). As can clearly
be seen, 2-photon imaging is able to
give much better images deep into the
specimen.
Comparison of confocal and 2-photon
imaging (JPEG-100K)
Images from: Multi-Photon Excitation Fluorescence
Microscope Coordinator, Madison, WI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 48 t:/classes/BMS 602B/lecture 5 602_B.ppt
Comparison of XZ images taken by confocal and 2-photon imaging. The images
were obtained by sequentially scanning a single horizontal XY line while
stepping the focus into the specimen. The sample is a safranine stained zebra
fish (prepared by B. Amos). The 2-photon system (left) is able to reveal
structural information in regions where nothing can be seen in the confocal
system (right).
Comparison of XZ images of confocal and 2-photon imaging (JPEG-25K)
2-photon
confocal
Images from: Multi-Photon
Excitation Fluorescence
Microscope Coordinator,
Madison, WI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 49 t:/classes/BMS 602B/lecture 5 602_B.ppt
Double labeled 3t3 cell in anaphase showing microtubules (Green FITC) and actin
staining (red rhodamine phalloidin). This is a fixed specimen and is included to
demonstrate that double labeling is possible with the 1047 nm excitation wavelength
used in the 2-photon imaging system.
Double labeled 3t3 cell in anaphase showing "green" microtubules and "red" actin
staining (JPEG-15K)
Images from: Multi-Photon
Excitation Fluorescence
Microscope Coordinator,
Madison, WI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 50 t:/classes/BMS 602B/lecture 5 602_B.ppt
2-photon -Bacterial Studies
•
An other example of the use of two-photon excitation microscopy is the imaging of Dental Biofilm. It
consists of various aerobic and anaerobic bacteria embedded in a matrix of polysaccharides and proteins
and can reach thicknesses of several hundred micrometers. The pH is an important property of the biofilm
with respect to the effect on dental enamel. Using a carboxy-fluoresceine staining the pH of the biofilm
was monitored after the addition of sucrose. The lifetime of the probe is sensitive to the local pH.
Calibration of the fluorescence lifetime in biofilm at several pH values allows a determination of the local
pH in the measured images.
In the image (right) a fluorescence intensity image of
biofilm is shown. Several types of bacteria can be
distinguished.
http://www.phys.uu.nl/~wwwmbf/ResJV.htm
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 51 t:/classes/BMS 602B/lecture 5 602_B.ppt
http://www.phys.uu.nl/~wwwmbf/ResJV.htm
In the above image a fluorescence intensity image of biofilm is shown. Several types of bacteria can be
distinguished. Below (left) another intensity image of biofilm is shown. After supplying the biofilm with a
sucrose solution the bacterial metabolic activity increases which results in the production of H+. The
fluorescence lifetime images before (middle) and 70 minutes after the addition of sucrose (right) show a clear
drop in pH. Here, the lifetime range in the images runs from pH 6.5 (black) to pH 2 (white).
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 52 t:/classes/BMS 602B/lecture 5 602_B.ppt
Spectral Imaging
• Increasing the number of
spectral channels collected
• Allows more advanced
classification systems
• Takes more time to image
• Much more complex
analysis
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 53 t:/classes/BMS 602B/lecture 5 602_B.ppt
Multispectral microscopy – Not more
colors!!!
Greyscale
image
Color image
Multispectral
image
Expansion/rebirth of the Landsat Concept from the 1970s
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 54 t:/classes/BMS 602B/lecture 5 602_B.ppt
Multispectral microscopy
Intensified
camera
Intensified camera
CCD camera
AOTF
Camera controller
AOTF
AOTF controller
Monitor
Microscope controller
Microscope
PC computer
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Purdue Spectral
Imaging Project
Slide 55 t:/classes/BMS 602B/lecture 5 602_B.ppt
Enabling Technology: Liquid tunable filters
Single bandpass
Measured center wavelength (nm)
Lyot filter (static)
750
700
650
600
550
500
450
400
400
450
500
550
600
650
700
750
Wavelength “dialed-in”
LCTF (randomly tunable)
High precision and accuracy
Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 56 t:/classes/BMS 602B/lecture 5 602_B.ppt
High-resolution cytology segmentation
Characteristic
Spectra
Conventional
RGB Image
Spectrally
segmented Image
Wavelength (nm)
High spectral resolution increases utility of spectrally responsive indicator dyes
Purdue University Cytometry Laboratories
Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com
© 1995-2004 J.Paul Robinson, Purdue University
Slide 57 t:/classes/BMS 602B/lecture 5 602_B.ppt
Multispectral Imaging – Zeiss Meta
Ability to select a range of wavelengths
As desired by the user
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 58 t:/classes/BMS 602B/lecture 5 602_B.ppt
Nuance-Micro
Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com
© 1995-2004 J.Paul Robinson, Purdue University
Slide 59 t:/classes/BMS 602B/lecture 5 602_B.ppt
Purdue University Cytometry Laboratories
Lecture Summary
• Live cell applications are relatively common using confocal
microscopy
• Correct use of fluorescent probes necessary
• Temperature and atmosphere control may be required
• Thick specimens often require advanced image processing
• Exotic applications are potentially useful
• A limited window of time is available to image live cells before cells
deteriorate
• 2-photon microscopy can penetrate greater tissue depth
• 2-3 photon has advantages for excitation of lower wavelengths (UV)
• 2-photon is very complex technology
• 2-photon is very expensive
• Possibly the future replacing confocal imaging
• Spectral Imaging will be next major change in biological imaging
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson, Purdue University
Slide 60 t:/classes/BMS 602B/lecture 5 602_B.ppt