slide 1 t:/classes/bms 602b/lecture 5 602_b.ppt purdue university cytometry laboratories ©...

t:/classes/BMS 602B/lecture 5 602_B.pptPurdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University

Week 5Live Cell Imaging in Confocal Microscopy

Multiphoton MicroscopySpectral 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.


Lecture Summary

• 1. Live cell confocal microscopy

• 2. Live cell applications and examples

• 3. Multiphoton microscopy

• 4. Spectral Imaging


Specific Organelle Probes

BODIPY Golgi 505 511

NBD Golgi 488 525

DPH Lipid 350 420

TMA-DPH Lipid 350 420

Rhodamine 123Mitochondria 488 525

DiO Lipid 488 500

diI-Cn-(5) Lipid 550 565

diO-Cn-(3) Lipid 488 500

Probe Site Excitation Emission

BODIPY - borate-dipyrromethene complexesNBD - nitrobenzoxadiazoleDPH - diphenylhexatrieneTMA - trimethylammonium


Organelle Function

• Mitochondria Rhodamine 123

• Endosomes Ceramides

• Golgi BODIPY-Ceramide

• Endoplasmic Reticulum DiOC6(3) Carbocyanine


Calcium Related Applications• Probe Ratioing

– Calcium Flux (Indo-1)

– pH indicators (BCECF, SNARF)

Molecule-probe Excitation EmissionCalcium - Indo-1 351 nm 405, >460 nmCalcium- Fluo-3 488 nm 525 nmCalcium - Fura-2 363 nm >500 nmCalcium - Calcium Green 488 nm 515 nmMagnesium - Mag-Indo-1 351 nm 405, >460 nmPhospholipase A- Acyl Pyrene 351 nm 405, >460 nm


Probes for Ions

• INDO-1 Ex350Em405/480

• QUIN-2 Ex350 Em490

• Fluo-3 Ex488 Em525

• Fura -2 Ex330/360 Em510


Ionic Flux Determinations• Calcium Indo-1• Intracellular pH 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

Time (Seconds)0 36 72 108 144 180

RAT

IO [s

hort

/long

]0

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1000

StimulationStimulation0

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0 50 100 150 200

Rat

io: i

nten

sity

of 4

60nm

/ 40

5nm

sig

nals

Time (seconds)

Flow Cytometry Image Analysis


Oxidative Reactions

• Superoxide Hydroethidine

• Hydrogen Peroxide Dichlorofluorescein

• Glutathione levels Monobromobimane• Nitric Oxide DAF-FM (4-amino-5-

methylamino-2',7'-difluorofluorescein)


DCFH-DA DCFH DCFDCF

COOHH

Cl

O

O-C-CH3

O

CH3-C-O

Cl

O

COOHH

Cl

OHHO

Cl

O

COOHH

Cl

OHO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’-dichlorofluorescin

2’,7’-dichlorofluorescin diacetate

2’,7’-dichlorofluoresceinCellular Esterases

H2O2

DCFH-DA

DCFH-DADCFH-DA

DCFHDCFH

DCF

H OH O 2 22 2

Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence.1

1000

100

10

1

0

20

40

60

cou

nts

PMA-stimulated PMNControl

80


HydroethidineHE EB

NCH2CH3

NH2H2N

H Br-NCH2CH3

NH2H2N

+

O2-

Phagocytic Vacuole

SODH2O2

NADPH

NADP

O2

NADPH Oxidase

OH-

O2-

DCFDCF

HE

OO22--

HH22OO22

DCFDCF

Example: Neutrophil Oxidative Burst


Macrovascular Endothelial Cells in Culture

Time (minutes)0 60


Hydrogen peroxide measurements with DCFH-DA

0

200

400

600

800

1000

1200

1400

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0 500 1000 1500 2000 2500 3000Time in seconds

cell 1

cell 2

cell 3

cell 4

cell 5

% c

hang

e (D

CF

fluo

resc

ence

)

525 nm

1 23

45

Step 6B: Export data from measured regions to Microsoft Excel

Step 7B: Export data from Excel data base to Delta Graph

Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.


Superoxide measured with hydroethidine

Export data from Excel data

base to Delta Graph

Export data from measuredregions to Microsoft Excel

cell 1

cell 2

cell 3cell 4

cell 5

Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.

%ch

ange

(D

CF

fluo

resc

ence

)

-200

0200

400600

8001000

12001400

16001800

cell 1

cell 2

cell 3

cell 4

cell 5

Time in seconds

1000 1200 1400 1600 1800600 800 200 400


H2O2 stimulation and DCF & EB loading in Rat Pulmonary Artery Endothelial Cells

ENDO HBSSENDO HBSS TNFa

ENDO L-argENDO/ L-arg TNFaENDO/ D-arg

ENDO/ D-arg TNFaEndo + 200uM H2O2Endo + 200uM H2O2Endo + 200uM H2O2

Endo / TNFa + 200uM H2O2Endo / TNFa + 200uM H2O2Endo / TNFa + 200uM H2O2

Endo / L-arg + 200uM H2O2Endo / L-arg + 200uM H2O2Endo / L-arg + 200uM H2O2

Endo / L-arg TNFa + 200uM H2O2Endo / L-arg TNFa + 200uM H2O2Endo / L-arg TNFa + 200uM H2O2Endo / D-arg + 200uM H2O2Endo / D-arg + 200uM H2O2Endo / D-arg + 200uM H2O2

Endo / D-arg TNFa + 200uM H2O2Endo / D-arg TNFa + 200uM H2O2Endo / D-arg TNFa + 200uM H2O2

0

20

40

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0 20 40 60 80 100 120 140Time (minutes)

Me

an

EB

Flu

ore

sc

en

ce

.

200uM H2O2

added

Time (seconds)

DC

F F

luo

resc

ence

Confocal System - Fluorescence Measurements

200uM H2O2

added

24 treatments - 5000 cells each


pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620

440/488 525[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

Probe Excitation Emission


Exotic Applications of Confocal Microscopy

• FRAP (Fluorescence Recovery After Photobleaching)

• Release of “Caged” compounds

• Lipid Peroxidation (Parinaric Acid)

• Membrane Fluidity (DPH)


“Caged” Photoactivatable Probes

• Ca++: Nitr-5

• Ca++ - buffering: Diazo-2

• IP3

• cAMP

• cGMP

• ATP

• ATP--S

Available Probes

Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm


Release of “Caged” Compounds

UV Beam

Release of “Cage”

Culture dish


Time (seconds) after UV FLASH

Release of Caged Nitric Oxide inAttached PMN

0

50

100

150

200

250

0 20 40 60 80 100 120 140 160Flu

ores

cenc

e E

mis

sion

at 5

15 n

m

Release of Caged Compounds

CDUV excited

Control Region

Time (seconds) CONTROL

0

50

100

150

200

250 CONTROL STUDY

Fluo

resc

ence

Em

issi

on a

t 515

nm

0 100 200 300 400


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.


1

2

33

2

1

405/35 nm460 nm

Calcium ratios with Indo-1

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 Export data from Excel data base to Delta Graph

50 100 150 2000

0.1

0.20.3

0.40.5

0.60.7

0.8

0

cell 1 cell 2 cell 3

Ratio: intensity1 (460nm) / intensity2 (405/35nm)


FRAPIntense laser BeamBleaches Fluorescence

Recovery of fluorescence

10 seconds 30 secondsZero time

Time

%F


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


Thick Tissue - Bone and Cartilage

• Very difficult to image thick specimens

• Can use live specimens if appropriately stained

• Special preparation techniques


Multi-Photon Microscopy

An introduction


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


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.


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.


2-Photon ExcitationThe 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.


3-Photon Microscopy

Advantages UV fluorophore excitation without UV irradiation Similar resolution to 2 photon excitation of UV fluorophores

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.


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.


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.


Multi-Photon Fluorescence MicroscopyThe 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


Instrument Setup


InstrumentationTypical Instrumentation for Multi-Photon Time-

Resolved 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)



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.



2-photon Vs single photon (confocal)

From Current Protocols in Cytometry OnlineCopyright © 1999 John Wiley & Sons, Inc. All rights reserved.

Photo from Brad Amos

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 Nd-doped 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.


Comparison of One-Photon Excitation vs. Two-Photon Excitation

One- 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.

Two- Photon Excitation



Lasers


Dye Excitation Spectra


Lasers and ProbesPulsed 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



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)


Ref. J. R. Lakowicz and I. Gryczynski, "Topics in Fluorescence Spectroscopy", volume V, Plenum Press, 1997Calcium dependent emission spectra of Indo-1 for one-, two- and 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 two-photon. Hence the calcium bound or free form of Indo- 1 can be selectively sense by two- or three-photon excitation respectively.

Selective Detection of Fluorophores in Multi-Photon Excitation



ExamplesDNA 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)

Images from: Multi-Photon Excitation Fluorescence Microscope Coordinator, Madison, WI

3-photon image of a DAPI stained Caenorhabitis elegans worm


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)


1047 nm 488nm


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


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)



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.

http://www.phys.uu.nl/~wwwmbf/ResJV.htm

In the image (right) a fluorescence intensity image of biofilm is shown. Several types of bacteria can be distinguished.


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).

http://www.phys.uu.nl/~wwwmbf/ResJV.htm


Spectral Imaging

• Increasing the number of spectral channels collected

• Allows more advanced classification systems

• Takes more time to image

• Much more complex analysis


Multispectral microscopy – Not more colors!!!

Color imageMultispectralimage

Greyscaleimage

Expansion/rebirth of the Landsat Concept from the 1970s


Multispectral microscopy

Camera controller

AOTF controller

Microscope controller

PC computer

Monitor

Intensified camera

CCD camera

AOTF

Microscope

Intensifiedcamera

AOTF

Purdue Spectral Imaging Project


Lyot filter (static) Single bandpass

LCTF (randomly tunable)

400

450

500

550

600

650

700

750

400 450 500 550 600 650 700 750Mea

sure

d c

ente

r w

avel

eng

th (

nm

)

Wavelength “dialed-in”

High precision and accuracy

Enabling Technology: Liquid tunable filters

Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com


High-resolution cytology segmentation

ConventionalRGB Image

Spectrallysegmented Image

Wavelength (nm)

CharacteristicSpectra

High spectral resolution increases utility of spectrally responsive indicator dyes



Multispectral Imaging – Zeiss Meta

Ability to select a range of wavelengthsAs desired by the user


Nuance-Micro



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

slide 1 t:/classes/bms 602b/lecture 5 602_b.ppt purdue university cytometry laboratories ©...

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