slide 1 t:/classes/bms602 b/lecture 2 602_b.ppt© 1995-2004 j.paul robinson - purdue university...

t:/classes/BMS602 B/Lecture 2 602_B.ppt© 1995-2004 J.Paul Robinson - Purdue University Cytometry Laboratories

Week 2Excitation, 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

UPDATED February 2004

JThese 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.


Overview of lecture 21. Excitation Sources2. Fluorescence3. Raman & Raleigh Scatter4. Photobleaching5. CCD cameras for fluorescence6. Fluorescent probes for biological material7. The structure of a confocal microscope8. Optical properties of confocal systems9. Confocal principals11.Resolution, gray scales and image structure12 Sampling theory and electronic zoom13.Reflection Imaging.


Excitation Sources

Excitation SourcesLamps

XenonXenon/Mercury

LasersArgon Ion (Ar)Krypton (Kr)Helium Neon (He-Ne)Helium Cadmium (He-Cd)Krypton-Argon (Kr-Ar)


Fluorescence

• Chromophores are components of molecules which absorb light

• They are generally aromatic rings


FluorescenceE

NE

RG

Y

S0

S1

S2

T2

T1ABS FL I.C.

ABS - Absorbance S 0.1.2 - Singlet Electronic Energy LevelsFL - Fluorescence T 1,2 - Corresponding Triplet StatesI.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence

IsC

IsC

PH

[Vibrational sublevels]

Jablonski Diagram

Vibrational energy levelsRotational energy levelsElectronic energy levels

Singlet States Triplet States


Simplified Jablonski Diagram

S0

S’1

En e

r gy

S1

hvex hvem


FluorescenceThe longer the wavelength the lower the energy

The shorter the wavelength the higher the energyeg. UV light from sun causes the sunburn

not the red visible light

Intensity related to the probability of the event

Wavelengththe energy of the light absorbed or emitted


FluorescenceStokes Shift

– is the energy difference between the lowest energy peak of absorbence and the highest energy of emission

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Flu

ores

cnec

e In

tens

ity

Wavelength


Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines


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


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)


How many Photons?

• Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e2 intensity is 0.25m 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, FITCFITC would have 63% of its molecules in an excited state and 37% in ground state at any one time


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 excitation488 nm excitation this would give emission at 575-595575-595 nm nm


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)


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)


Photobleaching example

• FITCFITC - at 4.4 x 1023 photons cm-2 sec-1 FITCFITC bleaches with a quantum efficiency Qb of 3 x 10-5

• Therefore FITCFITC 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


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, p-phenylenediamine 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)


Excitation - Emission Peaks

Fluorophore EXpeak EM peak

% Max Excitation at488 568 647 nm

FITC 496 518 87 0 0Bodipy 503 511 58 1 1Tetra-M-Rho 554 576 10 61 0L-Rhodamine 572 590 5 92 0Texas Red 592 610 3 45 1CY5 649 666 1 11 98

Note: You will not be able to see CY5 fluorescence under the regular fluorescent microscope because the wavelength is too high.


Fluorescent Microscope

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination


Fluorescence Microscope withColor Video (CCD) 35 mm Camera

camera

Camera viewer

ocular

filters

objectives

stage

condensor


Cameras and emission filters

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).

Camera goes here

Cooled color CCD camera


Types of Probes

•Proteins•Nucleic Acids•DNA•Ions•pH Sensitive Indicators•Oxidation States•Specific Organelles


Probes for Proteins

FITC 488 525

PE 488 575

APC 630 650

PerCP™ 488 680

Cascade Blue 360 450

Coumerin-phalloidin 350 450

Texas Red™ 610 630

Tetramethylrhodamine-amines 550 575

CY3 (indotrimethinecyanines) 540 575

CY5 (indopentamethinecyanines) 640 670

Probe Excitation Emission


Probes for Ions

• INDO-1 Ex350 Em405/480

• QUIN-2 Ex350 Em490

• Fluo-3 Ex488 Em525

• Fura -2 Ex330/360 Em510


pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620

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

Probe Excitation Emission


Probes for Oxidation States

• DCFH-DA(H2O2) 488 525

• HE (O2-) 488 590

• DHR 123 (H2O2) 488 525

Probe Oxidant Excitation Emission

DCFH-DA - dichlorofluorescin diacetateHE - hydroethidineDHR-123 - dihydrorhodamine 123


Specific Organelle Probes

BODIPY Golgi 505 511

NBD Golgi 488 525

DPH Lipid 350 420

TMA-DPH Lipid 350 420

Rhodamine 123 Mitochondria 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


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


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


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


Fluorescence

Resonance Energy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2


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


Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Confocal Microscope


MRC 1024 System

UV Laser

Kr-Ar Laser

Optical Mixer

ScanheadMicroscope


Bio-Rad MRC 1024


MRC 1024 System

Light Path

PMT


Optical Mixer - MRC 1024 UVArgon Laser

Argon-KryptonLaser

Fast Shutter

UV CorrectionOptics

FilterWheels

To Scanhead

UV Visible

353,361 nm

488, 514 nm

488,568,647 nm

Beam Expander


MRC 1024 Scanhead

From Laser

To and from Scope

32

1PMTGalvanometers

EmissionFilterWheel


To Scanhead

From Scanhead


Scanning Galvanometers

xy

Laser in

Laser out

Point Scanning

ToMicroscope


The Scan Path of the Laser Beam767, 1023, 1279

511, 1023

00Start

Specimen

Frames/Sec # Lines1 5122 2564 1288 6416 32


How a Confocal Image is Formed

CondenserLens

Pinhole 1 Pinhole 2

ObjectiveLens

Specimen

Detector

Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989


Fundamental Limitations of Confocal Microscopy

FromSource

To Detector

. x,y,z

2

n2 photons2

1

n1 photons

1

z

y

xVOXEL

PIXEL

From: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989


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!


Pixels

T

• Pixels & image structureHardcopy 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.


320x240 x 24

Zoom x 2Zoom x 8

Zoom x 4

Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.

Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.

The final image appears to be very “boxy” this is known as “pixilation”.

The final image appears to be very “boxy” this is known as “pixilation”.


541x600x8(2,596,800) 1.5x)

361x400x8

(1,155,200) 2x

180x200x8

(288,000) 1X

Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!

Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!

Socrates?….well perhaps not...

Socrates?….well perhaps not...


320x240 x 24

1500x1125x24

Originals collected at high resolution - compared to a low resolution image magnified

Originals collected at high resolution - compared to a low resolution image magnified


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


Digital Zoom

1 x1024 points

2 x1024 points

4 x1024 points

Note that we have reduced the field of view of the sample


Reflection Imaging

Backscattered light imaging

Same wavelength as excitation

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

CD-ROM pits

Increasingmag

Collagen


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


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)

slide 1 t:/classes/bms602 b/lecture 2 602_b.ppt© 1995-2004 j.paul robinson - purdue university...

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