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Optical, Optical, ConfocalConfocal, , Fluorescence, and TwoFluorescence, and Two--

Photon MicroscopyPhoton Microscopy

Dr. McShaneMSE 505

April 28, 2004

LOUISIANA TECH UNIVERSITYINSTITUTE FOR MICROMANUFACTURING / BIOMEDICAL ENGINEERING Outline

• Optical Microscopy• Conventional Microscopes• Fluorescence Microscopy• Scanning Microscopes• Confocal Systems• Two-Photon Microscopy• Near-field Scanning Optical Microscopy• Comparisons

NOTE

• This presentation is not mathematically rigorous…

• Discussion and comparison of optical microscopy can be done by considering the instrumentation (optical/ mechanical/ electrical system configuration used to form images) and impact on resolution

Magnification

The aim of using a microscope is not to magnify an imagebut to see finer details in the image.

Image: scales on the wing of a mosquito – identical mag

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Resolving Ability

The performance of the microscope is expressed as its “resolving power”, the ability to separate (“resolve”) fine details.

Components of Microscope

1. Foot2. Limb3. Tube4. Objectives5. Eyepiece6. Coarse focus7. Fine focus8. Stage9. Turret10.Mirror11.Condenser12.Condenser iris

diaphragm13. Swing-out filter ring14. Control for oblique illumination

Modern Light Microscope Magnification in MicroscopesMagnification of the image takes place in 2 stages: in the objectives and in the eyepieces. Both have their proper magnification engraved.

Magnification of objectives ranges from 3X to 100X, and that ofeyepieces from about 5X to about 15X. The total magnificationthus ranges from about 15X to 1500X.

Exercise: at maximum magnification, how large will a10nm structure appear?

If you have two 10nm structures separated by 10nm,will you be able to see them?

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Optical Aberrations - Chromatic Corrections for Chromatic Aberrations

Spherical Aberrations Longitudinal / Transverse Aberrations

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Astigmatism Astigmatism Aberration

Coma - Geometrical Aberration Note on Aberrations

THE SMALLER THE OBJECT, THE GREATER THE IMPACT OF ABERRATIONS ON IMAGE QUALITY

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Wave Nature of Light Constructive Interference

Young’s Double Slit Experiment Diffraction of Light - Effects of Apertures

The wave pattern that passes the slit can be constructed by representing the wave front in the slit as a collection of point sources all emitting inphase. Diffraction can be considered as “interference” between this collection of point sources.

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Fringe Separation

Fringe separation is expressed in terms of angular separation.Sin θ = λ / W (as W decreases, θ increases)

θ = λ / W, when W is not too small

For circular apertures, the diffraction pattern is also circular.The angular separation between the central maximum and thefirst dark ring is given by,

Sin θ = 1.22 λ / D (D is the diameter of the aperture)

θ = 1.22 λ /D (for large D or small L/D)

Diffraction and Light Intensity Distribution

Numerical Aperture Numerical Apertures

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Numerical Aperture - Airy Disc size The Point Spread Function

• Mathematically, airy disks can be described as a PSF– PSF – output of an imaging

system for an input point source

– General Form

• J1 = Bessel function

www-inst.eecs.berkeley.edu/~ee243/ sp03/lectures/ps_mc.pdf

Airy Disks and the Point Spread Function

• Pinhole at x1

– E-field at any x2 is dependent only on distance:

– Light intensity = (above function) x (complex-conjugate) = airy image intensity distribution function

Rayleigh Criterion

A point source imaged by a lenswill not be imaged to a point, butto a diffraction pattern (Airy disk)of that point source withthe first dark ring having aradius of 1.22 λf/D.

θR = 1.22 λ/D

Rayleigh criterion:Two point sources are just resolvable if the maximum of the diffraction pattern of one point source falls on the first dark ring of the pattern of the second point source.

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Resolution

� The limit of resolution is the smallest separation at whichtwo points can be seen as distinct entities.

� Resolution in light microscope is limited primarily by thewave nature of the light.

The relationship between resolution and wavelength is given by Abbe’s equation: d = 0.612 λ / NA

� There are several equations that have been derived to express the relationship between Numerical Aperture, wavelength, and resolution:

R = λ / 2(NA)R = 0.61 λ / NAR = 1.22 λ / (NA(obj) + NA(cond))

Resolution Versus Wavelength

NA~0.95

Depth of Field/ Image Depth

One important aspect to resolutionis the axial resolving power of anobjective, which is measured parallel to the optical axis, and isoften referred to as DOF (d).

Thickness of object space in focus is inversely proportional to resolution

2

22

)()(

NA

NAnd

−= λ

Conventional Microscopy

• Illuminator– light source and condenser

• Collecting Lens (Objective)• Eye Piece• Detector (Eye, Camera)• No scanning req’d

– Simple– Inexpensive

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Fluorescence Microscopy

• Sometimes we cannot resolve the tiny nanostructures that we make or use, do to size or optical contrast…

• …but if we can specifically “tag” them with another molecule that provides a unique signature, we can use that to determine presence and image distributions

Fluorescence SpectroscopyFluorescence Spectroscopy

• Emission of Photons from Excited States

• Sensitive, Specific• Large choice of dyes

– “Labeling,” “tagging”– Immunostaining, etc.

hhννexex

RelaxationRelaxation

hhννemem

EXEX EMEM

WavelengthWavelength

Ext

inct

ion

Ext

inct

ion

Fluor. IntensityFluor. Intensity

Quantum Yield (QY)Quantum Yield (QY)

# fluorescent photons emitted# fluorescent photons emitted

# photons absorbed# photons absorbed

Epi-illumination

• Illuminate from same side as view

• Less loss due to propagation through turbid samples

Fluorescence Microscopy

Epifluorescent Illumination of Upright Microscope

Epifluorescent Illumination of Inverted Microscope

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“Hot” and “Cold” Mirrors Fluorescence Microscopy

http://micro.magnet.fsu.edu/primer/java/lightpaths/ix70fluorescence/index.html

Point Imaging Microscopy

• Illuminator– light source and condenser

• Collecting Lens (Objective)

• Single detector with pinhole, scanned across fully illuminated sample

• Slower, but only single detector required for digital image acquisition

Scanning Microscopy

• Illuminator– Point source and

objective (projector)– Spot scanned across

specimen

• Collecting Lens• Single Detector

• Slower image formation

• Reduced overall exposure

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Confocal Scanning Microscopy

• Illuminator– Point source and

objective (projector)– Scanned across sample

• Collecting Lens (Objective)

• Point Detector• Pinhole matches source

pinhole (conjugate)

• Both lenses play key roles in image quality

• Slower, more complex instrumentation

• Higher resolution, optical sectioning

The Confocal Principle - Summary

• Provides…– Increased resolution– Optical sectioning capability

• Confocal Necessities– Laser, Light Source Pinhole, Dichromatic

Mirror, Objective Lens, Specimen, Detector Pinhole, Photodetector

Theory of a Confocal System

• Light is directed through objective to object points on specimen near the focal point of lens

• Light is reflected back through objective, or through a second lens, to a pinhole aperture covering a photodetector.

• The pinhole is CONjugate to the FOCAL point of the lens

– Therefore, only the part of the specimen located at the focal point of the lens is detected

• Scanning is achieved via galvanometer mirrors, or translating the stage of the specimen

Theory of a Confocal System

• In-focus plane reaches detector through conjugate pinhole• Out-of-focus plane not able to reach detector

Tilted IC� Blurred Image

Confocal� only in-focus

light accepted

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Resolution in Confocal Microscopy

• Lateral resolution: small advantage– Pinhole � 0– R�

• Axial resolution: major advantage– Reflected light:

– Photoluminescence: 2

77.1

NA

λ

2sin

22.0

2 θλ

n

NA2

22.1

14

1 λ

Comparing with Standard Optical

Standard ConfocalLateral

Best (µm) Typical (µm)

0.013640.0151550.01705

0.0189440.0208390.0227330.0246270.026522

0.1252170.13913

0.1565220.1739130.1913040.2086960.2260870.243478

Axial

resolution (�m)wavelength (nm) Conventional Confocal

360 0.54442344 0.381096400 0.604914934 0.42344450 0.680529301 0.476371500 0.756143667 0.529301550 0.831758034 0.582231600 0.907372401 0.635161650 0.982986767 0.688091700 1.058601134 0.741021

Confocal provides ~30% better resolution, in addition to optical sectioning capability

Confocal Layouts - Minsky

• From Minsky (US Patent 03013467).

Confocal Layout

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Typical CLSM Layout The Need for a Fluor-Confocal System

• Out-of-focus fluorescence overwhelms in-focus details.

• CLSM uses high intensity laser/pinhole.– Intensity decreases with 3rd potency above

and below the focal plane.– Pinhole further reduces out of-focus light.

Confocal Fluorescence Microscopy

• http://www.loci.wisc.edu/images/movies/lpath.mpg

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Ultimate Visualization The Leica TCS SP

Confocal Detection Pinhole Spectrophotometer

Prism

Photomultiplier Tubes

The Effect of Slit on PMTs

The Photomultiplier

• Measures intensity• Slit which allows specific detection

increments, while reflecting other λ’s to other PMTs

• PMT-readings are depicted on monitor

• Slits can be controlled from software:

Slits control increment of detectable wavelengths

The Dichromatic Mirror

• Dichroic mirror can split different colors for simultaneous detection of different fluorophoresin same focal plane

+ =

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Optical Sectioning in CM

• The capability of observing selected thin layers– Less fluorescence blur– Less out-of-focus light

• Only features located within thin plane are seen Conventional

Microscopy

Optical Sectioning in CM

• Optical sectioning depends on axial resolution.– Parts of specimen several axial resolutions away

from focal plane will not be detected– Parts of specimen at different z coordinates within

one axial resolution are different intensities– Extended focus image is the sum of all optical

sections• IEF(x,y) = ∫ I(x,y,z)dz This is the

key factor for optical

sectioning

Optical Sectioning in CM Optical Sectioning in CM

Focal Plane 1: Fig. BDetects S2

Focal Plane 2Nothing Detected

Focal Plane 3: Fig. CDetects S3 and S4Note Intensities

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Optical Sectioning in CM 3D Reconstruction of Optical Sections

Optical Sectioning in CM Optical Sectioning Examples

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Sectioning / Reconstruction IC imaging

• http://micro.magnet.fsu.edu/primer/java/confocal/nikon/index.html

Advantages of CM

• Clearer Images – only in-focus light is detected

• Optical Sectioning– Allows 3D reconstruction

• Increased Sensitivity – PMT for low intensities– Controlled excitation intensity to control

photobleaching (fluorescence)

• Reduced Photobleaching– reduced due to point-illumination

Advantages of CM

• Three-four Dimensional Measurements– through optical sectioning

• Scan through z by changing focal plane

– can use time as a variable

• More Accurate Quantification• Multiple Simultaneous Analyses

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Photobleaching in CM

• CM Enemy #1• Confocal Pattern of Photobleaching

– Photobleaching is proportional to illumination at thatplane

• Illumination level = (photons/µm2) x time• Therefore, assuming absorption of photons is low (number of

photons is constant/plane), as µm2 increases, severity of bleaching decreases.

Photobleaching in CM

• However, as the illuminating cone scans, less severely bleached planes are naturally illuminated for longer periods of time– Volume of space that is equally photobleached is in the shape of

an octahedron, defined by the convergence angle of the beam (half-angle of objective), α

• Note, during reversal of scan, increased time causes increased photobleaching

Focal Plane

Resolution in CM

• Via Point of Illumination

• Via Pinhole

Influence of Pinhole Size in CM

• Airy Disk – disk seen around image of a point source due to diffraction of light– Governs size of pinhole

• Confocal pinhole allows elimination of out-of-focus light!

• Pinhole too big – interference by out-of-focus light

• Pinhole too small – loss of in-focus light � dimmer image

Image obtained at focal plane

Images obtained away from focal plane

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Airy Disks and Pinhole Size in CM

• Intensity of Light as a Function of Radius

Overexposed picture of airy disk; note secondary ring

Optimization of light

Secondary ring

Airy Disks and Resolution

• 2 image points are resolvable if their distance is larger than the radius of the airy disk– Therefore, smaller airy radii allow smaller

distances to be resolved

• Confocal rAiry = 0.4λo/NAobj

– NAobj = numerical aperture of the objective lens– λo = wavelength of light in a vacuum

• If pinhole…– <0.5 rAiry, x-y resolution is improved by 40%, but

signal level reduced by 95%!• As pinhole increases, resolution is reduced

– = rAiry, total focused signal captured, but resolution about 10%

PSF – Key to confocal resolution A Virtual Confocal Instrument

http://micro.magnet.fsu.edu/primer/virtual/confocal/

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Cellular Imaging Confocal Applications

• Latex or silica nanoparticlesserve as template

• Thin films assembled on surface• Inner core removed (acid)• Polyion “shell” acts like cell

membrane Confocal & AFM images5µm shell, 20nm wall (PSS/PAH)4

Shells Produced by Core Removal

Template Dissolution Fragment migration

5µm

5µmNanocapsule from SiO2 core

Decomposition Expts/Results• Capsule walls: {PVS/PAH}4 and

{PVS/PAH}2/{PSS/PAH}2

• Monitored over 15 min w/ 45 sec between images

{PVS/PAH}4

{PVS/PAH}2{PSS/PAH}2

Added stability through the addition of PSS/PAH layers

Confocal Transmission Imaging

• Fluorescent Recovery After Photobleaching(FRAP)– After bleaching area of interest, replacement of the

fluorochrome is a measure of diffusion.

Confocal Applications

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FRAP analysis for diffusion measurements

• Shells loaded 24hr prior to CLSM

• Cover slip sealed to prevent loading solution evaporation

• Data collected over various time periods, depending on Mw.

• Area selected for analysis• 4 data sets collected for

each capsule/Mw combination

FRAP PVS/PAH 4400mw

0

50

100

150

200

250

0 100 200 300 400

Time (sec)

Flu

ore

sc

enc

e In

ten

sity

(AU

)

Capsule Interior

Capsule Exterior

Confocal Applications

• Fluorescence Resonance Energy Transfer (FRET)– Excitation does not release photon, but transfers

energy to nearby molecule– Detects changes in molecular proximity– Can detect changes in conformation of a protein

Two-Photon Microscopy

• Elimination of out of-focus fluorescence• Two photons’ energies combine to excite a fluorophore

1-Photon Excitation 2-Photon Excitation

λ1

λ2

λ3

λ3 = (1/ λ1 + 1/ λ2)-1

Multiphoton Physics

• http://microscopy.fsu.edu/primer/java/multiphoton/jablonski/index.html

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Two-Photon Microscopy

• Minimization of Photobleaching

http://micro.magnet.fsu.edu/primer/java/multiphoton/excitationbleaching/index.html

Two-Photon Microscopy

• 2-photon absorption leads to more localized excitation• Note:1-P intensity constant above and below focal plane• Note: 2-P intensity maximized more locally (A)

A

http://microscopy.fsu.edu/primer/java/multiphoton/excitationregion/index.html

Multi-photon Excitation

• Superposition of energy of photons arriving simultaneously• 2-P Absorption cross-section ~10-50 cm4s/photon-molecule

• C = concentration (molecules/cm3)

• δ = 2-P cross section• P = average laser power (photons/sec)• l = absorption pathlength (cm)• A = cross-sectional area of beam (cm2)

• ΦF = Fluorescence quantum yield (QY/2)

econd)(photons/s 2

22 A

lPKCFl F

h

Φδ=ν

Multi-photon Excitation

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Pawley. Handbook of Biological Confocal Microscopy. 1995

Two-Photon Microscopy

• Pulsing increases two-photon absorption probability• Probability of absorption:

na ≈ δ⟨P⟩2Fp-1(πANA

2/hcλ)2ξ

δ = 2-photon cross section

⟨P⟩ = average power

Fp = repetition frequency

ξ = “advantage” factor

ξ = ⟨p2⟩/⟨p⟩2 = (t1 – t2) ∫ p2(t)dt / (∫ p(t)dt)2

(Integrals over t1 to t2)

Advantages of TPE

Decreased photobleaching: bleaching only occurs on the focal plane

True optical section: fluorescence excitation/emission only from focal plane

Deeper penetration: elimination of using lower wavelength light (higher scattering)

Versatility: tunable excitation wavelength (720-930nm)..one laser for many fluorophores

TPE Manufacturers/Vendors

• Conneaut Lake Scientific, Hartstown, PA 16131 Phone: 814-382-1604, Fax: 814-382-8349 http://www.conneautlakescientific.com

• JASCO, Inc., 649 Commerce Dr., Easton, MD 21601 Phone: 410-822-1220, Fax: 410-822-7526 http://www.jascoinc.com

• Micro Photonics, P.O. Box 3129, Allentown, PA 18106-0129 Phone: 610-366-7103, Fax: 610-366-7105 http://www.microphotonics.com

• VayTek, Inc, 305 West Lowe Ave. P.O. Box 732, Fairfield, IA 52556 Phone: 641-472-2227, Fax: 641-472-8131 http://www.vaytek.com

• WITec Instruments Corp, 101 Tomaras Ave., Savoy, IL 61874 , Fax: 217-352-6655 http://www.WITec-Instruments.com

Near-Field Scanning Optical Microscopy

(NSOM, SNOM)

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Introduction

Near-field scanning optical microscopy (NSOM) is a technique that can achieve spatial resolution performance beyond the

classical diffraction limit by employing a sub-wavelength light source or detector positioned in close proximity to a specimen.

NSOM is also one of the scanning probe techniques…It is a combination of optical microscopy and SPM.

NSOM probes – used for imaging and metrology

Optical Diffraction Limit

Optical systems of any kind that use lenses or mirrors to form an image are limited in their spatial resolution, even with the best designs. Diffraction limit or Abbé's limit:

To increase resolutionDecrease wavelength- X-ray, electron microscopyIncrease NA- immerse the lens into oil

The typical resolution of conventional optical microscopy is about

half the wavelength; for argon laser light (blue/green line, λ= 488 nm)the resolution is ~250nm.

NA/61.0 λδ =

Near-field Optical Microscopy

Light in the visible range is diffraction limited on lengthscales of about 1 micron.

To circumvent this diffraction limit and obtain true nm-scalespatial resolution, a near-field system is used to scan a small 100 nm aperture positioned very close to the surface of interest.

This aperture couples to the high spatial-frequency (evanescent)modes of light that decay exponentially from the surface and that are thus never seen with traditional optical methods.

Principles of NSOM

Fourier optics analysis shows that the diffraction limit on resolution in

optical microscopy is the fundamental constraint for far-field light…

but has little effect on the near-field light.

So, if we can “see” the near-field part of light, we can get images with

resolution dependent on the probe size and the probe-to-sample separation.

That is the key aspect of NSOM.

Both probe size and probe-to-sample separation can, in principle, be made

much smaller than the wavelength of light by micromachining technology.

So we can get resolution beyond the diffraction limit.

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Principle of NSOM

Light from an object can consist of two components: the far-field and the near-field light.

Conventional optical microscopy can only “see” the far-field light. So we can also call it “far-field microscopy.”

NSOM (or SNOM?)

Uses the interaction of a sharp (sub-λ) probe tip with near-fieldlight on a sample or sub-λ aperture near-field light source and/or detector, in order to image the surface at sub-λ opticalresolution.

The spatial resolution is determined by the size and shape of the probe tip.

SNOM employs nanometer precision piezoelectric raster-scanning together with nm-sized sharp probes to obtain lightoptical images at higher resolution

Far-field vs. near-field

Since SNOM can provide very high spatial resolution, one canapply this method to chemical and structural characterizationby the addition of spectroscopic analysis.

The light field at the surface of an object actually contains moreinformation – higher spatial frequencies- than we can imageby using a far-field lens system. Only the spatial frequenciesthat reach the imaging lens (pass through the NA) are “seen”.These are the propagating, low frequencies.Higher spatial frequencies exist at the sample surface, but decay exponentially within a distance less than the wavelength,so never reach the detector.

General NSOM System

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Internal Configuration NSOM Probe – Sample Interaction

http://www.chem.ukans.edu/rdunngroup/nsom.html

Fabrication of NSOM Probes Modes of Operation

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Sample-Probe-Detector Configurations Topographic and Near-field Images

NSOM – Modified Configuration SNOM vs. STM and AFM

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NSOM Manufacturers/Vendors

• Conneaut Lake Scientific, Hartstown, PA 16131 Phone: 814-382-1604, Fax: 814-382-8349 http://www.conneautlakescientific.com

• JASCO, Inc., 649 Commerce Dr., Easton, MD 21601 Phone: 410-822-1220, Fax: 410-822-7526 http://www.jascoinc.com

• Micro Photonics, P.O. Box 3129, Allentown, PA 18106-0129 Phone: 610-366-7103, Fax: 610-366-7105 http://www.microphotonics.com

• VayTek, Inc, 305 West Lowe Ave. P.O. Box 732, Fairfield, IA 52556 Phone: 641-472-2227, Fax: 641-472-8131 http://www.vaytek.com

• WITec Instruments Corp, 101 Tomaras Ave., Savoy, IL 61874 , Fax: 217-352-6655 http://www.WITec-Instruments.com

Other Resources

• http://micro.magnet.fsu.edu/primer/resources/confocal.html

• http://microscopy.fsu.edu/primer/resources/multiphotonweb.html