1 Optical, Optical, Confocal Confocal , , Fluorescence, and Two Fluorescence, and Two- Photon Microscopy Photon Microscopy Dr. McShane MSE 505 April 28, 2004 LOUISIANA TECH UNIVERSITY INSTITUTE 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 image but to see finer details in the image. Image: scales on the wing of a mosquito – identical mag
28
Embed
Optical, Confocal, Fluorescence, and Two- Photon Microscopyramu/msnt505/lec_notes/McShane/MSE_505… · • This presentation is not mathematically rigorous… • Discussion and
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
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
• 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
2
Resolving Ability
The performance of the microscope is expressed as its “resolving power”, the ability to separate (“resolve”) fine details.
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?
3
Optical Aberrations - Chromatic Corrections for Chromatic Aberrations
THE SMALLER THE OBJECT, THE GREATER THE IMPACT OF ABERRATIONS ON IMAGE QUALITY
5
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.
6
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
7
Numerical Aperture - Airy Disc size The Point Spread Function
• Mathematically, airy disks can be described as a PSF– PSF – output of an imaging
– 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.
8
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:
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
• 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
• 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
18
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
19
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
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.
25
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.