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Microscopes
Varieties and Applications
Presented by Jeff Butler, Quorum Technologies
Resolution Continuum and
different resolving
Techniques
THE FIRST QUESTION
WHAT DO YOU WANT TO SEE?
Consider:
Experimental design
Controls
Imaging Constraints
• We rarely see things directly
• So we often rely on indicators
(fluorescent probes or refractive
properties)
• these may not reflect function
Imaging Constraints
The more we manipulate the sample, the
less it reflects physiological processes
Imaging Constraints
Real Life
Temporal Resolution
Spatial Resolution
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Imaging Continuum
Microscopy Techniques
Overview
‘Classical’ Techniques
• Electron Microscope:
– Transmission Electron Microscope (< 1 nm)
– Scanning Electron Microscope (~ 1-5 nm)
• Confocals (~150 nm lateral, ~350 nm axial):
– Point Scanning Confocal
– MultiPhoton Confocal
– Spinning Disk Confocal
• Widefield Fluorescent Microscopy including:
– Structured Illumination Microscopy (~180 nm lateral)
– Deconvolution Microscopy (~180 nm lateral)
• Transmitted Light Microscopy:
– Brightfield, Phase, DIC, Darkfield (~200 nm)
‘New’ Techniques
• Scanning Tunneling Microscope
(0.1nm lateral, 0.01 nm axial - surface technique)
• Atomic Force Microscope
(0.1nm lateral, 0.01 nm axial - surface technique)
• 4Pi or I3M (2 objectives) (< 100 nm)
• STED (stimulated emission depletion)
(< 100 nm)
• OMX (structured illumination)
(~ 50 nm?)
• TIRF (100 nm axial)
Resolution
The ability to resolve two separate spots
Resolution
Resolution
Airy Disks
Rayleigh criterion
• The peak to peak
minimum distance that
allows separation of two
distinct signals
• Rayleigh criterion: the
radius of one Airy disk:
measured from its point
of maximum intensity to
its first ring of minimum
intensity.
Resolution measurements
Raleigh Equation:
sin Θ = 1.220 λ
D
where:
• Θ is the angular resolution
• λ is the wavelength
• D is the lens aperture diameter
Raleigh Equation
Derivations:
1. Δl =
λ
2 n.a.
2. Δl = 0.61 λ
n.a.
3. Δl =
1.22
(n.a.(obj) + n.a.(cond))
.
.
where:
• Δ l is the minimal spatial resolution
• λ is the wavelength
• n.a. is the lens numerical aperture
Resolution
Spatial resolution is the first determinant
in microscope choice
Electron Microscopes
High resolution
Electron Microscope
• Uses electrons to image the sample
• Electromagnets act as lenses
(though very weak)
• Electron λ ~ 0.0055 nm
• Thus resolutions of < 1 nm
• WONDERFUL RESOLUTION BUT:
Electron Microscope
•
•
•
•
•
•
E.M. requires nonphysiological
conditions including:
very high vacuum
Dehydration
Fixation
Very thin samples (50 - 100 nm)*
exposure to electron opaque heavy
metal salts (Ur, Pb, Os)
Ab staining (immunogold)
Optical Microscopy
Good resolution
Transmitted Light Microscopy
The classical technique
Transmitted light microscopy
• Considered the simplest approach
• All transmitted light microscopes in use
are designed for Kohler illumination
Kohler illumination
Basic format for all
transmitted light
systems
Ensures that:
1.
2.
3.
The lamp is out of focus at
the specimen
The field diaphragm is in
sharp focus at the
specimen plane
The n.a. of the illuminating
beam can be adjusted
without affecting the size of
the illuminated field
Contrast Techniques
• Does not require any stain, but they
enhance contrast:
– Phase Contrast
– Darkfield
– DIC
• All require an initial Kohler setup
Transmitted Light Techniques
Transmitted Light
Advantages:
Disadvantages:
• Simple hardware
requirements:
• Minimal axial
resolution
• Difficult to identify
specific cellular
components (e.g.
proteins)
– Microscope
• Excellent sensitivity
and speed (with the
right camera)
Fluorescent Microscopy
Imaging labeled molecules
Fluorescent Microscopy
• One of the most prevalent techniques
• Allows (requires) a probe specific to
molecule of interest
• BUT: you are visualizing the probe, not
the object of study
– This is even true of GFP-labeled proteins the GFP moiety is often considerably
smaller than the protein
Fluorescence
• Short wavelength
absorbed
• Longer wavelength
emitted
• But this occurs over
over all possible
excitation states
Fluorescence Excitation
• The complexity of
multiple possible
excitation and
emission energies
yields a spectrum
for excitation and
emissions
Fluorescence
There is a symmetrical distribution of excitation
and emission wavelengths
Fluorescence Microscope
• Generally in an epifluorescence format
• Require excitation filter, dichroic &
emission filter
Transmitted vs. epifluorscence
Haze/Blur
• Where does it come from?
Convolution through optics
Convolution through optics
Why?
Convolution through optics
Why?
Perfect Optics
Convolution through optics
Why?
Perfect Optics
DIFFRACTION
Convolution through optics
Why?
Perfect Optics
DIFFRACTION
Real world
Convolution through optics
Why?
Perfect Optics
DIFFRACTION
Real world
Aberrations caused by the objectives
What are some of the solutions?
• Deconvolution
• Structured Illumination (Grid Confocal)
• Confocal Systems
– Point Scanners (classical method)
– Nipkow Spinning Disk (live-cell imaging)
– MultiPhoton (deep tissue imaging)
Fluorescent Microscopes
Why not just use one solution:
• Different microscopy techniques offer
different advantages regarding:
– Speed
– Sensitivity
– Resolution
– Viability
– Penetration
Deconvolution
Removal of haze/blur using math
Point spread function
Calculated PSF
Measured PSF
• The point spread
function is a
description of the
convolution of light
for a given objective
or wavelength
• Dependent on
wavelength, n.a.
and refractive index
DEBLURRING ALGORITHMS
•
•
•
•
Nearest-neighbor
Multi-neighbor
No-neighbor
Unsharp mask
Treats each 2-D plane of a 3-D image
separately
Nearest Neighbor Deblurring
• Operates on plane ‘z’ by blurring its
neighboring planes (z±1) using a digital
blurring filter
• Subtracts the blurred planes from z
• Multi-neighbor methods extend this
method
From: Wallace et al, 2001. BioTechniques 31:1076-1097
IMAGE RESTORATION:
INVERSE FILTERS
Division of the Image FT by the PSF FT
Algorithms:
• Wiener deconvolution
• Regularized least squares
• Tikhonov-Miller regularization
From: Wallace et al, 2001. BioTechniques 31:1076-1097
IMAGE RESTORATION:
CONSTRAINED ITERATIVE
• Nonlinear method that produces quantifiable data
• Uses iterations to test different image estimates
• Adds constraints or rules to each Iteration
–
–
–
–
Distortion removal (smoothing)
Non-negativity
Boundary constraints (non-saturation)
Noise removal
From: Wallace et al, 2001. BioTechniques 31:1076-1097
IMAGE RESTORATION:
CONSTRAINED ITERATIVE
CONSTRAINED ITERATIVE ALGORITHMS:
• Jansson-VanCittert (JVC) algorithm
• Maximum Likelihood
• Expectation maximization
• Maximum Entropy
• Blind Deconvolution*
From: Wallace et al, 2001. BioTechniques 31:1076-1097
Constrained Iterative
Estimate of deconvolved image
Apply PSF*
Compare
to original
image
*blind vs. non-blind
Deconvolution examples
Deconvolution examples
Deconvolution
Advantages:
Disadvantages:
• Simple hardware
requirements:
• Less Axial resolution
• Requires post
processing
– Microscope
– Z-drive
– Camera
• Excellent sensitivity
and speed (with the
right camera)
Structured Illumination
Defining the focal plane
Structured Illumination
• The in focus sample plane is defined by
a grid (grating) in a conjugate focal
plane
• The grid is only in clear focus when the
sample is in focus
Structured Illumination
http://www.microscopyu.com/articles/confocal/confocalintrobasics.html
Image A
Image B
Image C
Image B
Image A
Grid off in Lower Focal Plane
Grid on in Lower Focal Plane
Structured Illumination
• How does it work?
• A combination of optical elements
(the grid) and basic math
Structured Illumination
• When the Sample is
in Focus the Grid is
in Focus
• The Sharper the
Image the Sharper
and Stronger the
Grid
• Weak or out of
Focus information is
removed in the Math
The Math!
Image A
Image B
Image C
(A-B)2
(B-C)2
(A-C)2
Image
OptiGrid
1
1
0
0
1
1
1.414
0.8
0.8
0.2
0
0.36
0.36
0.848
0.5
0.5
0.5
0
0
0
0
Widefield
Structured Illumination
QuickTime™ and a
Cinepak decompressor
are needed to see this picture.
Structured Illumination
Advantages:
• Simple hardware
requirements:
–
–
–
–
Microscope
Z-drive
Camera
Grid Paddle
• No lasers needed
• Excellent axial
resolution
• No post image
processing needed
Disadvantages:
• Slow acquision:
requires 3 captured
frames for one
complete image
Confocal
Removing out of focus light
Convolution through optics
Why?
Perfect Optics
DIFFRACTION
Real world
Aberrations caused by the objectives
Confocal
Out of focus emission light is removed by the
pinhole
Confocal
Out of focus emission light is removed by the
pinhole
Confocal
Out of focus emission light is removed by the
pinhole
Confocal
• The laser aperture minimizes haze by
reducing excitation outside of the point of
interest
Point Scanning Confocal
http://www.microscopyu.com/articles/confocal/confocalintrobasics.html
Point Scanning Confocal
Laser Source
PMT
X Y Mirrors
Dichroic mirror
Pinhole
Raster Scan
http://www.microscopyu.com/articles/confocal/confocalintrobasics.html
Point Scanning Confocal
Point Scanner
Advantages:
Disadvantages:
• Excellent Axial and
Spatial resolution
• Pinholes adjusted
for each wavelength
and objective
• Not so fast, typically
around 5 fps ( though this
can be faster with resonant
scanners - but these are less
accurate)
• Phototoxicity is a
problem - not ideal
for living cells
Multiphoton Confocal
• Uses multi-photon
excitation - no pinholes
needed
• Requires powerful laser
to generate multiphoton
effect
Multiphoton Confocal
• Inherently confocal (no pinhole needed)
• Less toxic to thick living samples
(because it uses lower-energy longer
wavelength excitation light)
• More penetrating (because infrared light
is scattered less going into the sample).
Multiphoton Confocal
Advantages:
Disadvantages:
• Less phototoxicity
• Excellent tissue
penetration
• Ideal for living tissue
• Requires highly
specialized laser
• Not optimal for all
fluorophores
• Slow for many
physiological
processes
Spinning Disk Confocal
Laser beam
Collector disc
Microlens
CCD Camera
Pinhole disc
Dichroic
Objective lens
Sample
Pinhole
Spinning Disk Confocal
QuickTime™ and a
Sorenson Video 3 decompressor
are needed to see this picture.
Spinning Disk Confocal
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Spinning Disk Confocal
Advantages:
Disadvantages:
• Excellent speed
(~30 fps)
• Less phototoxicity:
laser power divided
between pinholes
• Fixed pinhole
QUESTIONS ????
Resources
• Molecular Expressions website:
http://micro.magnet.fsu.edu/
• Microscopy U website:
www.microscopyu.com
• Wallace et al. A Workingperson’s Guide to
Deconvolution in Light Microscopy
BioTechniques, 2001. 31:1076-1097
• James Pawley, Handbook of Biological
Confocal Microscopy