Introduction – the optical microscope in cell biology
Brief historical overview from Robert Hooke (cells, 1685) through Swammerdam, van Leeuwenhoek (sperm, bacteria, blood cells), Robert Browne (nucleus), Schwann & Schleiden (cell theory), Lister (corrected objectives), Abbe, etc.
Chapter 1 - The light microscope
Lenses and microscopes
The Back Focal Plane of a lens
Good resolution
Resolution - Rayleigh’s approach
Abbe
Add a drop of oil ...
Köhler Illumination
References
Chapter 2 – Optical contrasting techniques
Darkfield
Phase contrast
Differential interference contrast
Hoffman Modulation Contrast
Which technique is the best?
Polarization
References
Chapter 3 - Fluorescence and fluorescence microscopes
What is fluorescence?
How molecules fluoresce
What
makes a molecule fluorescent?
Pathways for de-excitation – photobleaching
Quantum
yield and extinction coefficient
The fluorescence microscope
Optical arrangement
Light source
Filter sets
References
Chapter 4 – Image capture
Optical layout for image capture
Color recording
Additive color model
Subtractive color model
CCD cameras
Frame transfer
Interline transfer
Back illumination
Binning
Capturing color
Filter wheels
Filter
mosaics
Three CCD
with beamsplitters
References
Chapter 5 - The confocal microscope
The scanning optical microscope
The confocal principle
Resolution and Point Spread Function
Lateral resolution in the confocal microscope
Practical confocal microscopes
The light source – lasers
Gas lasers
Solid state lasers
Semiconductor lasers
Supercontinuum lasers
Laser delivery
The primary beamsplitter
Beam Scanning
Pinhole and signal channel configurations
Detectors
References
Further
reading
Chapter 6 – The
digital image
Pixels and voxels
Contrast
Spatial sampling - the Nyquist criterion
Temporal sampling - signal to noise ratio
Multichannel images
References
Further
reading
Chapter 7 – Aberrations and their consequences
Geometrical aberrations
Spherical aberration
Coma
Astigmatism
Field curvature
Chromatic aberrations
Axial chromatic aberration
Chromatic
difference of magnification
Practical
consequences
Apparent depth
References
Further
reading
Chapter 8 - Non linear microscopy
Multiphoton microscopy
Principles of multiphoton fluorescence
Theory and practice
Lasers for non-linear microscopy
Advantages of two-photon excitation
Construction of a multiphoton microscope
Fluorochromes for multiphoton microscopy
Second harmonic microscopy
Summary
References
Further
reading
Chapter 9 - High-speed confocal
Tandem scanning (spinning
disk) microscopes
Petràn system
One-sided
TSM
Microlens array: the Yokogawa system
Costs
and benefits of disk-based systems
Slit scanning
microscopes
Multi-point array
scanners
Structured illumination
References
Further
reading
Chapter 10 - Deconvolution and image
processing Guy Cox & Nuno
Moreno
Deconvolution
Deconvolving confocal images
Image processing
Grayscale operations
Image
arithmetic
Convolution:
smoothing and sharpening
References
Further
reading
Chapter 11 - Three dimensional imaging - stereoscopy & reconstruction
Surfaces – 2½ dimensions
Perception of the 3D world
Motion parallax
Convergence
and focus of our eyes
Perspective
Concealment
of one object by another
Our
knowledge of the size and shape of everyday things
Light and
Shade
Limitations of confocal microscopy
Stereoscopy
Three-dimensional reconstruction
Techniques which require objects to be identified
Techniques which create views directly from intensity data
Simple projections
Weighted projection (alpha blending)
References
Chapter 12 – Green Fluorescent Protein
Structure and properties
of GFP
GFP variants
Applications of GFP
Heat shock
Cationic
lipid reagents
DEAE-dextran
and polybrene
Calcium
phosphate co-precipitation
Electroporation
Microinjection
Gene
gun
Plants:
Agrobacterium
References
Chapter 13 – Fluorescent staining Guy
Cox, Teresa Dibbayawan & Eleanor Kable
Immunolabelling
Types of antibodies
Raising antibodies
Labeling
Fluorescent stains for
cell components and compartments
References
Chapter 14
- Quantitative fluorescence
Fluorescence intensity measurements
Linearity
Calibration
Measurement
Colocalization
Ratio
imaging
Cell
loading
Membrane potential
Fast response dyes
Slow
response dyes
Fluorescence recovery
after photobleaching
References
Chapter 15 – Advanced fluorescence techniques: FLIM, FRET and FCS
Fluorescence Lifetime
Practical Lifetime Microscopy (FLIM)
Frequency domain
Time domain
Fluorescence Resonant Energy Transfer (FRET)
Why use FRET?
Identifying and quantifying FRET
Increase in brightness of acceptor emission
Quenching of emission from the donor
Lifetime of donor emission
Protection from bleaching of donor
Fluorescence correlation spectroscopy (FCS)
Raster Image Correlation Spectroscopy
References
Further reading
Chapter 16 – Evanescent Wave Microscopy
The near field and
evanescent waves
Total
internal reflection microscopy
Near field microscopy
References
Chapter 17 – Beyond the Diffraction Limit
4-pi and multiple
objective microscopy
Stimulated emission
depletion (STED)
Structured illumination
Stochastic techniques
Super-resolution summary
References
Appendix A – Microscope care and maintenance
Cleaning
The fluorescent illuminator
Appendix B – Keeping cells alive under the microscope. Guy Cox & Eleanor Kable.
Chambers
Light
Movement
Finally
Appendix C –Antibody labeling of plant and animal cells: tips and sample schedules. Eleanor Kable & Teresa Dibbayawan
Antibodies: Tips on Handling and Storage
. Pipettes: Tips on
Handling
Antibodies
and Antibody Titrations
Example
Immunofluorescence Protocol
Methods
Multiple labeling and different samples
Plant material
Protocol
Diagram showing position of antibodies on multiwell slide
Appendix D – Image processing with Image J. Nuno Moreno.
Introduction
Different windows in Image J
Image levels
Colors and look-up tables
Size calibration
Image math
Quantification
Stacks and 3D representation
FFT and image processing
Macro language in Image J
Index