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
Chapter 2 – Optical contrasting techniques
Darkfield
Phase contrast
Differential interference contrast
Which technique is the best?
Polarization
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
Chapter 4 – Image capture
Optical layout for image capture
Film
Monochrome
Additive vs.
subtractive color
CCD cameras
Frame transfer
Interline transfer
Back illumination
Binning
Recording colour
Chapter
5 - The confocal 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
Laser delivery
The primary beamsplitter
Beam
Scanning
Pinhole and
signal channel configurations
Detectors
Chapter
6 – The digital image
Pixels and voxels
Contrast
Spatial sampling - the Nyquist criterion
Temporal
sampling - signal to noise ratio
Multichannel images
Chapter
7 – Aberrations and their consequences
Geometrical aberrations
Spherical aberration
Coma
Astigmatism
Curvature of Field
Chromatic aberrations
Axial chromatic aberration
Chromatic difference of magnification
Practical consequences
Apparent depth
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
Harmonic microscopy
Chapter 9 - High-speed confocal
Spinning disk
Tandem scanning microscope
One-sided
TSM
Microlens
spinning disk systems
Costs
and benefits of disk-based systems
Slit scanners
True slit scanning systems
Spot
scanning, slit detection
Multifocus
Structured illumination
Chapter 10 - Deconvolution and image
processing Guy Cox & Nuno Moreno
3D deconvolution of wide-field
images
Overview – the problem
Inverse
filters
Iterative
approaches
Improving the confocal
image
Convolution filters for
smoothing and sharpening
Chapter
11 - Three dimensional imaging - stereoscopy & reconstruction
Surfaces – 2½ dimensions
Perception of the 3D world
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)
Chapter 12 - GFP, its relatives and
derivatives
The discovery of GFP
GFP – structure
and function
GFP variants
Man-made mutants
Wild
variants
Techniques for
transfection
Chapter 13 – Fluorescent staining
Guy Cox, Teresa Dibbayawan & Eleanor Kable
Immunolabelling
Basics of the immune system
Types
of antibodies
Raising
antibodies
Labelling
Other fluorescent
probes, stains and labels
Vital dyes
Cell-permeant dyes for organelles and
compartments
Membrane
tracer dyes
Chapter 14 - Quantitative fluorescence
Fluorescence measurement
Calibrating
levels
Calibrating
linearity
Limitations
of fluorescence cytometry
Colocalization
The fluorogram
Manders
colocalization coefficient
Ratio imaging
Excitation
ratiometry
Emission
ratiometry
pH
indicators
Calcium
dyes
Membrane
potential
FRAP and other tools for
cell dynamics
Fluorescence recovery after photobleaching
Fluorescence loss in photobleaching
Color
changing labels
Chapter 15 – Advanced fluorescence techniques
Fluorescence Lifetime
Practical Lifetime Microscopy
Frequency domain
Time domain
Fluorescence Resonant Energy Transfer (FRET)
What is FRET useful for?
Identifying and quantifying FRET
A practical worked example
Correction by acceptor bleaching
Estimation of FRET by FLIM
Fluorescence
correlation spectroscopy (FCS)
Chapter 16 - Breaking the diffraction limit
Near field
NSOM
TIRF
Far field approaches
4-pi
Theta
STED
Appendices
Appendix 1 – Guy Cox. Microscope care and maintenance
Appendix 2 – Guy Cox & Eleanor Kable. Keeping cells alive under the microscope.
Appendix 3 – Teresa Dibbayawan & Eleanor Kable. Sample protocols for immunolabelling of plant and animal cells
Appendix 4 – Nuno Moreno. Image
processing with Image J