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Biology 370: Principles and Practice of Microscopy
Outline of semester by week
The course is mainly theory-based as it is designed to complement regular
use of microscopes in your research. Sessions that are mainly
practical are marked below in orange and
will involve small groups using the equipment. The times for
the practical work may be slightly different to the normal course
hours. Click on the links to see the slides.
Week |
General theme |
Topics covered |
1
8/24/09
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|
Introduction to the course •
Basic review of some relevant physics •
Optical principles of the compound microscope •
Microscope components for forming an image - objective, eyepiece,
condenser •
Infinity optics •
Conjugate planes and Kohler illumination •
Magnification, resolution and contrast •
Numerical Aperture •
Optical aberrations •
Objectives in more detail •
Contrast in brightfield •
Darkfield •
Phase contrast •
Polarized light •
DIC |
2
8/31/09 |
|
Principles of fluorescence •
Fluorescence energy diagrams and spectra •
Advantages of fluorescence •
How a fluorescence microscope works •
Macroscopes and Stereoscopes •
Objectives for fluorescence •
Resolution in terms of fluorescence •
Illumination sources and control •
Filters and dichroics - principles and terminology •
Single and multichannel imaging •
Capturing a fluorescence image •
CCD cameras - how they work, qualities •
EMCCD cameras •
Binning •
Color cameras •
Software and computer control |
3
9/7/09
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|
Kohler illumination •
Setting up phase contrast, DIC •
General fluorescence imaging •
Stereoscope •
Cleaning objectives •
Aligning an arc lamp and halogen bulb •
Questions about everything so far |
4
9/14/09
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|
The confocal principle • How a
point scanning confocal works • Lasers and their control
• Scanning mirrors
and speed • The pinhole • Photomultiplier tubes
as detectors • Gain and offset • Averaging
• Multi-channel confocal imaging • Zoom and
pixel density • Resolution
in 2D • Nyquist sampling
rate • 3D acquisition • The point spread function
and resolution in 3D • Spectral imaging • Transmitted
light and reflected
light images
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5
9/21/09
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Confocal practice |
Imaging with a Leica
SP5 confocal • Review and questions from the
confocal theory class |
6
9/28/09
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|
Limitations of point-scanning confocals •
Making standard confocals better •
Fast point-scanners •
The photo-bleaching and -toxicity advantage •
Spinning disks and similar •
Comparison of photodetectors •
Line scanning confocals •
Non-confocal means of sectioning •
Deconvolution •
Structure illumination •
TIRF •
Selective Plane Illumination Microscopy and similar
|
7
10/5/09
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|
Principles of MPE •
Differences between 1-Photon and 2-Photon systems •
Excitation advantage •
Emission advantage •
Fluorophore and MPE spectra •
Photobleaching and toxicity in MPE •
Resolution in multi-photon •
How 2P systems work and are constructed •
Tunable pulsed lasers •
Pre-chirping •
NDDs
Review and comparison of imaging modalities
Strengths and weaknesses of the instruments •
How much things cost •
Commercial choices |
8
10/12/09 |
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File storage and transfer • File formats
- bitmap or raster; uncompressed or compressed • Metadata and
image informatics • Bit depths • Digital image contrast and
scaling • Gamma • Lookup tables • Color - perception and color
space • Image display - screen, print, publication • Movies
• Presentation of 3D and 4D data |
9
10/19/09
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|
Principle • Convolution
and deconvolution • Image formation - PSF and OTF • Nyquist
and sampling rates • Deconvolution algorithms • Estimated
and measured PSF • Process of deconvolution • The
benefits of deconvolution for different modalities • Noise • Resolution • Uses
and limitations • Trusting deconvolution |
10
10/26/09
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|
Immunofluorescence •
Properties of a good fluorophore •
Photobleaching •
The green fluorescent protein •
Development of fluorescent proteins •
The fluorescent protein gamut •
Choosing fluorescent protein(s) to use •
Luminescence |
11
11/09/09
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|
Common quantifications •
What’s in an image •
Limitations and non-linearities •
Imaging for quantification •
Software – Commercial and ImageJ •
Getting grey levels •
ROI measurements •
Thresholding and segmentation •
Measurements in 3D •
Colocalization •
Tracking |
12
11/23/09
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|
High throughput/content imaging •
Live cell imaging •
Minimizing phototoxicity •
FRAP/FLIP •
Photoactivation •
Photoconversion •
Protein-protein interactions •
Ratiometric probes •
FRET reporters •
Lifetime measurements •
FCS |
13
11/30/09
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Super-resolution |
The most important concepts •
AFM and EM •
4Pi •
STED •
Structured illumination •
PALM/STORM •
The future . . . |
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