Advanced Optical Microscopy and its Applications in Biomedicine

Course Name: Advanced Optical Microscopy and its Applications in Biomedicine

Lecturer: Dr. Natan T. Shaked
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Level: Graduate (MSc/PhD/Postdoc) and advanced undergraduate (4th year BSc)
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Course No: 0553-5510-01

Course Grade: 80% final exam, 20% student presentation (last two meetings), up to 5% bonus for simulation/experimental results

Course Subjects:
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1. Introduction and history

Optical microscopy goal.
Microscope basic structure.
Optical microscopy main considerations.
Optical microscopy limitations.
Comparison to other microscopy methods: atomic force microscope (AFM) and electron microscopy (SEM/TEM).
History overview and milestones in optical microscopy for biomedical applications.

2. Optical principles – overview

Complex field.
Radiometry.
Geometric optics.
Thin and thick lenses.
Scalar diffraction theory.
Propagation through a lens.
Fourier optics.
Polarization states.

3. Microscopy basics

The compound microscope.
Resolution limit, depth of field, field of view.
Microscope objective design, numerical aperture, infinity correction, immersion medium.
Digital imaging and camera selection.
Optical aberrations and their corrections.
Light paths in the microscope.
Critical and Kohler’s illuminations.
Reflection and epi-illumination, transmission mode.
Inverted microscope.

4. Special considerations in imaging live biological samples

Incubator and heating stages.
Special slides and coverslips.
Flow and heated chambers.
Water-immersion objectives.

5. Label-free microcopy

Dark-field illumination.
Phase microscopy and the meaning of phase.
Phase-contrast microscopy.
Differential interference contrast (DIC) microscopy.
Digital holographic microscopy and quantitative phase microscopy

6. Label-based microcopy

Fluorescent microscopy
- Epi-illumination scheme.
- Fluorescence lifetime, quantum yield.
- Light source for fluorescent microscopy.
- Fluorescent filters.
- Photobleaching.
- Fluorescent dyes and proteins.
- Calcium probes.
- Optical molecular highlighters.
- Fluorescence lifetime imaging microscopy (FLIM).
- Fluorescence correlation spectroscopy (FCS).
Nanoparticle-based microscopy (metal nanoparticles, quantum dots) and single molecule tracking.

7. Sectioning and selectivity in microscopy

Confocal microscopy: laser scanning and spinning disk.
Fluorescence (Forster) resonance energy transfer (FRET) microscopy.
Total-internal-reflection fluorescence (TIRF) microscopy.
Two-photon and multi-photon microscopy.
Second harmonic generation microscopy.
Deconvolution microscopy and digital deconvolution algorithms.
Optical coherence microscopy.

8. Resolution-enhanced microscopy and nanoscopy

Near-field microscopy.
Structured illumination and super-resolution microscopy.
Simulated emission depletion (STED) microscopy.
Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM).

Books:

1. J. C. Mertz, Introduction to Optical Microscopy, Roberts and Company Publishers (2009).
2. M. Pluta, Advanced Light Microscopy, Elsevier (1988).3. A. Diaspro, Nanoscopy and Multidimensional Optical Fluorescence Microscopy, CRC (2010).
3. A. Diaspro, Nanoscopy and Multidimensional Optical Fluorescence Microscopy, CRC (2010).