This course is aimed at providing with an understanding of a variety of advanced light microscopes as research tools. Students will be exposed to topics in basic transmitted light microscopes, fluorescent microscopes (upright and inverted) including wide field, confocal and two-photon microscopes, light-sheet microscope (SPIM) those adapted to high-throughput experiments, as well as live and intravital (in vivo) microscopy.
Some of the topics to be discussed in this course range from optics, the unique properties of each microscope, sample preparation, and up to a variety of applications. Importantly, the course will enable the participants to design work strategies for a variety of microscopes, with the goal of providing with a global understanding regarding how to choose the appropriate technology for ones needs.
Most of the topics in the theoretical part of the course will be presented by the students. Each student will present once for 30-40 minutes. Guidance and advice will be provided in advance. This theoretical part will be followed by hands-on demonstrations of microscope assembly and by 3x6 hours sessions at a microscope of choice (the practical part at the end of the first semester).
The practical part, which will be performed using microscopes located throughout the main campus, will take place at the level of a research group of choice (pending space). A given project will involve planning the experiment, preparing samples, collecting data, image analysis and eventually presenting data in class. During the project presentations students will define for a given microscope several applications and subsequently suggest the parameters and specifications needed to make the microscope suitable for achieving the tasks of interest.
DETAILED SYLLABUS
Introduction
- Microscope basic structure.
- Optical microscopy limitations.
- History overview and milestones in optical microscopy for biomedical applications.
- Basics of optical microscopy image acquisition
- Digital imaging and camera selection.
Microscopy basics
- The compound microscope.
- Resolution limit, depth of field, field of view.
- Microscope objective design, numerical aperture, infinity correction, immersion medium.
- Optical aberrations and their corrections.
- Light paths in the microscope.
- Critical and Kohler’s illuminations.
- Reflection and epi-illumination, transmission mode.
- Inverted microscope.
Label-free microcopy
- Dark-field illumination.
- Phase-contrast microscopy.
- Differential interference contrast (DIC) microscopy.
- Digital holographic microscopy and quantitative phase microscopy
- Polarization microscopy
Label-based microcopy (Fluorescent Microscopy)
- Epi-illumination scheme.
- Fluorescence lifetime, quantum yield.
- Light source for fluorescent microscopy.
- Fluorescent filters.
- Photobleaching.
- Fluorescent dyes and proteins.
- Fluorescence lifetime imaging microscopy (FLIM).
- Fluorescence correlation spectroscopy (FCS).
Optical sectioning 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.
- Light sheet fluorescent microscopy
Super Resolution microscopy
- Near-field microscopy.
- Structured illumination and super-resolution microscopy.
- Stimulated emission depletion (STED) microscopy.
- Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM).
Special Techniques
- Live cell imaging
- Intravital microscopy
- Computational microscopy
- High throughput and robot assisted microscopy