Course Identification

Advanced light microscopy

Lecturers and Teaching Assistants

Dr. Vyacheslav Kalchenko, Dr. Yoseph Addadi, Dr. Eduard Korkotian, Dr. Reinat Nevo

Course Schedule and Location

First Semester

Field of Study, Course Type and Credit Points

Life Sciences: Laboratory; Elective; Regular; 2.00 points
Life Sciences (Molecular and Cellular Neuroscience Track): Laboratory; Elective; Regular; 2.00 points
Life Sciences (Brain Sciences: Systems, Computational and Cognitive Neuroscience Track): Laboratory; Elective; Regular; 2.00 points
Life Sciences (Computational and Systems Biology Track): Laboratory; Elective; Regular; 2.00 points


Lectures (room A at FGS):
1. 27.10/21 (14:15-16:00)
2. 03.11/21 (14:15-17:00)
3. 10.11/21 (14:15-17:00)
4. 17.11/21 (14:15-17:00)
5. 24.11/21 (14:15-17:00)
6. 08.12/21 (14:15-17:00)
7. 15.12/21 (14:15-17:00)
8. 22.12/20 (14:15-17:00)
9. 29.12/21 (14:15-17:00)
Practical part:
Concluding session 19/01/2022 (13:00-16:00)- FGS room A





Language of Instruction


Registration by


Attendance and participation


Grade Type

Numerical (out of 100)

Grade Breakdown (in %)


Evaluation Type


Scheduled date 1


Estimated Weekly Independent Workload (in hours)



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.




  • 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).
  • Expansion microscopy

Special Techniques

  • Live cell imaging
  • Intravital microscopy
  • Computational microscopy
  • High throughput and robot assisted microscopy
  • Deep Learning Microscopy 


Learning Outcomes

Upon successful completion of the course students will be able to:

  • Teach the principles of a microscope system of choice.
  • Demonstrate proficiency in common instruments in advanced light microscopy.
  • Analyze samples under variety of microscopes
  • Describe an array of methods in advanced light microscopy.
  • Evaluate their own experimental data.
  • Discuss the relevant methods, their potential and limitations in molecular cell biology

Reading List

1. Early History of Microscopy (Joseph Gall)

2. Lenses and Image Formation (Daniel Fletcher)

3.  Microscope Imaging and Koehler Illumination (Ron Vale)

4. Resolution in Microscopy (Jeff Lichtman)

5. Cameras and Digital Image Analysis (Nico Stuurman)

6. Introduction to Fluorescence Microscopy (Nico Stuurman)

7. Optical Sectioning and Confocal Microscopy (Kurt Thorn)

8. Super-Resolution Microscopy (Xiaowei Zhuang)

9.  Choosing the Right Microscopy Technique (Ron Vale)

10. Dark Field, Phase Contrast, Polarization and DIC (Edward Salmon)