Essentials II – Essentials of confocal microscopy (online)

Organizer: Manuel Gunkel, Aliaksandr Halavatyi and Marko Lampe

 Registration for EMBL staff only 

The course is covering the essential knowledge needed to conduct confocal microscopy-based experiments. Along with that, best practices of acquiring, handling and inspection of 3D image (volume) datasets with confocal microscopes are covered.

The prerequisite for attending this course is the knowledge covered in Essentials I course. The microscopy knowledge covered in this is mandatory for introductions into ALMF Confocal-microscopes.

Please be aware, that this course mainly cover practical aspects with only a limited amount of theory. Therefore, we strongly suggest to watch the following recommended tutorials in advance.

Essentials I (Widefield Microscopy):

1. Transmission light path and its alignment
   https://www.ibiology.org/talks/microscope-imaging-koehler-illumination/
2. Fluorescence Microscopy
   https://www.ibiology.org/talks/fluorescence-microscopy/
3. Bit depth and resolution in an image
   https://www.ibiology.org/talks/digital-image-analysis/
4. Resolution in Microscopy
   https://www.ibiology.org/talks/resolution-of-a-microscope/
5. Objectives and optical aberrations
   Objectives and Eyepieces
6. Point spread function
   https://www.youtube.com/watch?v=Tkc_GOCjx7E&list=PL7Y2NBzyw8AhVl5iJZIACsq-Rk8fmB5f9&index=2

Essentials II (Confocal Microscopy):

To follow the course properly you need to have the knowledge from the Essentials I course. If you did not take it, please check the videos above, to make sure you have the knowledge.

1. Basics of confocal microscopy (video)
   Optical sectioning and Confocal microscopy
2. Resolution in microscopy II (video)
   https://www.ibiology.org/talks/resolution-in-microscopy
3. Basics of confocal microscopy (online tutorial)
   https://myscope.training/#/LMlevel_2_14
4. Multicolor acquisition / bleedthrough (online text)
   https://www.olympus-lifescience.com/de/microscope-resource/primer/techniques/confocal/bleedthrough/

Course content and program outline:

Course content:

Practical point scanning confocal – part 1:
Why/when one needs optical sectioning
Lightpath
Why need to scan
Confocal principle
Pinhole size
3D data acquisition with confocals

Practical point scanning confocal – part 2:
Lateral and axial resolution
Selecting pixel size
Choosing an objective
Saturation and offset
SNR Averaging/Accumulation, Voltage, laser power

Practical point scanning confocal – part 3:
How to achieve higher frame rates
3D proer stack configuration
XZ scanning
Refractive index and spherical aberrations
Correcting for z-distortion
Minimizing spherical aberrations

Practical point scanning confocal – part 4:
Multicolor imaging – use of sequences and tracks
Multicolor imaging – configuration of detectors
Chromatic aberrations
Point scanning multicolor imaging – spectral detectors
Getting transmitted light image
Sample types

Image inspection and handling:
Using ImageJ, we will cover the following topics related to 3D image data:
-Orthogonal and arbitrary slice viewing
-3-D rendering
-Reslicing
-Projections

Program outline:

Practical session February 10th:

Image inspection and handling session February 14th:

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