[ALMF]: Essentials II – Essentials of confocal microscopy (online)

Registration for EMBL staff only

The course is covering the essential knowledge needed to conduct a confocal microscopy-based experiments. 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.

Teaching material:

1. Basics of confocal microscopy
2. Nikon Microscopy University – Introductory Confocal Concepts
3. Bit depth and resolution in an image
4. Choosing the right microscopy technique

Course outline

Practical point scanning confocal – part 1:

1. Why/when one needs optical sectioning
2. Lightpath
3. Why need to scan
4. Confocal principle
5. Pinhole size

Practical point scanning confocal – part 2 (stained cells – will use mainly one channel)

1. Lateral and axial resolution
2. Selecting pixel size
3. Saturation and offset
4. SNR Averaging/Accumulation, Voltage, laser power
5. How to achieve higher frame rates (e.g. time lapse): scan speeds, number of pixels, bi-directional scan

Practical point scanning confocal – part 3

1. 3D data acquisitions with confocals – proper stack configuration
2. Multicolor imaging – use of sequences and tracks
3. Multicolor imaging – configuration of detectors
4. Point scanning multicolor imaging – spectral detectors
5. Getting transmitted light image

Q&A and discussion round – alternatives to point scanning confocal for optical sectioning

1. TIRF
2. Spinning disk
3. Multiphoton
4. AryScan
5. SPIM

Bookings