Subject: MODULE C: Course on Optical Sectioning and 3D reconstruction

The module has been designed to teach fundaments of optical sectioning and 3D reconstruction from ultra-structure to function. Microscope components that allow optical sectioning and concepts such as structured illumination and channels will be explained as well as going deeper into image analysis.T

Talks will describe the various approaches for obtaining optical sections in the range of cellular dimensions. Hardware and software solutions will be presented. Principles of optical sectioning have been discussed in previous Modules in details and will be briefly summarized. Participants are invited to revisit the didactic material of Module A and B.

LANGUAGE OF THE COURSE: ENGLISH

CONFOCAL Microscopy – Dr. Carlos Sánchez (UAM-CSIC)

Confocal types

LSM/PSM (Line scanning Microscopes/Point scanning Microscopes)

Excitation sources: Laser, MP

Line and points scanning

Detectors: GaAsP, APD, PMT.

Spinning Disk

Variable pinhole

Detector: Camera

Important concepts:

Channels and laser lines

Size of sections

Size of pinhole

How to handle the zooming

Spectral and sequential acquisition

Filters and detectors

Total Internal Reflection Fluorescence Microscopy (TIRFM)– Dr. Valeria Caiolfa (CNIC)

Principle of TIRFM and microscope set up

Applications

Structured illumination Microscopy - Speaker to be confirmed (Zeiss)

Excitation source: White light

Principle of imaging by structured illumination: grid, pattern

Detector: Cameras

LIGHT SHEET Microscopy- Franziska Klein (Zeiss)

Excitation source and “sheet formation”: Principles of the technique

Target samples: Advantages and disadvantages

Image post-processing, 3D rendering,

TOMOGRAPHY: Scanning Electron microscopy applied to biological samples - David Jiménez (Zeiss)

SEM Principles:

Excitation source: electrons

How an image is formed in EM

Detectors

FE-SEM and FIB-SEM

Correlative microscopy

TOMOGRAPHY: X-Ray Microscopy applied to biological samples Antonio Casares (Zeiss)

Excitation source

How an image is formed in XRM

Detectors (cameras)

Applications in biology/examples