Imaging Technologies

Virtual Beginner

Imaging Technologies modules are dedicated to theoretical basis of imaging technologies including light and electron microscopy, and biomedical imaging.

01
Light Microscopy Technologies (17 topics)
User beginner 8 hours 15 mins 45 secs
Last updated 4 months ago
Introduction to Microscopy & Contrast Enhancement and Considerations in Objective Design

Presenters: Manoj Mathew, Carl Zeiss, Singapore and Steven Ross, Nikon, USA

Introduction to Microscopy & Contrast Enhancement and Considerations in Objective Design - Topics

Introduction to Microscopy

  • Reflection, refraction, and how does a lens work?
  • What is Imaging?
  • Magnification, microscope tube length, aberrations, and microscope objectives
  • Köhler illumination
  • Microscope optical train
  • Abbe's resolution limit
  • Diffraction angle, resolution and numerical aperture

Contrast Enhancement

  • Specimen properties effect on light: absorption, phase shift, and other interactions
  • Why do we need contrast?
  • Phase contrast microscopy: concept, phase rings, phase components, and limitations of phase contrast
  • Dark-field microscopy
  • Differential interference contrast (DIC) microscopy
  • Light polarity, polarizers, and birefringence
  • Polarized light microscopy

Considerations in Objective Design

  • The purpose of a microscope objective lens: magnification, resolving details, and minimizing aberrations
  • How lens functions
  • Common optical aberrations: chromatic, axial chromatic, spherical, coma, and astigmatism
  • Correcting spherical aberration with a correction collar
Fluorophores, Detectors and Imaging Automation pt.1

Presenter: Nico Stuurman, University of California, San Francisco, USA

Fluorophores, Detectors and Imaging Automation pt.1 - Topics
  • Advantages of fluorescence microscopy
  • What is fluorescence? (excitation and emission, Jablonski diagram, fluorescence spectra)
  • Types of fluorescent dyes
  • Photobleaching (mechanism, minimizing photobleaching)
  • Fluorescent labeling (direct immunofluorescence, indirect immunifluorescence, site specific labeling)
  • Single molecule imaging
  • Fluorescent proteins (nonoligomerizing fluorescent proteins, swtichable fluorescent proteins, fluorescent proteins as sensors)
  • Fluorescent filters (excitation and emission filters, interference filters, matching filters and fluorophores)
  • Multiple band pass filters
Fluorophores, Detectors and Imaging Automation pt.2

Presenter: Nico Stuurman, University of California, San Francisco, USA

Fluorophores, Detectors and Imaging Automation pt.2 - Topics
  • Photomultiplier tube (PMT)
  • Imaging cameras (CMOS, CCD, EMCCD)
  • CCD cameras (CCD architecture, binning, color CCDs, magnification and pixel size, Nyquist–Shannon sampling theorem)
  • Quantum Efficiency (QE)
  • Sources of noise, noise-to-signal ratio (SNR)
  • Scientific CMOS
  • Distinguishing intensity levels, intensity scaling, and histogram
Introduction to Laser Scanning Confocal Microscopy

Presenter: Jasmina Dikic, Carl Zeiss

Introduction to Laser Scanning Confocal Microscopy - Topics
  • Introduction to confocal microscopy and the power of optical sectioning
  • Detecting the signal in laser scanning confocal microscope using PMT and GaAsP detectors
  • How to read fluorescence spectra
  • Overview of multifluorescence imaging
  • Overview of FRAP, Photoconversion, FCS, and RICS
  • Understanding your imaging needs - speed, resolution, and sensitivity
  • Airyscan imaging - enhanced resoluion and sensitivity
Multi-colour Imaging

Presenter: Kalliopi Arkoudi and Chris Power, Carl Zeiss

Multi-colour Imaging - Topics
  • Properties of fluorescence microscopy
  • Basic properties of light
  • Spectral separation, excitation & emission crosstalk, and bleed through
  • Multicolor labelling and sequential image acquisition
  • What is a spectral system?
  • Spectral separation
  • Acquisition of lambda stack
  • Linear unmixing
Super-resolution Microscopy

Presenter: Georg Wieser, Carl Zeiss

Super-resolution Microscopy - Topics
  • Resolution and circumvention of diffraction limit
  • Overview of laser scanning confocal microscopy and optical sectioning
  • What is deconvolution?
  • Methods to improve resolution (pinhole, Airyscan, STED, structured illumination)
  • Stimulated Emission Depletion (STED)
  • Structured Illiminaion Microscopy (SIM)
  • Lattice SIM
  • Single Molecule Localization Microscopy (SMLM)
Fluorescence Correlation Spectroscopy (FCS)

Presenter: Thorsten Wohland, National University of Singapore, Singapore

Fluorescence Correlation Spectroscopy (FCS) - Topics
  • Concept: fluctuation
  • Concept: correlation
  • Autocorrelation function
  • Confocal set-up for FCS
  • Model for 1 particle in confocal volume
  • Model for 2 particles in confocal volume
  • Imaging Fluorescence Correlation Spectroscopy
  • Fluorescence Cross-Correlation Spectroscopy (FCCS)
Fluorescence Lifetime Imaging (FLIM)

Presenter: Deborah Barkauskas, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

Fluorescence Lifetime Imaging (FLIM) - Topics
  • The difference between FLIM and confocal imaging
  • How does a detector convert photons into an image?
  • Photon counting in FLIM: time-correlated single photon counting
  • Fluorescene lifetime decay curve
  • Different fluorophores give rise to different decay curves
  • Mathematical modelling of the fluorescence decay curve
  • What is mean fluorescence lifetime?
  • Advantages of fluorescence lifetime analysis
  • What are parametric maps?
  • Environmental factors affect fluorescence lifetime
  • NAD(P)H & FAD metabolism and metabolic ratio
  • Phasor analysis fluorescence lifetime
  • Controls are the key to interpretation of phasor analysis
  • Phasor analysis of FRET
Source material
  1. Introduction to Microscopy & Contrast Enhancement and Considerations in Objective Design: Global BioImaging in collaboration with India BioImaging Consortium
  2. Fluorophores, Detectors and Imaging Automation: Global BioImaging in collaboration with India BioImaging Consortium
  3. Introduction to Laser Scanning Confocal Microscopy: Global BioImaging in collaboration with Zeiss Microscopy
  4. Multi-colour Imaging: Global BioImaging in collaboration with Zeiss Microscopy
  5. Super-resolution Microscopy: Global BioImaging in collaboration with Zeiss Microscopy
  6. Fluorescence Correlation Spectroscopy (FCS): Global BioImaging in collaboration with India BioImaging Consortium
  7. Fluorescence Lifetime Imaging Microscopy (FLIM): Institute for Molecular Bioscience Microscopy, The University of Queensland, Brisbane, Australia