ISAC Pre-congress imaging course
Fundamentals of Digital Microscopy

 

Short course description

Modern optical microscopy provides powerful tools for studying biological samples varying from sub-cellular structures through cells and tissues to whole species. Effective use of these techniques, especially where quantitative imaging is important, requires in-depth knowledge about optics, spectroscopy, and image processing. The goal of this workshop is to provide that necessary background.

The workshop is intended for students and scientists who uses optical microscopy and would like to strengthen their knowledge and understanding of optical microscopy. It will provide the participants with the required tools for quantitative understanding of imaging and microscopy experiments. The course does not require thorough background in mathematics or physics. The program is based on courses that are being taught for graduate students as well as on a broad range of scientific background.

Topics covered by the workshop

  • Physics of light and color
  • Optical image formation and magnification
  • Diffraction limit, numerical aperture, point spread function
  • Principles of fluorescence microscopy
  • Imaging detectors: PMTs and CCD, ICCD, and EM-CCD cameras.
  • 3-D imaging methods (confocal microscopy, 2-photon, 4p )
  • Fundamentals of quantitative imaging and image processing: digitization, dynamic range, sampling, feature extraction, shape descriptors

Course synopsis

This one-and-a-half day course is comprised of five modules, covering (1) fundamentals of light, color and optical imaging, basic and three-dimensional microscopy, (2) optical detectors used for imaging (PMTs and CCDs), (3) advanced imaging techniques, (4) quantitative imaging, and image processing, and (5) practical demonstration of popular imaging tools (fluorescence microscopy, confocal microscopy, automated high-content screening system).

The first part of the course will provide an introduction to the fundamental principles of optics, microscopy and spectroscopy

The physical limitations of biological optical microscopy are well established. However, considerably less attention is paid to the fact that the biological nature of the objects studied imposes additional constraints on microscopic imaging of cells and tissues. The course instructors will present the factors that limit the accuracy, resolution, and reproducibility of microscopic imaging of biological objects. The imaging techniques covered include transmitted light bright-field microscopy, wide-field fluorescence microscopy, confocal microscopy, as well as 2-photon imaging.

The second module will discuss theory and praxis of electronic imaging. Special emphasis will be placed on CCD cameras used for microscopy applications. Participants will learn the most important characteristics of CCD cameras and the way they effect the measurements. It will also cover a set of procedures for evaluation of CCD cameras in the context of cell studies.

The third module will introduce techniques for imaging molecular interactions and protein behavior in live cells. The module will discuss advanced techniques such as FRET, FRAP, FLIP, and FLIM.

In the fourth part, the course will cover the basics of quantitative imaging and image analysis for biological microscopy. After introduction of basic terminology and concepts, some advanced image analysis techniques routinely employed for microscopy and high-content screening will be emphasized.

The last module of the workshop will allow the attendees to see the practical demonstration of the discussed technologies. Widefield and confocal fluorescence microscopy, as well as automated high-contents screening system will be presented.

Modules

The workshop will be composed of the following five modules:

Module I. Optical Microscopy

  1. Introduction to Optical Microscopy - presented by Yuval Garini
    • The physics of light and color
    • Poisson noise and signal-to-noise ratio
    • Microscope image formation
    • Diffraction limit, PSF, resolution, and MTF
  2. 3-D Optical Microscopy - presented by Bartek Rajwa
    • Principles of fluorescence microscopy
    • Confocal system, 2D and 3D imaging
    • Alternative 3-D techniques: 2-photon imaging, 4p, image deconvolution

Module II. Electro-optical detection - presented Bart Vermolen

  • Grayscale & color CCD chips, chip architecture
  • Dynamic range, readout noise, dark noise, cooling
  • ROI, binning, SNR, sampling density, ICCD & EM-CCD
  • Evaluating & selecting a CCD (parameters) per given application

Module III. Quantitative techniques and methods in advanced optical imaging - presented by György Vereb

  • Fluorescence (Forster) Energy Transfer (FRET)
  • Fluorescence recovery after photobleaching (FRAP and iFRAP) and Fluorescence loss in photobleaching (FLIP)
  • Fluorescence lifetime imaging microscopy (FLIM and FLIM-FRET)

Module IV. Quantitative image analysis

  1. Introduction to image analysis - presented by Gustavo Rhode
    • Digital images, pixels, voxels, sampling
    • Histogram manipulations
    • Binary images, segmentation, area estimation, basic shape parameters, scale invariant shape parameters
    • Morphological filters, convolution in spatial domain
    • Averaging and noise reduction
  2. Advanced image analysis, from images to features - presented by Ted Young
    • Filtering in frequency domain (convolution, removal of periodic noise)
    • Feature extraction, shape and texture descriptors (geometric moments, Zernike moments, Haralick GLCMs)
    • Live demo with a microscope + Matlab package

Module V. Practical demonstrations - Zeiss Hungary and BD Bioscience (with participation of Yuval Garini, Bart Vermolen, Gyorgy Vereb, and Bartek Rajwa)

  • Basic microscopy: optical pathways in brightfield microscope Koehler illumination, fluorescence microscopy
  • CCD cameras: setup, image collection, setup, basics of operations
  • Confocal microscopy: principles of operation, image collection
  • HCS system, principles of operations

Learning outcomes

This course will enable you to:

  • Understand the physical fundamentals of optical microscopy and its limitations
  • Understand modern 3-D imaging modalities: methods such as deconvolution, confocal microscopy and 2-photon microscopy
  • Understand the concept of shot noise, read noise, quantum yield and efficiency, signal to-noise camera performance, and dynamic range
  • Understand the basics of imaging detectors and how to select them
  • Understand the basics of image analysis and quantitative imaging

Intended audience

This material is intended for graduate students and young biological research scientists who want to improve their understanding of fundamentals of optical microscopy used in biological research, and who do not have extensive light microscopy experience. No previous experience in advanced light microscopy is required. Participants do not need to have training in physics, optical engineering, or image processing.

Course faculty

Course advisors

  • Ian T. Young, Faculty of Applied Sciences, Department of Imaging Science and Technology, The Netherlands. E-mail: i.t.young@tnw.tudelft.nl
  • Robert Zucker, United States Environmental Protection Agency. E-mail: zucker.robert@epa.gov