PH5631: Light Microscopy and Image Processing

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Course Information

L-T-P-C3-0-0-3
Prerequisites None

Syllabus

Module 1: Foundations of Image Formation

Lecture hours: 8 · Tutorial hours: 0 · Lab hours: 0

Geometrical optics in imaging systems: thin lens formula, magnification, Numerical aperture (NA) and resolving power, Diffraction from circular aperture, Abbe theory of image formation, Rayleigh criterion .

Module 2: Architecture of Light Microscopes

Lecture hours: 6 · Tutorial hours: 0 · Lab hours: 0

Infinity-corrected optical systems, Objective lenses: NA, working distance, immersion optics, Tube lens and image plane formation, Condenser optics and Köhler illumination, Optical aberrations (spherical, chromatic, astigmatism) .

Module 3: Contrast Microscopy

Lecture hours: 6 · Tutorial hours: 0 · Lab hours: 0

Bright-field and dark-field microscopy, Phase contrast microscopy, Differential interference contrast (DIC), Polarization microscopy .

Module 4: Fluorescence Microscopy

Lecture hours: 5 · Tutorial hours: 0 · Lab hours: 0

Fluorescence microscopy: electronic transitions, Stokes shift, quantum yield, photobleaching, Basic filter cube design, Fluorophores .

Module 5: Advance Microscopy Techniques

Lecture hours: 5 · Tutorial hours: 0 · Lab hours: 0

Confocal microscopy: optical sectioning principle, Spinning disk confocal microscopy, Total internal reflection fluorescence (TIRF) microscopy, Structured illumination microscopy (SIM), Introduction to super-resolution techniques (STED, PALM/STORM), Light-sheet microscopy .

Module 6: Image Detection and Noise

Lecture hours: 6 · Tutorial hours: 0 · Lab hours: 0

Point spread function (PSF) and convolution description of imaging, Fourier optics approach to imaging, CCD and CMOS detectors, Noise sources: shot noise, read noise, dark noise, Signal to noise ratio, Nyquist sampling criterion in microscopy .

Module 7: Fundamentals of Digital Image Processing and Analysis

Lecture hours: 6 · Tutorial hours: 0 · Lab hours: 0

Digital image representation and bit depth, Histogram analysis and contrast enhancement, Spatial filtering (mean, Gaussian, median), Edge detection (Sobel, Canny), Frequency-domain filtering, Basic deconvolution concepts, Thresholding, segmentation, and particle tracking fundamentals, Open-source image processing tools .

Study Materials

Textbooks

  1. Fundamentals of Light Microscopy and Electronic Imaging, D. B. Murphy and M. W. Davidson, Wiley, 2nd Edition (2013), ISBN: 9780471692140
  2. Digital Image Processing, R. C. Gonzalez and R. E. Woods, Pearson, 4th Edition (2018), ISBN: 978-0133356724

References

  1. Introduction to Fourier Optics, J. W. Goodman, Roberts & Company, 3rd Edition (2005), ISBN: 978-0974707723
  2. Handbook of Biological Confocal Microscopy, J. B. Pawley (Ed.), Springer, 3rd Edition (2006), ISBN: 978-0387455242
  3. Principles of Fluorescence Spectroscopy, J. R. Lakowicz, Springer, 3rd Edition (2006), ISBN: 978-0387312781
  4. Principles of Optics, M. Born and E. Wolf, Cambridge University Press, 7th Edition (1999), ISBN: 978-1108769914