PhD & MS programs > Medical Biophysics: Biomolecular Imaging > Ph.D. Minor in Biomolecular Imaging > Advanced Cellular Imaging

Advanced Cellular Imaging, G613

Course Description

The course goal is to introduce the imaging methods and concepts that are used to extract information about cellular structure and function. The course emphasizes general principles of light microscopy, electron microscopy and digital imaging as modalities of cellular imaging. Applications and examples relate to analysis of cellular properties. Ken Dunn is the Course Director.

Prerequisites

F592 Introduction to Biomolecular Imaging.

Course Instructors

Instructor Department
Atkinson Nephrology
Bacallao Nephrology
Bohlen Physiology
Dunn Nephrology
Elmendorf Physiology
Gattone Anatomy & Cell Biology
Montrose Physiology
Moreno Physiology
Yu Nephrology

Required and Recommended Texts

Fundamentals of Light Microscopy and Electronic Imaging, by Douglas Murphy (Wiley-Liss, 2001) is recommended but not required.

Evaluation and Grading

Two closed book exams will take ~2 hours each. The final is non-cumulative. Laboratory reports will be graded and contribute 10% of the final grade. Exam grades weighted by number of lectures covered in each exam.

Syllabus

Lecture Topic Faculty

Basics of Optical Microscopy

1

Refresher: nature of light

  • Light as a particle, light as a wave
  • Resolution limit of light microscopy

Light path of optical microscope

  • Idealized lenses and geometric optics         
  • Optics of magnification
  • Koehler illumination and conjugate planes

Dunn

2
3

Light sources

  • Monochromatic versus polychromatic
  • Polarization
  • Collimated versus divergent

Optical Components

  • Material properties (glass vs. silica)
  • Lens design, aberration types
  • Multi-coatings
  • Objective, filters, mirrors, 1/4 plates

Montrose

4

 Laser Physics

  • Stimulated emission and amplification
  • Coherence
  • Continuous output versus pulsed output

Yu

5

Transillumination Techniques

  • Inducing and using interference
  • Phase contrast
  • interference contrast

Atkinson

6

Epi-Illumination Techniques

Reflection

  • Fluorescence
  • Review physics and chemistry of fluorescence

Limits to fluorescence detection

  • Shot noise
  • Dynamic range
  • Photobleaching
  • Photon flux

Montrose

Modes of Optical Microscopy

7

Cellular architecture review

  • Organelle and membrane biogenesis
  • cytoskeleton

Atkinson

8
9

Wide field versus confocal

  • studies of localization (immunofl, GFPs)
  • Principle
  • Alternative design Advantages and drawbacks

Multiphoton

  • Principle
  • Advantages and drawbacks

Optical limits to light microscopy

  • Resolution
  • Optical aberrations
    • Spherical aberration
    • Chromatic aberration
  • Imaging at depth in biological tissues
    • Refraction (mounting medium and heterogeneity)
    • Scattering (illumination and detection)
    • Absorbance (illumination and detection)

Montrose

10

Confocal versus Multiphoton Microscopy
Laboratory Session I

Dunn/Montrose

11

Ion imaging
Caged probes

Moreno

12

Reflection interference contrast microscopy
Optical traps
Evanescent wave induced fluorescence

Dunn

13

FRET

Dunn

14

FRAP

Dunn

15
16

FLIM
Fluorescence correlation spectroscopy

Yu

17

FRET &  FRAP Microscopy
Laboratory Session II

Dunn

Mid-Term Exam

18

Mid-Term Exam

  

Digital Image Collection

19

Illumination and collection control

Comparison of detector designs

Maximizing sensitivity, signal-to-noise & dynamic range

Dunn

20

Practice of digital imaging – collecting images

  • Prioritizing speed, resolution, phototoxicity, photobleaching & signal-to-noise ratio
    • Integration time
    • Detector gain
    • Illumination level
    • Resolution (confocal aperture)
  • Resolution and sampling statistics
  • Living within a dynamic range
  • Contrast – truth and beauty

Dunn

21

Controlling Signal-to-noise ratio
Laboratory session III

Montrose/Dunn

22

Multicolor microscopy –

  • sensitivity & discrimination
  • Probe choice and probe design
  • System design
  • Image collection considerations

Elmendorf

23

Multichannel imaging
Laboratory session IV

Montrose/Dunn

Imaging Cellular Specimens

24

Fixed specimens

  • Fixation, permeabilization, mounting techniques
  • Refractive index matching
  • Labeling techniques (Immunofluorescence)

Bacallao

25

Fixed specimen preparation
 laboratory session V

Bacallao/Dunn

26
27

Physiological maintenance of living cells

  • Cultured cells
  • Isolated tissues
  • Labeling approaches (GFP, ligands, surface epitopes)

Montrose

28

Live specimens

  • Intravital imaging
  • The fourth dimension: Time
  • Issues of life, death and focus

Bohlen

29

In vivo imaging
Laboratory session VI

Montrose/Dunn

Electron microscopy

30

Physics and principles of electron microscopy

Atkinson

31
32
33

Modes of electron microscopy

Gattone

34

Electron Microscopy
Laboratory session VII

Gattone

Final Exam

35

Final Exam

  

 

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