Imaging According to Biologists

1
Imaging According to Biologists James
West Marnett Lab Department of Biochemistry Septe
mber 25, 2003 email jim.west_at_vanderbilt.edu Roo
m 860 Robinson Building (MRB1) 343-7330
2
Lecture Overview

• I. Introductory Remarks
• -Autoradiography/fluorography
• -Phosphorimaging
• -Chemiluminescent and fluorescent imaging
• II. Technical Details of Each Imaging Method
• Data Analysis
• IV. Applications

3
Review of Previous Lectures The Western Blot
Paradigm
4
Old-Fashioned Autoradiography
Autoradiography the direct exposure of film to
radiation (e.g., light, b-particles, or g-rays)
leading to activation of silver bromide
crystals on the film emulsion Fluorography the
exposure of film to secondary light that
was generated by the excitation of a fluor upon
activation by a b-particle
5
Emerging Replacements for Traditional Isotopic
Imaging
Phosphorimaging the direct exposure of
isotopically-labeled samples to a Phosphor screen
and detection by a scanner Chemiluminescent and
Fluorescent Imaging exposure of a sample giving
off light or fluorescence to film or
visualization of this signal by a
computer-operated detector
6
Technical Details of Traditional Autoradiography

• Points to remember
• Never expose film directly to light. Work
• using an infrared light only.
• 2. Always tape down membranes/gels.
• Never feed cardboard into an
• automated film developer.

7
Developing Autoradiograms Manual Processing or
What Developing Machines Do For Us
8
Automated Film Developing A Convenient
Alternative
9
Fluorography An Advanced Autoradiography

• is useful for 3H, 14C, and 35S
• uses organic scintillants (e.g.,
  2,5-diphenyloxazole (PPO)) to
• give off a fluorescent signal when encounter by a
  weak b-emitter
• must soak fluorescent scintillant into gel before
  drying it

10
Making a Quality Impression Using an
Intensifying Screen

• For various film types and isotopes, it may be
• appropriate to use an intensifying screen.
  Screens
• work best with 125I and 32P.

11
Making a Quality Impression Choosing the
Correct Film

• Companies offer different films depending upon
  the
• application, isotope being used, special coating
  chemicals, etc. Consult their catalog to know
  which
• type of film to purchase.

12
Technical Details of Phosphorimaging

• Points to remember
• Do not expose Phosphor screen to high intensity
• white light unless it is to blank it.
• 2. Always tape down membranes/gels.
• Be careful not to scratch the coating off of the
• Phosphor screen.
• 4. Erase the screen when finished.

13
Technical Details of Fluorescent
and Chemiluminescent Imaging
14
Traditional Densitometry Analysis of
Autoradiograms

• used to quantify data on autoradiograms
• compares the intensity of light transmitted
  through
• a sample with the intensity of incident light
• determines an optical density similar to the way
• a spectrophotometer would
• is often computerized (much quicker and easier)

15
Narrow Linear Range of Exposure when Using Film
Why most scientists now prefer to use
Phosphorimagers for radioactive analysis
16
(No Transcript)
17
Applications Which Utilize Autoradiography

• blotting applications Southerns, Northerns, and
  Westerns
• radioactive thin layer chromatography (TLC)
• detection of radiolabeled DNA or proteins
  directly (e.g.,
• immunoprecipitation of a protein from
  radiolabeled cells)
• etc

18
Supplementary reading (not required) Current
Protocols in Molecular Biology, Appendix
Sectionon Detection and Quantitation of
Radiolabeled Proteins and DNA in Gels and Blots
(1999) contributed by Daniel Voytas Ning Ke,
A.3A.1-A.3A.10