ECM Macromolecules: Rotary Shadowing and Scanning Transmission Electron Microscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 121

Fig. 6. Main components of a digital STEM system. Bright-field detector (BF),
annular dark-field detector (ADF), analog-to-digital (A/D) and digital-to-analog
(D/A) convertors.

(ZP). ZP defines the residual signal from bright- and dark-field detectors in the
absence of an incident electron beam. Note the objective lens setting.

2. Extract a rectangular region centered on a TMV particle ( Fig. 8 , panel B).

3. Project (average) the extracted region along the long axis to form a 1D picture

( Fig. 8 , panel C).

4. Calculate the pixel intensity because of the filament ( pi ) ( Fig. 8 , panel C) from

the filament mass ( fm ), left background ( lbg ), and right background ( rbg ).

pi = fm - lbg + rbg / 2 (1)

5. Correct the pixel intensity for fluctuations in probe intensity and detector effi-

ciency where mlc = 1, bf = bright-field panel and zp = zero probe level ( Fig. 8 ,
panel A). Correct for the loss of electrons due to the carbon film, for a thin
(2-3 nm) film cfi = 1.015.

pi' = pi × mlc / ( bf - zp ) × cfi (2)