第11章单分子光谱_502604452

染料进人DNA 前后发光变化

0306090k H z

(a)

TIR-FM( NR MCB, 4:ss1-5)

Fig. 1. (A) The wtGFP chromophore, consisting of a cyclized tripeptide

FIG. 2. Excitation and emission spectra for the BFP–GFP and BFP–YFP combinations. (A) The BFP (P4-3 variant) and GFPS65T excitation and emission spectra are shown, illustrating the overlap in BFP emission with GFP absorption, a critical requirement for FRET. (B) The overlap in BFP emission with the YFP excitation spectra. The filter sets used to detect the signals from these fluorescent protein pairs and minimize the spectral cross talk are also illustrated (gray lines). FIG. 4. FRET microscopy with BFP and GFP. Pituitary GHFT1-5 cells were cotransfected with expression vectors encoding GFP–and BFP–

C/EBP D244. (A) A reference image showing the nucleus of a cell coexpressing these two proteins was acquired using the GFP filter set to establish the expression level for GFP–C/EBP D244 (bar 5 10 m m). (B) A second digital image was obtained at the same focal plane using the donor filter set to detect the BFP–

C/EBP D244 signal. (C) By changing only the emission filter and using identical conditions as for (B), the acceptor (FRET) image was then acquired. The histograms shown demonstrate that the signal in the FRET channel exceeds the donor signal, and is greater than the signal expected for spectral cross talk alone (see Fig. 3A).

SM analysis of cellular events (NR MCB, 4:ss1-5)

a. Science, 294:864;

b. EMBO J, 19:892;

c. Science, 295:1083; Nat CB, 2:168

Speckle analysis Figure 7 Phosphorylation of EGF–EGFR complexes. A431 cells

were perforated using streptolysin O and stimulated with 10 ng

Cy5–EGF for 1 min. Phosphorylation of the EGFR was detected with

Myosin V1

Ion channel

Combinations of SM techniques for in vitro protein analysis

NR MCB, 4:ss1-5) a, b, BBRC, 290-311; c,d, Single Molecule, 3-33

ZnS-capped CdSe Quantum Dot

A. QD covalently coupled to protein through mercaptoacetic acid

量子点

Luminescence

images of cultured HeLa cells

A. Incubated with

Mercapto-QDs B. QD-transferrin

Conjugates Antibody-induced

agglutination of

QDs that were

labeled with

Human IgG

•In the presence of

BSA (0.5 mg/ml)

B. Aggregated QDs induced

by a specific polyclonal

antibody (0.5ug/ml)

Science, 264:415 (1994)

How Strong Is a Covalent Bond?

The rupture force of single covalent bonds under an

external load was measured with an atomic force

microscope (AFM). Single polysaccharide molecules were

covalently anchored between a surface and an AFM tip

and then stretched until they became detached. By using

different surface chemistries for the attachment,

it was found that the silicon-carbon bond ruptured at 2.0 6

0.3 nanonewtons, whereas the sulfur-gold anchor ruptured

at 1.4 6 0.3 nanonewtons at force-loading rates of 10

nanonewtons per second. Bond rupture probability