Reducing background contributions in fluorescence fluctuation time-traces for single-molecule measurements in solution.
We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.
Földes-Papp
Zeno
Z
ISS, Champaign-Urbana, IL 61822, USA. Zeno.Foldes-Papp@medunigraz.at
Liao
Shih-Chu Jeff
SC
You
Tiefeng
T
Barbieri
Beniamino
B
D015502:Absorptiometry, Photon; D000465:Algorithms; D005453:Fluorescence; D007834:Lasers; D012996:Solutions / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014867:Water
D012996:Solutions; D014867:Water