Ultrafast fluorescence spectroscopy via upconversion applications to biophysics

Abstract

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.

Figure 8.1

Schematic diagram of upconversion.

Figure 8.2

A schematic of upconversion spectrophotofluorometer.

Figure 8.3

A typical response time profile of upconversion spectrophotofluorometer.

Figure 8.4

Trp emission at very early times in water. Reproduced with permission from J. Phys. Chem. B. 105 (2001) 6260. Copyright © 2007 American Chemical Society.

Figure 8.5

Representative fluorescence intensity decay for dipeptides in water (Trp-Leu) within 100 ps.

Figure 8.6

(A) Representative upconverted fluorescence intensity decay for Trp in water (red lines) and in monellin (black lines) at wavelengths 339, 355, and 390 nm using excitation at 295 nm. Reproduced with permission of J. Am. Chem. Soc. 128 (2006) 1214. Copyright © 2007 American Chemical Society.

Figure 8.7

Rawdecay-associated spectra of Trp in monellin extracted from upconversion data (circles) superposed with their polynomial fits (lines) (1.2 ps, blue;16 ps, green; and 2 ns, red). Reproduced with permission of J. Am. Chem. Soc. 128 (2006) 1214. Copyright © 2007 American Chemical Society.

Figure 8.8

Fluorescence decays of five uracils in room-temperature aqueous solutions (~2.5 × 10⁻³ mol/dm³) at 330 nm: (in increasing order) uracil, 6-methyluracil, 1,3-dimethyluracil, 5-methyluracil (thymine), and 5-fluorouracil. Also shown is the 330 fs (FWHM) Gaussian apparatus function. The inset shows the same curves on a semilog scale. Reproduced with permission of J. Am. Chem. Soc. (2006) 607. Copyright © 2007 American Chemical Society.