Excitation spectra and brightness optimization of two-photon excited probes

Abstract

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced--resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.

Figure 1

(a) Setup to record molecular brightness and action cross section based on fluorescence correlation spectroscopy (FCS). PBS, polarizing beam splitter; PD, photodiode; SP, shortpass emission filter; APD, avalanche photodiode. (b) Autocorrelation curve G(τ) resulting from FCS measurement. The amplitude G(0) is proportional to the inverse of the number of molecules in the focal volume. (c) Plot of the molecular brightness versus the square of the average excitation power. (Red line) Quadratic dependence at low excitation powers (linear in the double logarithmic plot against squared power). (d) Comparison of cross section of fluorescein obtained by Xu and Webb (12) and Makarov et al. (14) (plotted on left axis), and the unscaled two-photon absorption cross section for fluorescein as determined by this work using FCS (right axis). (e) Plot of peak brightness spectra (ε_max, left axis) and scaled action cross section (σ₂η₂, right axis) for fluorescein, where the action cross section has been scaled by 1.9× from the measured (unscaled) value based on comparison to literature values for fluorescein.

Figure 2

Two-photon fluorescence excitation spectra of AlexaFluor dyes in PBS. Data (every 10 nm, line added to connect data points) represent peak molecular brightness.

Figure 3

Peak molecular brightness spectra of rhodamine dyes in PBS.

Figure 4

Peak molecular brightness spectra of genetically encoded and organic Ca²⁺ indicators in 39 μM free Ca²⁺ calibration buffer (30 mM MOPS, 100 mM KCl, 10 mM CaEGTA, pH 7.2). Peak molecular brightness spectra of OGB-1 and OGB-5N were also determined at 0 μM Ca²⁺ (30 mM MOPS, 100 mM KCl, 10 mM EGTA; OGB-1 APO, OGB-5N APO).

Figure 5

Two-photon action cross section and peak brightness are correlated. Action cross section (red circles, line connects data points) and peak brightness (blue boxes) spectra of fluorescein, BODIPY 492/515, BODIPY-TR, rhodamine 110, 5C-TMR, Sulforhodamine 101, AlexaFluor 430, and Resorufin, as determined by FCS. Measurements were performed in 39 μM Ca²⁺ MOPS, pH 7.2, buffer, except for fluorescein and BODIPY 492/515, which were measured in H₂O at pH 11.0 and pH 7.0, respectively. Action cross-section values were normalized to the peak of fluorescein from Makarov et al. (14). The Pearson correlation coefficient r, and the associated p-test value, are given for each curve pair.

Figure 6

Effect of passive pulse splitter on Rhodamine 110. (a) Brightness per molecule as a function of squared excitation intensity divided by the splitting ratio N (N = 1 or 8). (b) 8× splitting as well as ascorbic acid reduce the effects of bleaching. Apparent residence time (normalized to the initial values) decreases with increasing illumination intensity. Addition of ascorbic acid or the splitter increases the bleaching effect thresholds.