Optically modulated fluorescence bioimaging: visualizing obscured fluorophores in high background

Abstract

Fluorescence microscopy and detection have become indispensible for understanding organization and dynamics in biological systems. Novel fluorophores with improved brightness, photostability, and biocompatibility continue to fuel further advances but often rely on having minimal background. The visualization of interactions in very high biological background, especially for proteins or bound complexes at very low copy numbers, remains a primary challenge. Instead of focusing on molecular brightness of fluorophores, we have adapted the principles of high-sensitivity absorption spectroscopy to improve the sensitivity and signal discrimination in fluorescence bioimaging. Utilizing very long wavelength transient absorptions of kinetically trapped dark states, we employ molecular modulation schemes that do not simultaneously modulate the background fluorescence. This improves the sensitivity and ease of implementation over high-energy photoswitch-based recovery schemes, as no internal dye reference or nanoparticle-based fluorophores are needed to separate the desired signals from background. In this Account, we describe the selection process for and identification of fluorophores that enable optically modulated fluorescence to decrease obscuring background. Differing from thermally stable photoswitches using higher-energy secondary lasers, coillumination at very low energies depopulates transient dark states, dynamically altering the fluorescence and giving characteristic modulation time scales for each modulatable emitter. This process is termed synchronously amplified fluorescence image recovery (SAFIRe) microscopy. By understanding and optically controlling the dye photophysics, we selectively modulate desired fluorophore signals independent of all autofluorescent background. This shifts the fluorescence of interest to unique detection frequencies with nearly shot-noise-limited detection, as no background signals are collected. Although the fluorescence brightness is improved slightly, SAFIRe yields up to 100-fold improved signal visibility by essentially removing obscuring, unmodulated background (Richards, C. I.; J. Am. Chem. Soc. 2009, 131, 4619). While SAFIRe exhibits a wide, linear dynamic range, we have demonstrated single-molecule signal recovery buried within 200 nM obscuring dye. In addition to enabling signal recovery through background reduction, each dye exhibits a characteristic modulation frequency indicative of its photophysical dynamics. Thus, these characteristic time scales offer opportunities not only to expand the dimensionality of fluorescence imaging by using dark-state lifetimes but also to distinguish the dynamics of subpopulations on the basis of photophysical versus diffusional time scales, even within modulatable populations. The continued development of modulation for signal recovery and observation of biological dynamics holds great promise for studying a range of transient biological phenomena in natural environments. Through the development of a wide range of fluorescent proteins, organic dyes, and inorganic emitters that exhibit significant dark-state populations under steady-state illumination, we can drastically expand the applicability of fluorescence imaging to probe lower-abundance complexes and their dynamics.

Figure 1

(A) Typical fluorescence blinking from a single silver nanodot (900 nm emitter). (B) Jablonski diagram illustrating the reverse intersystem crossing between bright and dark states of silver–DNA nanodots. (Reproduced with permission from ref (20). Copyright 2013 American Chemical Society.)

Figure 2

Schematic of SAFIRe using FRET pairs on hairpin-forming ssDNA. (A) Typical energy transfer from donor to acceptor with donor excitation only. (B) Saturation of the acceptor excitation can block (or modulate) energy transfer, thereby increasing donor emission through frustrated FRET. (Reproduced with permission from ref (15). Copyright 2010 American Chemical Society.)

Figure 3

(A) Fluorescence response of an optically modulatable blue fluorescent protein (modBFP/H148K) with constant primary excitation and modulated secondary excitation. The insets show Jablonski diagrams representing dark/bright populations. (B) Time trace of aqueous modBFP/H148K with 372 nm primary excitation and 514 nm secondary excitation modulated at 13 Hz. The inset shows the fast Fourier transform (FFT) of the bulk intensity trajectory, recovering the modulation frequency encoded in the fluorescence signal. (C) Analysis of optically modulated image stacks acquired with SAFIRe by taking the Fourier transform of each pixel’s intensity trajectory. The demodulated image is formed from the FFT amplitude at each pixel.

Figure 4

Modulation frequency dependence of AcGFP enhancement at 561 nm for immobilized (red) and diffusing (black) molecules. The modulation depth at each frequency was computed by normalizing the doubled Fourier transform of each time trace by the number of data points and by its DC component. The inset shows the Fourier transform of the square-wave-modulated fluorescence of AcGFP at 1 Hz. (Reproduced with permission from ref (9). Copyright 2012 American Chemical Society.)

Figure 5

(A) Frequency response curves from Cy5, AcGFP, and modBFP/H148k in solution showing the span of the cutoff frequencies due to different photophysical on/off times. (B) Power-dependent cutoff frequency of Cy5 molecules in aqueous solution with constant 710 nm secondary excitation intensity (12 kW/cm²) and varied 594 nm primary excitation intensity.

Figure 6

Selective fluorescence recovery of mitochondria-targeted AcGFP in the presence of high nuclear-targeted EGFP fluorescence for (A) fixed and (B) live NIH 3T3 cells: (leftmost images in (A) and (B)) raw fluorescence of AcGFP-labeled mitochondria and EGFP; (rightmost images in (A) and (B)) demodulated AcGFP fluorescence. SAFIRe efficiently eliminates the heterogeneous, unmodulatable EGFP signal and reveals a >10-fold improved contrast in the demodulated fluorescence images. For all of the images, the primary laser intensity was held at 5.9 kW/cm² and the secondary intensity (64 kW/cm²) was modulated at 300 Hz. All scale bars are 10 μm. (Reproduced with permission from ref (9). Copyright 2012 American Chemical Society.)

Figure 7

(A) Highly nonlinear titration of the numbers of Cy5 molecules with and without constant Texas Red background when FCS was used to determine the numbers of fluorophores. (B) Plots of FFT amplitude vs the number of Cy5 molecules, showing that the modulation amplitude is independent of the presence of the unmodulatable Texas Red background.

Figure 8

Live-cell demodulation of mitochondria-targeted modBFP/H148K. Upon 405 nm illumination, blue fluorescence was collected from modBFP/H148K-mito mixed with high background emission. Coillumination at 514.5 nm modulated at 2 Hz (secondary illumination only within the white circle) recovered only the modBFP/H148K-mito signal on a greatly reduced background (lower circle). The scale bar is 20 μm. (Reproduced with permission from ref (13). Copyright 2013 American Chemical Society.)

Figure 9

(A) Comparison of experimental and computed emission signals of Cy5 buried within high-background skin-tissue-mimicking phantoms. (B) Schematic of an emitter 4 mm deep within the tissue phantom. (C) Experimentally determined and finite element-calculated FFT signals at the 100 Hz modulation frequency when the 0.5 mm thick Cy5-loaded phantom was fixed at a depth of 4 mm. (Reproduced with permission from ref (10). Copyright 2013 American Chemical Society.)