High-fidelity hydrophilic probe for two-photon fluorescence lysosomal imaging

Abstract

The synthesis and characterization of a novel two-photon-absorbing fluorene derivative, LT1, selective for the lysosomes of HCT 116 cancer cells, is reported. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for two-photon fluorescence microscopy (2PFM) lysosomal imaging. The cytotoxicity of the probe was investigated to evaluate the potential of using this probe for live two-photon fluorescence biological imaging applications. Colocalization studies of the probe with commercial Lysotracker Red in HCT 116 cells demonstrated the specific localization of the probe in the lysosomes with an extremely high colocalization coefficient (0.96). A figure of merit was introduced to allow comparison between probes. LT1 has a number of properties that far exceed those of commercial lysotracker probes, including higher two-photon absorption cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. 2PFM was used to demonstrate lysosomal tracking with LT1.

Scheme 1. Synthesis of Fluorene Derivative LT1

Figure 1

(a) Normalized absorption (solid line) (in PBS), emission (dashed line) (in PBS), and anisotropy (dotted line) spectra (in pTHF) and two-photon absorption cross section (squares) (in toluene) of LT1. (b) Viability of HCT 116 cells with LT1.

Figure 2

Colocalization images of HCT 116 cells incubated with LT1 (20 μM, 2 h) and LysoTracker Red (LT Red, 75 nM, 2 h) and photostability comparison of LT1 and LTRed as lysosome markers in HCT 116 cells. Row A: (a) differential interference contrast (DIC) image, (b) one-photon confocal probe LT1 fluorescence image using a custom-made Fluor out filter cube (Ex 377/50, DM 409, Em 525/40), (c) one-photon confocal probe LT Red fluorescence image using a Texas Red filter cube (Ex 562/40, DM 593, Em 624/40), and (d) merged DIC image and two channels of fluorescence images. Rows B and C show one-photon confocal microscopy images of HCT 116 cells co-stained with (B) LT1 and (C) LT Red. The images were taken at (a) 0, (b) 6, (c) 12, and (d) 15 min under successive irradiation; the power on the focus plane was ∼9 mW. All images were acquired with a 60× oil immersion objective.

Figure 3

Images of HCT 116 cells incubated with fluorescence probe LT1 (20 μM, 2 h), all taken with a 60× oil immersion objective: (a) DIC, 500 ms; (b) one-photon fluorescence image, 150 ms (filter cube Ex 377/50, DM 409, Em 525/40); (c) 3D reconstruction from overlaid two-photon fluorescence images (Ex, 700 nm; Em, long-pass filter, 690 nm), 5 μm grid; and (d) two-photon fluorescence image (Ex, 700 nm; Em, short-pass filter, 690 nm). A movie showing a 3D rotation of the image in panel c is available in the HTML version.