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

Article abstract of DOI:10.1021/ja1057423

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.

Full text of DOI:10.1021/ja1057423

Published on Web 08/16/2010
High-Fidelity Hydrophilic Probe for Two-Photon Fluorescence Lysosomal
Imaging
Xuhua Wang,† Dao M. Nguyen,† Ciceron O. Yanez,† Luis Rodriguez,† Hyo-Yang Ahn,†
Mykhailo V. Bondar,§ and Kevin D. Belfield*,†,‡
Department of Chemistry and CREOL, The College of Optics and Photonics, UniVersity of Central Florida,
Orlando, Florida 32816, and Institute of Physics, Prospect Nauki, 46, KieV-28, 03094 Ukraine
Received June 29, 2010; E-mail: belfield@mail.ucf.edu
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This paper contains enhanced objects available on the Internet at http://pubs.acs.org/jacs.
highly localized excitation and prolonged observation time.7
A
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 imag-
ing. 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.
biocompatible, photostable, and water-soluble lysosomal marker
with high 2PA cross section will provide a strong tool to study
lysosomal trafficking and its role in invasion with high-resolution
spatial images.
Scheme 1. Synthesis of Fluorene Derivative LT1
Herein, we describe a symmetric hydrophilic fluorene derivative,
4,4′-[9,9-di(2,5,8,11,14,17,20,23,26,29-decaoxahentriacontan-31-yl)-
9H-fluorene-2,7-diyl]bis(ethyne-2,1-diyl)bis(N,N-dimethylaniline)
(LT1), and demonstrate its use as an efficient 2PA fluorophore to
perform noninvasive labeling of lysosomes in Vitro. The new
fluorene derivative LT1 was specifically designed to contain a pair
of 10-unit polyethylene glycol (PEG) groups in the 9-position of
the fluorene ring, which made the probe highly water-soluble and
imparted very low cytotoxicity. The hydrophilic fluorene derivative
LT1 was synthesized from the 2,7-diiodofluorene 2 and 4-ethynyl-
N,N-dimethylaniline 3 through a Sonogashira cross-coupling reac-
tion using a PdCl2P(Ph3)2/CuI catalytic system in 72% yield
(Scheme 1).8
The linear and nonlinear photophysical properties of the probe
are reported to investigate specific fluorescence characteristics that
are important for nonlinear optical applications. The absorption and
emission spectra of LT1 were sensitive to solvent polarity, and the
emission spectra exhibited large bathochromic shifts in the order
of toluene < THF < EtOH < H2O (Figure S1 and Table S1,
Supporting Information). The emission spectra showed much greater
solvatochromic shifts than absorption spectra (79 nm vs 7 nm),
suggestive of the potential of LT1 as a polarity-sensitive probe.
In addition, the excitation anisotropy spectra and anisotropy
values, r, for LT1 in polytetrahydrofuran (pTHF) are shown in
Figure 1, along with the linear absorption spectra for LT1 in
phosphate-buffered saline. Probe LT1 displayed a constant value
of r ≈ 0.37 for excitation in the spectral range λ ≈ 374-455 nm,
corresponding to the first electronic transition S0 f S1. A higher
electronic transition, S0 f S2, was observed in the wavelength range
λ ≈ 289-326 nm (r ≈ 0.24). Because the pH of lysosomes is ca.
4.8, pH stability in acidic environments is essential for lysosomal
probes. The pH stability of LT1 was investigated by measuring
the absorbance and emission of LT1 in a series of buffers with
different pH values from 4.16 to 10.0. The results, illustrated in
Figure S2 (Supporting Information), indicated that LT1 was very
stable over this entire pH range.
Lysosomes are membrane-bound organelles of ∼500 nm diam-
eter that are terminal degradative compartments of mammalian
cells.1 Lysosomes are involved in numerous physiological processes,
such as bone and tissue remodeling, plasma membrane repair, and
cholesterol homeostasis, along with cell death and cell signaling.2
In addition, tumor invasion and metastasis are largely associated
with altered lysosomal trafficking, as are increased lysosomal
enzyme expression and activity.3 Effective techniques to fluores-
cently label lysosomes of cancer cells and solid tumor models are
therefore of significant interest to study lysosomal trafficking and
its role in invasion.4
In order to understand the biological activities of lysosomes, a
limited number of fluorescent probes derived from Neutral Red
(toluylene red) and Acridine Orange (N,N,N′,N′-tetramethylacridine-
3,6-diamine) have been developed.5 However, most of the com-
mercial probes require a rather short excitation wavelength, seriously
limiting their use in tissue imaging due to low penetration depth,
the need for biomolecular conjugation, pH sensitivity, poor water
solubility, and poor photostability.6 These limitations motivated us
to search for two-photon-absorbing dyes for two-photon fluores-
cence microscopy (2PFM) imaging, since the longer wavelength
(700-1000 nm) light and quadratic dependence on laser intensity
of two-photon absorption (2PA) both provide advantages such as
† Department of Chemistry, University of Central Florida.
‡ College of Optics and Photonics, University of Central Florida.
§ Institute of Physics, Prospect Nauki.
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C O M M U N I C A T I O N S
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.
By employing a standard two-photon-induced fluorescence
method with a femtosecond laser system, probe LT1 afforded a
maximum 2PA cross section of ∼1100 GM (1 GM ) 10-50
cm4 ·s·photon-1) at 700 nm (Figure 1a, squares), which is much
higher than for commercial probes or those reported by others
(<10-200 GM for 2PFM bioimaging).9 In addition, the photosta-
bility and 2PA cross sections of LT1 were compared with those of
the commercially available lysosomal markers LysoTracker Red
(LT Red) and LysoTracker Green (LT Green) via photodecompo-
sition experiments and 2PA cross section measurements. Herein,
we define a figure of merit (FM) by which probes for 2PFM can be
compared, calculated as the product of their fluorescence quantum
yield (Φ) and 2PA cross section (δ) normalized by their photode-
composition quantum yield (η), i.e., FM ) Φδ/η. The FM of LT1
was 2 orders of magnitude higher than those of LT Green and LT
Red (Table 1), providing strong support for the fidelity of LT1
relative to commercial lysotracker probes for 2PFM biological
imaging.
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.
5, 10, 20, and 30 µM LT1 for 2 h. The results demonstrate that
HCT 116 treated with 20 µM provides bright images with negligible
toxicity.
In addition, to determine the location of the probes in the cells,
a colocalization study of LT1 with several well-known one-photon
fluorescence probes in HCT 116 cells was conducted. 1PFM images
of HCT 116 cells co-stained with LT1 and LT Red, mitochondrion
marker MitoTracker Red FM (MT), or Golgi apparatus marker
Alexa Fluor 555 (AF) (Figure 2 and Figure S6, Supporting
Information) individually demonstrated that the localization of LT1
in the cells was nearly identical to that of LT Red. The 1PFM
images of HCT 116 cells were obtained with two different channels:
Fluor out [excitation filter (Ex) 377/50, where 377 means the
transmission maximum is 377 nm and 50 represents bandwidth of
50 nm, i.e., 377 ( 25 nm; dichroic mirror (DM) 409 (this is a
long-pass filter/mirror, with a cutoff at 409 nm, thereby reflecting
all light <409 nm and transmitting light >409 nm); emission filter
(Em) 525/40, i.e., transmission maximum 525 ( 20 nm] for LT1
and Texas Red [Ex 562/40; DM 593; Em 624/40) for commercial
markers under the same conditions. The colocalization coefficient,
A, was calculated by using Slidebook 5.0 software with Pearson’s
method to evaluate the colocalization of LT1 relative to the
commercial probes. The data in Table S2 (Supporting Information)
show that the colocalization coefficient of LT1 with LT Red was
much higher than the others (0.96 vs 0.45, 0.46), indicating that
LT1 possesses lysosomal specificity.
Moreover, the photostability of LT1 and LT Red in the HCT
116 cells was also compared (Figure 2 and Figure S9, Supporting
Information) and was consistent with the photodecomposition
experiments in PBS buffer. The fluorescence intensity of LT Red
decreased by ca. 50% in the first 3 min under successive irradiation
using a 100 W mercury lamp (the power on the focal plane was
∼9 mW), resulting in only 10% of the initial fluorescence intensity
after 15 min irradiation. In contrast, the fluorescence intensity of
LT1 decreased very slowly to ca. 70% of the initial fluorescence
intensity after 15 min irradiation. In order to evaluate if LT1 can
be used as a long-term lysosome tracking probe, a long-term
Table 1. Photophysical Data for LT1, LysoTracker Green, and
LysoTracker Reda
b
f
h
(fl)
max
probe
λ(m1a)x/λ
εc
Φd
δe
η × 106
Φδg
FM × 10-6
LT1
LT Green 502/510 6.3 1.0
LT Red 575/591 4.9 0.07
387/501 7.7 0.38 1135
0.94
3.70
5.31
431.3
17
2.3
458.8
4.6
0.4
17
33
a Except for 2PA cross section measurements, all other measurements
b
were performed in PBS buffer. λmax values of the one-photon
c
absorption and emission spectra in nm. Molar absorbance in 1 × 104
M-1 ·cm-1 d Fluorescence quantum yield, (15%. e 2PA cross section
.
(LT1 in toluene at 700 nm, LT Green in DMSO at 700 nm, and LT Red
in DMSO at 740 nm) in 10-50 cm4 ·s·photon-1 (GM), (15%.
f Photobleaching decomposition quantum yield. g Product of fluorescence
quantum yield and 2PA cross section in GM. h Figure of merit in GM.
In order to demonstrate the potential utility of LT1 for 2PFM
cellular imaging, its cytotoxicity or cell viability must be assessed.
To address this, viability assays in an epithelial colorectal carcinoma
cell line, HCT 116, were conducted via the MTS assay.10 Figure
1b shows the viability data for HCT 116 cells after treatment with
several concentrations of LT1 for 24 h. The data indicate that LT1
has low cytotoxicity (∼85% viability) over a concentration range
from 1 to 20 µM, appropriate for cell imaging. This bodes well for
the utility of this hydrophilic probe, particularly in live cell imaging
applications for lysosomal tracking via 2PFM.
Subsequently, in order to assess whether LT1 can be efficiently
taken up by cancer cells, the uptake of LT1 by HCT 116 cells was
evaluated. The one-photon fluorescence microscopy (1PFM) and
2PFM images showed LT1 can be effectively taken up by HCT
116 cells, and the optimum concentration of LT1 for cellular uptake
was determined by comparing the images of HCT 116 treated with
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C O M M U N I C A T I O N S
lysosome tracking assay was conducted. The results (Figure S10,
Supporting Information) revealed that 9 h after incubation the
images still showed the probe was lysosome-specific, with a
Pearson’s colocalization coefficient of 0.78. This result demonstrates
the potential for LT1 as a stable lysosome marker for two-photon
fluorescence microscopy. An endocytosis process is proposed
(Figure S8, Supporting Information) as a possible pathway of LT1
cellular uptake, consistent with the literature.11
In a next step, to get an initial sense of the lysosomal specificity
of LT1 in other cell lines, COS-7 (African green monkey kidney
fibroblast-like) cells were employed using the same method
described above for the HCT 116 cells. The colocalization images
(Figure S7, Supporting Information) and colocalization coefficient
data (Table S2, Supporting Information) indicated that LT1 can
also specifically label lysosomes of other cells and significantly
avoid staining other organelles such as the Golgi apparatus and
mitochondria, suggesting broad, general utility for this new probe.
To demonstrate the advantage of using probe LT1 as a lysosomal
marker for 2PFM imaging, two-photon fluorescence imaging of
HCT 116 cells was conducted. The HCT 116 cell images (Figure
3) indicate that the 1PFM and 2PFM images are similar, with higher
3D resolution and contrast realized by 2PFM, to the point of
visualizing individual lysosomes, a feature rarely observed with
other lysosomal probes.
far exceed those of commercial lysotracker probes, including higher
2PA cross section, high lysosomal selectivity, 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, paving the way for future studies
with LT1 to detect aberrant lysosomal trafficking. This may
eventually lead to a new agent for studying lysosome-related
diseases such as Tay-Sachs disease, mucopolysaccharidosis III B,
and Niemann-Pick disease.12
Acknowledgment. This work was supported by the National
Institutes of Health (1 R15 EB008858-01), the National Science
Foundation (CHE-0832622 and CHE-0840431), the U.S. Civilian
Research and Development Foundation (UKB2-2923-KV-07),
and the Ministry of Education and Science of Ukraine (grant
M/49-2008).
1
Supporting Information Available: Experimental details, H and
13C spectra, pH sensitivity measurements, photostability measurements,
2PA cross section determination, cytotoxicity assay, cell incubation
conditions, colocalization studies, in Vitro photostability, long-term
lysosome tracking, and 2PFM lysosomal imaging (Figures S1-S10,
Tables S1 and S2). This material is available free of charge via the
Internet at http://pubs.acs.org.
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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).
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A movie showing a 3D rotation of the image in panel c is available in
the HTML version.
In conclusion, we present a very effective hydrophilic fluorene
derivative, LT1, as a lysosomal marker for two-photon fluorescence
cell imaging. A figure of merit was introduced to allow comparison
between probes. The new probe has a number of properties that
JA1057423
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J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010 12239