Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals

Abstract

Upconversion fluorescence imaging technique with excitation in the near-infrared (NIR) region has been used for imaging of biological cells and tissues. This has several advantages, including absence of photo-damage to living organisms, very low auto-fluorescence, high detection sensitivity, and high light penetration depth in biological tissues. In this report we demonstrate the use of a new upconversion fluorophore, lanthanide doped nanocrystals, for imaging of cells and some deep tissues in animal. Polyethyleneimine (PEI) coated NaYF₄:Yb,Er nanoparticles were synthesized, which produce very strong upconversion fluorescence when excited at 980 nm by a NIR laser. The nanoparticles were shown to be stable in physiologic buffered saline (PBS), non-toxic to bone marrow stem cells, and resistant to photo-bleaching. The nanoparticles delivered into some cell lines or injected intradermally and intramuscularly into some tissues either near the body surface or deep in the body of rats showed visible fluorescence, when exposed to a 980 nm NIR laser. To the best of our knowledge, this represents the first demonstration of use of upconversion fluorophores for cellular and tissue imaging.

Introduction

Fluorescence imaging is a very important technique for biological studies and clinical applications due to high temporal and spatial resolutions [1]. Conventional fluorescence imaging is based on single-photon excitation, emitting low energy fluorescence when excited by high energy light. Using high energy excitation light has some limitations like DNA damage and cell death caused by long-term irradiation, significant auto-fluorescence from biological tissues resulting in low signal-to-background ratio, and short penetration depth in biological tissues [2]. Upconversion fluorescence imaging, on the other hand, involves conversion of two or more low energy photons—usually near infrared (NIR)—to higher energy visible emissions [3]. The upconversion fluorophores are generally phosphor nanoparticles with a crystalline matrix doped with lanthanide ions. The rare earth elements used in synthesis of the particles have a lower toxicity than semiconductor elements of quantum dots (QDs, LD₅₀ is approximately a thousand times more than that of QDs) [4], [5], [6]. Infrared excitation is less harmful to cells, minimizes auto-fluorescence from biological tissues and penetrates tissues to a greater extent [7].

Upconverting phosphor particles have been used in immunohistochemistry in lateral flow (LF) assay formats, or in immunochromatographic assays for human chorionic gonadotropin (hCG) [13], [14], [15], [16], [17]. A host of in vitro nucleic acid assays have also been described [18], [19]. Furthermore, 150 nm sized particles have been inoculated into live C. elegans and imaged in the intestines of the worms [20]. However, these particles are not suitable for imaging of cells and animals because of their large size and unsuitable surface characteristics. Yb/Er (or Yb/Tm) co-doped NaYF₄ nanoparticles have been reported as the most efficient infrared-to-visible upconversion fluorescent material [21]. Colloidal Yb/Er and Yb/Tm co-doped NaYF₄ nanoparticles with strong upconversion fluorescence seven orders of magnitude higher than that of CdSe–ZnS QDs have been prepared [5], [6]. These nanoparticles are usually synthesized in organic solvents or at high temperatures [22], [23], [24]. Ethylenediamine tetraacetic acid (EDTA) has been used as a chelating agent to control the growth of NaYF₄ nanocrystals, and thermal decomposition of mixed trifluoroacetates has also been used to produce high quality cubic- and hexagonal-phase NaYF₄ nanocrystals [5], [24], [25], [26]. Very recently, we reported a method to use polyvinylpyrrolidone (PVP) as a chelating agent and surfactant to control size and stability of the nanoparticles but the surfactants used to control the nanoparticle growth do not lend themselves to easy modification with biomolecules, and further surface modifications of the nanoparticles are usually required [27]. Coating of the nanoparticles with a layer of silica is sometimes preferred but it is difficult to directly make silica coatings on hydrophobic nanoparticles unless they are previously made hydrophilic or some specially designed silane precursors are used [28]. Furthermore, it is very difficult to make uniform and thin silica coatings on individual nanoparticles and silica is easily coated on the aggregates of nanoparticles. Recently, a simple one-pot synthetic method was developed by our group using polyethyleneimine (PEI) to coat the nanoparticles and control the crystal growth [29]. PEI is a thermally stable and hydrophilic polymer with primary, secondary and tertiary amino groups, which render the nanoparticles water soluble. The amino groups can be used for conjugation of biomolecules to the nanoparticles. In this work, we explore the use of the PEI/NaYF₄ nanoparticles for upconversion fluorescence imaging of cells and animals in vitro and in vivo.