Biological applications of fluorescence lifetime imaging beyond microscopy

Transcription

1 Invited Paper Biological applications of fluorescence lifetime imaging beyond microscopy Walter J. Akers, 1 Mikhail Y. Berezin, 1 Hyeran Lee, 1 Kevin Guo, 1 Adah Almutairi 2, Jean M. J. Fréchet 3, Georg M. Fischer, 4 Ewald Daltrozzo, 4 Samuel Achilefu 1,5 1 Department of Radiology, Washington University School of Medicine, St. Louis, MO, 2 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 3 Department of Chemistry, University of California, Berkeley, CA, 4 University of Konstanz, Germany, 5 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO. ABSTRACT Fluorescence lifetime is a relatively new contrast mechanism for optical imaging in living subjects that relies on intrinsic properties of fluorophores rather than concentration dependent intensity. Drawing upon the success of fluorescence lifetime imaging microscopy (FLIM) for investigation of protein-protein interactions and intracellular physiology, in vivo fluorescence lifetime imaging (FLI) promises to dramatically increase the utility of fluorescencebased imaging in preclinical and clinical applications. Intrinsic fluorescence lifetime measurements in living tissues can distinguish pathologies such as cancer from healthy tissue. Unfortunately, intrinsic FLT contrast is limited to superficial measurements. Conventional intensity-based agents have been reported for measuring these phenomena in vitro, but translation into living animals is difficult due to optical properties of tissues. For this reason, contrast agents that can be detected in the near infrared (NIR) wavelengths are being developed by our lab and others to enhance the capabilities of this modality. FLT is less affected by concentration and thus is better for detecting small changes in physiology, as long as sufficient fluorescence signal can be measured. FLT can also improve localization of signals for improved deep tissue imaging. Examples of the utility of exogenous contrast agents will be discussed, including applications in monitoring physiologic functions, controlled drug release and cancer biology. Instrumentation for FLI will also be discussed, including planar and diffuse optical imaging in time and frequency domains. Future applications will also be discussed that are being developed in this exciting field that complement other optical modalities. Keywords: Time-resolved, molecular, optical, preclinical, near-infrared 1. INTRODUCTION 1.1. Optical molecular imaging Optical imaging using intrinsic and/or exogenous fluorescence contrast has made significant inroads to clinical applications in recent decades. Strategies for contrast-enhanced optical molecule imaging have relied on spectral resolution of fluorescent agents. Fluorescence microscopy is commonly performed using 3 or more fluorescent reporters Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications II, edited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 7576, SPIE CCC code: /10/$18 doi: / Proc. of SPIE Vol

2 that can be detected in the same tissue sample, then multiplexed with brilliant results, making scientific endeavors into works of art. Fluorescence imaging strategies have often been developed for microscopy applications then translated to preclinical optical imaging techniques. For example, immunolabeling of tissues to identify specific molecules within tissue slices was first demonstrated by fluorescence microscopy. 1 Since that time, labeling of antibodies with radionuclides and fluorescent reporters continues to be commonplace in preclinical molecular imaging. 2 Contrast agents for optical molecular imaging have been based on 3 main strategies: passive delivery, hybrid, and target-specific molecular probes. 3 In many diseases such as cancer and inflammation, reporter molecules may passively accumulate in pathologic tissues due to physiologic alterations such as enhanced bloodflow and vascular permeability. 4 Passive molecular probes can thus identify abnormal tissues, but often lack specificity and produce poor contrast. Hybrid contrast agents similarly accumulate via passive delivery, but upon interaction with disease-specific conditions or molecular events are activated to enhance contrast. 5 Targeted molecular probes, on the other hand, aim to improve disease-specific contrast by high affinity binding of specific molecular signatures within the diseased tissues, such as over-expressed cell-surface receptors. 6 Binding of these targeted molecular probes results in longer retention within the diseased tissues while unbound probe is quickly cleared from non-target tissues. Despite advances in these areas, contrast for diseased tissues is hindered by high nonspecific background Fluorescence lifetime imaging Fluorescence lifetime imaging microscopy (FLIM) is a relatively new field of microscopy which utilizes the sensitivity of excited state fluorophore to detect protein-protein interactions and for chemical sensing. 8 Fluorescence lifetime (FLT) is an intrinsic characteristic of fluorescent compounds including organic fluorophores and inorganic fluorescent materials. Like fluorescence intensity, FLT can change in response to physiochemical effects such as local polarity, ph, temperature and protein binding and photophysical effects such as fluorescence resonance energy transfer (FRET) and quenching. Unlike fluorescence intensity, FLT is generally immune to imaging artifacts due to changes in concentration or instrument effects. Many biomolecules in cells and tissues have fluorescent properties, including proteins, cofactors and others. 9 The combined fluorescence of diseased tissues has been found to differ from healthy tissues and has been proposed for diagnostic purposes via microscopy and clinical imaging systems using FLIM. 9, 10 Autofluorescence FLIM using UV excitation (355 nm/375 nm and 455 nm) can distinguish basal cell carcinoma for improving detection and surgical removal 11. Genetically engineered fluorescent protein reporters such as GFP and spectral derivatives have been used in microscopy for decades and are now used in living animals for cell tracking and to investigate molecular biology. 12 Fluorescent proteins also have distinct FLTs that may change depending on local conditions or due to intermolecular interactions such as fluorescence resonance energy transfer (FRET). FLIM offers an improved method for detecting FRET over spectral techniques that are more subject to background interference 13, 14. Proc. of SPIE Vol

3 Systems have been developed for measuring FLTs in thick tissue for in vivo applications McGinty et al demonstrated FLT DOT for in vitro detection of FRET using TN-L15, a calcium sensor consisting of cyan and yellow fluorescent proteins in turbid media 20. While reconstructed FLTs were underestimated, a clear difference between FRET and no FRET was observed. Cubeddu et al used gated imaging method to show that the fluorescence lifetime of hematoporphyrin derivative (HpD) is longer in xenograft tumor tissue than in surrounding normal tissue in living mice 21. This group also used this method to distinguish PPIX fluorescence in basal cell carcinomas from normal tissue autofluorescence in humans, showing that FLI is as effective for this purpose as multispectral approach 22. Like conventional intensity-based fluorescence imaging, FLI utilizes techniques developed for FLIM to answer biological questions in living animals. FLT contrast may come from intrinsic fluorophores and autofluorescence, fluorescent proteins or exogenous agents administered locally or systemically. In vivo FLI is currently in its infancy despite imaging systems being commercially available for a decade or more. This is due in part to the lack of appropriate contrast agents for in vivo applications and the lack of interest in the field. Here we report recent advances from our lab in contrast-enhanced NIR fluorescent lifetime imaging. Optical imaging applications which benefit from FLT contrast are demonstrated and discussed. FLT imaging has great potential and we are not just scratching the surface. 2. MATERIALS AND METHODS 2.1. In vivo FLI All animal experiments were conducted in accordance with protocols approved by the Animal Studies Committed at Washington University School of Medicine. For imaging and invasive procedures, mice were anesthetized by either intraperitoneal injection of ketamine and xylazine (87 mg/kg and 13 mg/kg, respectively) or 2% isoflurane in 100% oxygen delivered via nosecone. Fluorescent dyes were synthesized as reported previously Polystyrene NIR fluorescent nanoparticles (X-SIGHT 761) were provided by Carestream Health (New Haven, CT). Contrast agents were dissolved in PBS or 20% DMSO as noted below and administered intravenously via the lateral tail vein. Time-domain diffuse optical imaging was performed using the explore Optix system (Advanced Research Technologies, Montreal, Canada) as described previously. 26 The explore Optix system utilizes a 780 nm pulsed laser for excitation and TCSPC-equipped photodetection centered at 830 nm. The detection system is focused 3 mm distant from the excitation focus for diffuse optical spectroscopy and improved depth resolution relative to CCD-based planar reflectance systems. 27, 28 The single source, single detector system is raster-scanned over the subject by means of a translational heated platform on which the subject is placed. Briefly, the anesthetized animal was placed on the heated imaging platform. A region of interest (ROI) was manually selected over a top-view brightfield image to delineate the scanning area. Parameters for the imaging scan were chosen prior to optimize resolution and signal strength, including laser power, integration time and step size. In general, we found that an integration time of 0.3 s and step size of 1.5 mm was optimal and laser power was adjusted from 5-50 μw based on signal strength for each scan. Pre-injection scans were conducted using the highest laser power setting (500 Proc. of SPIE Vol

4 μw) to assess autofluorescence signal strength. Autofluorescence from tissues other than gastrointestinal organs was very low in the mice prior to contrast agent injection and was thus neglected in our analyses. 3. RESULTS AND DISCUSSION FLI using exogenous contrast agents has followed 2 main paths: static FLT probes and designer agents for chemical and biological sensing. Static FLT probes maintain stable FLT values regardless of changes in environment. Functional FLT probes are selected or synthesized with the aim of reporting on local physiologic conditions or biomolecular events by alterations of their FLTs Static FLT probes. We have previously demonstrated differential kinetics and tumor uptake of 2 organic NIR fluorophores using the explore Optix system described above, with 780 nm excitation and 830 nm emission detection. 29 NIR fluorescent reporters generally have relatively broad excitation and emission spectra which makes spectral differentiation in deep tissues difficult to impossible. The FLT resolution of this method is dependent on the system precision, the difference in FLTs between the probes and the variability of the FLT probe in different tissues. For example, fluorescent reporters with FLTs of 0.7 ns and 3.0 ns would be easier to differentiate using FLI than reporters with FLTs of 0.7 ns and 1.0 ns This is particularly true as few organic fluorophores are immune to FLT alterations. Inorganic fluorescent reporters such as quantum dots (QDs) may possess more static FLTs, but they have not been fully characterized at this time. It is also ideal that the fluorescence generated by the reporters be near the same level, preferably with near equivalent brightness. Precise FLT detection requires sufficient signal from both reporters. We have recently updated our library of fluorescent reporters for in vivo FLT imaging with FLTs ranging from 0.4 ns to almost 4 ns. 30, 31 With the addition of a long-lifetime dye, LS474 (Figure 1), FLT imaging with more than 2 dyes is feasible. Proc. of SPIE Vol

5 Figure 1: Fluorescence lifetime map overlaying brightfield image of mouse imaged after intravenous injection of long-lifetime NIR fluorescent dye, LS-474. The lifetime measured in vivo was greater than 3 ns similar to in vitro measurements Functional FLT probes. Perhaps the most promising applications of FLT imaging is in the field of functional imaging. Molecular probes that report on the biological status or biochemical function within living animals have demonstrated utility in medical imaging. Nuclear imaging probes such as 18 FDG and 18 FLT for detecting cancer by increased metabolism or increased cell proliferation, respectively, have made an enormous impact on cancer diagnosis and treatment. These probes actively detect diseased tissues based on biological activity. Fluorescent probes also have this ability and have been used in fluorescence microscopy and in vitro assays for many years. With the development of FLIM, some of these probes have been used as well with more quantitative success. These FLT probes have sensitivity to environmental conditions such as ph, polarity or to the presence of specific chemical species that is expressed as changes in FLT. We have demonstrated various uses for functional FLT probes for sensing protein binding in vitro 32 and in vivo 26, solvent polarity 23, controlled drug release 26, and biodegradation of polymeric nanoparticles 33. The primary mechanism for sensing protein binding is based on the polarity sensing capability of these NIR dyes. It is also well known that small MW compounds such as pharmaceuticals and fluorophores can bind to proteins by noncovalent interaction 32. This interaction commonly occurs with small hydrophobic compounds. Albumin preset in the blood of mammals generally has two such hydrophobic pockets for carrying compounds in the bloodstream, one of the mechanisms of clearing the body of waste metabolites and foreign compounds. 32 We showed that the FLTs of NIR Proc. of SPIE Vol

6 fluorophores are frequently sensitive to solvent polarity in vitro. 23 We then evaluated the correlation of NIR dye FLTs in protein solutions with that measured in living animals after intravenous administration. 26 The FLTs of NIR dyes varied with structure and correlated well with in vitro fluorescence in aqueous solutions of serum albumin. We screened a variety of NIR dyes, showing that the fluorescence lifetimes could be accurately measured in vivo and the route of administration could be clearly established by the changes in FLT of these dyes in the liver and kidneys, dependent on excretion route. 26 One application of protein binding studies is to assess renal function. As blood passes through the kidneys, metabolites and small molecular weight compounds are eliminated through glomerular filtration. High molecular weight proteins such as albumin (67 kd) are too large to pass through the glomerulus and are retained in the blood. Thus in healthy animals, the urine is relatively free of protein. In diseases such as diabetes, the renal function is compromised and proteins escape into the urine. We were able to detect protein in the urine of mice by changes in the FLT of a hydrophilic NIR dye after intravenous injection relative to normal mice. 34 The hydrophilic NIR dye, LS-288, was administered intravenously via the lateral tail vein to healthy nude mice and to mice in which albumin solution had been infused transcutaneously into the bladder. In untreated mice, the fluorescence lifetime in the bladder was lower than in the body. In the proteinuric mice, the FLT of LS-288 was close to that of the body, demonstrating the effect of protein binding and the utility of FLT imaging with LS-288 for non-invasive assessment of renal function in living animals. Protein binding of NIR dyes was also used to measure the rate of metabolism of biodegradable nanoparticles. Nondegradable NIR fluorescent nanoparticles were administered intravenously to mice. The FLT of these nanoparticles did not change significantly over 4 days except within the liver region. 35 On the other hand, biodegradable nanoparticles with a NIR fluorescent dye at their core showed an increase in measured FLT over time after injection. 33 We speculate that the FLT changed as the nanoparticles were degraded and the free NIR dye molecules were opsonized by plasma proteins such as albumin. In this manner, we were able to detect and estimate the rate of nanoparticles degradation (Figure 2). Proc. of SPIE Vol

7 Figure 2: Comparison of FLT changes measured after intravenous injection of nondegradable polystyrene (diamonds) or biodegradable dendrimer (squares) NIR fluorescent nanoparticles. The FLT remained stable for polystyrene nanoparticles while the biodegradable nanoparticle FLT increased in a linear fashion with time after injection for the first 48 hours before reaching a plateau about 5 days after injection for final FLT increase of about 80%. 33 Another source of alteration is FRET as described for fluorescent proteins above. In the case of FLT imaging, the acceptor molecule need not produce fluorescence. Systems based on multiple copies of the same fluorophore or on fluorophore-quencher systems can result in reduction of the detect FLT. Upon dequenching by separation of the donor and acceptor molecules, the fluorescence intensity and FLT should increase. Hybrid molecular imaging agents produce enhanced signal after activation by molecular events such as cellular internalization 36 or proteolytic cleavage 37, 38. Energy transfer between two molecules results in alteration of fluorescence intensity and in many cases, FLT 8. The reduction in FRET can be detected by this enhanced fluorescence. Application of FLT imaging to quenched fluorescent molecular probes is likely to significantly enhance the utility of both in the near future by direct visualization of dequenching via the related change in FLT rather than arbitrary increase in fluorescence intensity. In this case, complete quenching will not be required for high signal-to-background. Rather, incomplete quenching will allow detection of the probe by fluorescence intensity for biodistribution within the body while activation will be detected primarily by FLT imaging. In summary, we have demonstrated novel contrast agents and preliminary applications of FLT contrast for complementing optical molecular imaging techniques. Many new applications of FLT imaging await development and will significantly enhance the capabilities of optical molecular imaging. [1] Coons, A. H., The Beginnings of Immunofluorescence, J Immunol, 87(5), (1961). [2] Wu, A. M., and Olafsen, T., Antibodies for molecular imaging of cancer, Cancer J, 14(3), (2008). [3] Achilefu, S., Lighting up tumors with receptor-specific optical molecular probes, Technol Cancer Res Treat, 3(4), (2004). Proc. of SPIE Vol

8 [4] Greish, K., Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines, J Drug Target, 15(7-8), (2007). [5] Chen, J. Q., Tung, C. H., Mahmood, U. et al., In vivo imaging of proteolytic activity in atherosclerosis, Circulation, 105(23), (2002). [6] Eckelman, W. C., and Mathis, C. A., Targeting proteins in vivo: in vitro guidelines, Nucl Med Biol, 33(2), (2006). [7] Frangioni, J. V., The problem is background, not signal, Mol Imaging, 8(6), (2009). [8] Suhling, K., French, P. M., and Phillips, D., Time-resolved fluorescence microscopy, Photochem Photobiol Sci, 4(1), (2005). [9] Tadrous, P. J., Siegel, J., French, P. M. et al., Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer, J Pathol, 199(3), (2003). [10] Schweitzer, D., Hammer, M., Schweitzer, F. et al., In vivo measurement of time-resolved autofluorescence at the human fundus, Journal of Biomedical Optics, 9(6), (2004). [11] Galletly, N. P., McGinty, J., Dunsby, C. et al., Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin, Br J Dermatol, 159(1), (2008). [12] Villalobos, V., Naik, S., and Piwnica-Worms, D., Current state of imaging protein-protein interactions in vivo with genetically encoded reporters, Annu Rev Biomed Eng, 9, (2007). [13] Jares-Erijman, E. A., and Jovin, T. M., FRET imaging, Nat Biotechnol, 21(11), (2003). [14] Levitt, J. A., Matthews, D. R., Ameer-Beg, S. M. et al., Fluorescence lifetime and polarization-resolved imaging in cell biology, Curr Opin Biotechnol, 20(1), (2009). [15] Soloviev, V. Y., Tahir, K. B., McGinty, J. et al., Fluorescence lifetime imaging by using time-gated data acquisition, Appl Opt, 46(30), (2007). [16] Reynolds, J. S., Troy, T. L., Mayer, R. H. et al., Imaging of spontaneous canine mammary tumors using fluorescent contrast agents, Photochem Photobiol, 70(1), (1999). [17] Nothdurft, R. E., Patwardhan, S. V., Akers, W. J. et al., "Fluorescence Lifetime Tomography for Whole Body Small Animal Imaging." [18] Kumar, A. T., Raymond, S. B., Dunn, A. K. et al., A time domain fluorescence tomography system for small animal imaging, IEEE Trans Med Imaging, 27(8), (2008). [19] Godavarty, A., Sevick-Muraca, E. M., and Eppstein, M. J., Three-dimensional fluorescence lifetime tomography, Med Phys, 32(4), (2005). [20] McGinty, J., Soloviev, V. Y., Tahir, K. B. et al., Three-dimensional imaging of Forster resonance energy transfer in heterogeneous turbid media by tomographic fluorescent lifetime imaging, Opt Lett, 34(18), (2009). [21] Cubeddu, R., Canti, G., Pifferi, A. et al., Fluorescence lifetime imaging of experimental tumors in hematoporphyrin derivative-sensitized mice, Photochem Photobiol, 66(2), (1997). [22] Andersson-Engels, S., Canti, G., Cubeddu, R. et al., Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin, Lasers Surg Med, 26(1), (2000). [23] Berezin, M. Y., Lee, H., Akers, W. et al., Near infrared dyes as lifetime solvatochromic probes for micropolarity measurements of biological systems, Biophys J, 93(8), (2007). [24] Berezin, M. Y., Akers, W. J., Guo, K. et al., Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging, Biophysical Journal, 97(9), L22-L24 (2009). [25] Almutairi, A., Akers, W. J., Berezin, M. Y. et al., Monitoring the Biodegradation of Dendritic Near-Infrared Nanoprobes by in Vivo Fluorescence Imaging, Mol Pharm, (2008). [26] Akers, W. J., Berezin, M. Y., Lee, H. et al., Predicting in vivo fluorescence lifetime behavior of near-infrared fluorescent contrast agents using in vitro measurements, J Biomed Opt, 13(5), (2008). [27] de la Zerda, A., Bodapati, S., Teed, R. et al., A Comparison Between Time Domain and Spectral Imaging Systems for Imaging Quantum Dots in Small Living Animals, Mol Imaging Biol, (2009). [28] Keren, S., Gheysens, O., Levin, C. S. et al., A comparison between a time domain and continuous wave small animal optical imaging system, IEEE Trans Med Imaging, 27(1), (2008). [29] Akers, W., Lesage, F., Holten, D. et al., In vivo resolution of multiexponential decays of multiple near-infrared molecular probes by fluorescence lifetime-gated whole-body time-resolved diffuse optical imaging, Mol Imaging, 6(4), (2007). Proc. of SPIE Vol

9 [30] Berezin, M. Y., Akers, W. J., Guo, K. et al., Long fluorescence lifetime molecular probes based on near infrared pyrrolopyrrole cyanine fluorophores for in vivo imaging, Biophys J, 97(9), L22-4 (2009). [31] Berezin, M. Y., Lee, H., Akers, W. et al., Engineering NIR dyes for fluorescent lifetime contrast, Conf Proc IEEE Eng Med Biol Soc, 1, (2009). [32] Berezin, M. Y., Lee, H., Akers, W. et al., Ratiometric analysis of fluorescence lifetime for probing binding sites in albumin with near-infrared fluorescent molecular probes, Photochem Photobiol, 83(6), (2007). [33] Almutairi, A., Akers, W. J., Berezin, M. Y. et al., Monitoring the biodegradation of dendritic near-infrared nanoprobes by in vivo fluorescence imaging, Mol Pharm, 5(6), (2008). [34] Goiffon, R. J., Akers, W. J., Berezin, M. Y. et al., Dynamic noninvasive monitoring of renal function in vivo by fluorescence lifetime imaging, J Biomed Opt, 14(2), (2009). [35] Akers, W. J., Berezin, M. Y., Lee, H. et al., "In vivo imaging with near-infrared fluorescence lifetime contrast." 7190, 71900T-8. [36] Ogawa, M., Kosaka, N., Choyke, P. L. et al., In vivo molecular imaging of cancer with a quenching nearinfrared fluorescent probe using conjugates of monoclonal antibodies and indocyanine green, Cancer Res, 69(4), (2009). [37] Mahmood, U., and Weissleder, R., Near-infrared optical imaging of proteases in cancer, Molecular Cancer Therapeutics, 2(5), (2003). [38] McIntyre, J. O., and Matrisian, L. M., Optical proteolytic beacons for in vivo detection of matrix metalloproteinase activity, Methods Mol Biol, 539, 1-20 (2009). Proc. of SPIE Vol