Hyperspectral Imaging For Multiplexing Short-wave Infrared Emitting Nanoprobes In Biomedical Applications

Clinical imaging techniques,such as magnetic resonance imaging(MRI)or computed tomography(CT),greatly advanced the healthcare by facilitating early diagnosis of the life-threatening diseases.Nevertheless,growing need for safe,compact,low-cost alternatives promotes development of novel biological imaging modalities.Optical imaging emerges in clinical applications due to its advantageous features,such as superior spatial and temporal resolution,absence of ionizing radiation involvement and low cost.However,main drawback of the light-based modalities is low penetration depth of the light into biological tissues.Recently,photoluminescence(PL)bioimaging in near-infrared(NIR)and short-wave infrared(SWIR)spectral regions was introduced as a promising way to address this problem.Due to the lower absorbance and scattering of light by biological tissues as well as negligible autofluorescence in the SWIR spectral region,an availability of the SWIR PL signal can enable imaging of deeper tissues with superior resolution.With emerging development of PL bioimaging,an ability to simultaneously track several PL agents in vivo can be utilized for multiple purposes(e.g.,targeted imaging of the specific cells or organs along with imaging-guided drug delivery).To address this challenge,multiplexed imaging methods were developed.Most commonly used multiplexed imaging methods differentiate PL probes by spectral position of their emission,using appropriate optical filters.However,proper multiplexing in such an approach requires utilization of nanoprobes with spectrally narrow,non-overlapping PL spectra.In this regard,hyperspectral imaging(HSI)combined with spectral mixture analysis algorithms is a promising approach for multiplexing PL signals.In this thesis,SWIR HSI system was developed to integrate deep-tissue SWIR PL imaging with nanoprobes multiplexing capabilities of HSI.SWIR HSI system was developed with staring(band-sequential)acquisition mode,meaning 2D spectral images are sequentially acquired by varying spectral transmittance of the dispersive element.In contrast to other HSI modes,such approach allows for acquisition of spectrally resolved images in the specified spectral range,reducing total hypercube acquisition time.SWIR camera with sensitivity in the 900–1700 nm spectral range was selected as a detector for the system.Thermoelectric cooling of the camera sensor greatly improves signal to noise ratio and allows for detection of weak PL signals.Use of a liquid crystal tunable filter(LCTF)as a dispersive element enables tunability of the transmission wavelengths in the whole range of SWIR camera sensitivity.In addition,illumination optics was developed to implement both PL excitation and bright field illumination of the specimen.Moreover,acquisition software was developed in order to enable automatic hypercube acquisition and integrate all of the hardware controls in single computer program.Developed LABVIEW-based software combines hardware initialization,real-time system adjustment and acquisition of spectral images.The obtained sequence of the spectrally resolved images(spectral hypercube)contains mixture of multiple components(endmembers)with the distinctive spectral signatures.In order to extract abundances of these components,spectral unmixing algorithm have to be applied to the acquired hypercube.Specifically,supervised linear spectral mixture analysis(LSMA)algorithms can be applied to PL HSI data,using the previously obtained PL spectra of the endmembers.Thus,non-negativity constrained LSMA(NC-LSMA)algorithm was applied for analysis of the acquired SWIR HSI hypercube.Spectral unmixing software,which implements NC-LSMA algorithm,was created using MATLAB development environment.The software combines data import,rapid processing,intuitive visualization and export.The synthetic data tests demonstrated ability of the developed unmixing software to perform complex linear mixture analysis.A development of the efficient and sensitive SWIR imaging detectors promoted advances in PL bioimaging,which exploits beneficial optical properties of the biological tissues in the SWIR spectral region(?1000-1700 nm).Numerous photoluminescent nanoprobes are developed for SWIR PL imaging of the biological tissues,due to the absence of endogenous SWIR emitting contrast agents.Herein,two types of NIR-SWIR contrast nanoagents are introduced:dye-loaded polymeric nanoparticles(PNPs)and rare-earth ions doped nanoparticles(RENPs).The pre-synthesized polystyrene(PS)-poly-N-isopropylacrylamide(PNIPAM)core-shell nanoparticles were post-loaded with series of 2-azaazulene polymethine dyes.Obtained nanoformulations exhibit absorbance/emission in NIR-SWIR range,which allows for their application in high-contrast deep-tissue bioimaging.Subsequently,the pre-synthesized core-shell polystyrene(PS)-poly-N-isopropylacrylamide-co-acrylic acid(NIPAM-co-AA)nanoparticles were co-loaded with 2-azaazulene polymethine dye and2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide-a(HPPH)photosensitizer.Designed nanoformulation allows to combine SWIR PL imaging and photodynamic therapy(PDT)modes to implement“see and treat”concept.Cell imaging and viability assay demonstrated effective combination of PNPs uptake by cells and PDT action.In vivo studies with subcutaneously tumored mice demonstrated a possibility to image biodistribution of nanoformulation in visible-NIR-SWIR region to facilitate imaging guided PDT.RENPs are another class of SWIR emitting nanoprobes with superior photostability and large Stokes shift that can be effectively used as SWIR contrast agents.However,the majority of the reported SWIR RENPS are excited at 980 nm,which causes a biological heating effect.To address this problem,a class of erbium(Er³⁺)-sensitized core–shell nanocrystals of NaErF₄:Yb³⁺@NaLuF₄,which emit efficient SWIR luminescence peaked at 1525 nm under 808 nm excitation,without producing any local heating,were developed.Due to the effective suppression of upconverting pathways,designed nanocrystals exhibit a superior quantum yield of 11%.These bright SWIR nanoprobes were demonstrated to enable high contrast in vitro imaging of HeLa cells and in vivo through-skull imaging of blood vasculature in the mouse brain.Furthermore,a temperature dependence of the RENPs physical properties provides a possibility of the non-invasive temperature probing via assessment of the ratio between intensities of the specific PL peaks(luminescent intensity ratio,LIR).The pre-synthesized NaYbF₄:1%Tm³⁺@Na LuF₄:30%Nd³⁺core–shell nanoparticles were applied for the noninvasive probing and monitoring of the temperature during photopolymerization of dental materials.When excited at 808 nm,the synthesized nanoparticles emit NIR PL with two distinctive peaks at 865 and 980 nm,which correspond to radiative transitions from the doped Nd³⁺and Yb³⁺ions,respectively.Luminescence intensity ratio between these two bands was found to vary with temperature and allowed for the ratiometric evaluation of the in situ temperature during photopolymerization of resin cement(doped with nanoparticles)in a veneer placement procedure.In addition,the NIR emission also enables high-contrast imaging of the distribution of the adhesive under the veneer due to the reduced attenuation of light by dental ceramics in NIR spectral region.Finally,SWIR HSI combined with unmixing algorithms was applied for multiplexing of the SWIR-emitting nanoformulations by their PL spectra.Two types of the RENPs,NaErF₄:Yb@NaLuF₄ and NaErF₄:Y@NaLuF₄,which exhibit similar PL under single laser excitation,were applied for SWIR HSI imaging.Consequent unmixing of the obtained hypercube demonstrated effective multiplexing of two nanoprobes,as well as estimation of the abundance values in their mixture.By removing the noise component from the hypercube,contrast of the spectral images was shown to be greatly improved.Moreover,feasibility of the nanoprobes multiplexing through SWIR HSI was demonstrated at tissue depth of?5 mm.Subsequently,two types of nanoprobes(PNPs and RENPs)with SWIR emission in the same spectral range(900–1200 nm)were subcutaneously injected in the living animal(nude mouse)and the multiplexing of the nanoprobes by difference in their emission spectral shape was performed with use of SWIR HSI.A consequent reconstruction using developed unmixing software allowed to map the nanoprobes distribution in vivo.In conclusion,this thesis for the first time reports application of HSI for multiplexing SWIR photoluminescent nanoprobes via integration of the multiple innovative approaches.First,novel SWIR PL HSI system has been designed,which is capable of deep tissue PL acquisition.Second,the developed unmixing software enables high precision multiplexing of multiple nanoprobes from the HSI data.Moreover,four types of novel SWIR emitting nanoprobes have been designed for application in SWIR PL imaging and nanothermometry.Combinatorial application of these approaches enables in vivo multiplexing of NIR-SWIR PL nanoprobes with overlapping PL spectra,which is not possible in conventional PL imaging.