Field of the invention

The invention relates to near-infrared (NIR) chromophores, in particular tricarbocyanine N-triazoles compounds. The invention further relates to processes for the preparation of the compounds, and uses of the compounds in therapeutic, diagnostic, surgery and analytical applications.

Background of the invention

In vivo optical imaging has revolutionised the ability to visualise biological processes with high resolution in intact organisms. Most in vivo imaging fluorophores rely on near- infrared (NIR) chemical scaffolds as they allow deep penetration with minimal photodamage and low tissue autofluorescence. Many NIR fluorophores have been described for bioimaging applications, ranging from analyte detection to image-guided surgery.

Zwitterionic heptamethine cyanine analogues have for example been described as NIR agents with enhanced capabilities for cell, tissue and in vivo imaging. Among these structures, the sulfonated heptamethine cyanine dye Indocyanine Green (ICG) is the only clinically-approved NIR dye for studies in humans. The structurally-related IRDye®800CW fluorophores have recently entered clinical trials as biomarker- labelling molecules for fluorescence-assisted surgery. In both ICG and IRDye®800CW, the potential aggregation of the heptamethine cyanine scaffold is minimized by the incorporation of negatively-charged groups (i.e. sulfonates); however, these preclude cell uptake and impede long-term tracking of small populations of cells in vivo.

As an alternative, tricarbocyanine N- amines have been reported as cell-permeable NIR fluorophores, and they can be prepared via nucleophilic substitution of the IR780 tricarbocyanine core with amines (Figure 1 ). Their straightforward chemistry has enabled their adaptation to diversity-oriented studies, but most tricarbocyanines N- amines show low quantum yields and rapid photodegradation, which compromise their application for long-term NIR fluorescence imaging.

Cellular immunotherapies represent promising strategies for treating disorders driven by malfunctioning immune responses, including cancer, chronic infections and autoimmune diseases. Among these, T cell immunotherapies have shown great potential, in both experimental models and human patients. For instance, in cancer immunotherapy, tumour-responsive T cells are isolated from the peripheral blood of patients, expanded ex vivo and then transferred back to elicit anti-tumour immune responses. One important obstacle in the clinical translation of T cell immunotherapies is the lack of chemical agents to track post-transferred therapeutic cells in vivo.

Statement of the invention

These shortcomings are addressed by a tricarbocyanine /V-triazoles family of bright, photostable and cell-permeable NIR fluorophores containing neutral triazole groups. To date, tricarbocyanine /V-triazoles had not been demonstrated because of the lack of synthetic approaches that were compatible with the relatively unstable intermediate tricarbocyanine azide

The fluorophores of the invention can be also used to label other immune cells as well as stem cells for regenerative cell-based therapies, fluorescence-guided diagnostic and surgery and ophthalmic imaging.

The compounds of the invention are also suitable for other optical imaging modalities beyond fluorescence. For example, they can be used as multimodal reagents as they can be readily detected under Surface-Enhaced Raman Scattering upon conjugation to AuNPs, for optoacoustic imaging as they absorb NIR light and also for optical coherence tomography. The present invention provides a tricarbocyanine /V-triazoles family of bright, photostable and cell-permeable NIR chromophores containing neutral triazole groups. In particular, a compound of formula (I), a derivative or a salt thereof is provided:

Wherein R1 is absent or selected from the group consisting C1-C6 alkyl, and C1-C9 alkyl substituted with one or more of the following groups: halogen, hydroxyl, aryl, carboxylic acids; C3-C6 cycloalkyl, aryl groups substituted with one or more of the following groups: alkyl, cycloalkyl, naphthalenyl, phenanthrenyl, amino, aminoalkyl, aminocycloalkyl, formyl, trifluoromethyl, carboxyl, halogen, hydroxyl, hydroxyalkyl, alkyloxy, hydroxyaryl, nitro; thiophenyl, pyridinyl, imidazolyl, acetal and benzyl;

R2 and R3 are each independently selected from the group consisting of C1-C6 alkyl, and C1-C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, sulphonate groups, aryl, succinimidyl esters, maleimides, polyethyleneglycol-based substituents; R4, R5, R8 and R9 are each independently absent, C1 -C6 alkyl or H; R6 and R10 are each independently H; R7 and R11 are each independently selected from the group consisting of H, Me and sulphonate; or R6-R7 and/or R10-R11 form an aromatic ring and/or R5-R6 and/or R9-R10 form an aromatic ring as defined below;

R12 is selected from the group consisting of H, C1 -C6 alkyl, and C1 -C9 alkyl substituted with one or more of the following groups: hydroxyl, aryl, carboxylic esters, carboxylic acids; amine, aminoalkyl, aminocycloalkyl, aminoaryl, tosyl, hydroxyalkyl, hydroxyaryl, thioalkyl, thioaryl;

X and Y are each independently selected from the group consisting of C, S, O and N; and Z is either C or N;

According to one embodiment of the invention, Z in formula (I) is C.

According to one embodiment of the invention, Z in formula (I) is C and R2 and R3 are each independently selected from the group consisting of C1 -C6 alkyl, and C1 -C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, aryl, succinimidyl esters, maleimides, polyethyleneglycol-based substituents.

According to one embodiment of the invention, Z in formula (I) is C and R2 and R3 are each independently selected from the group consisting of C1 -C6 alkyl, and C1 -C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, succinimidyl esters, maleimides, and polyethyleneglycol-based substituents.

According to one embodiment of the invention, Z in formula (I) is C, X and/or Y is N and R2 and R3 are each independently selected from the group consisting of C1 -C6 alkyl, and C1 -C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, succinimidyl esters, maleimides, polyethyleneglycol-based substituents.

If X is N, preferably R4 or R5 is Me; if Y is N, preferably R8 or R9 is Me; if both X and Y are N preferably R4 or R5 and R8 or R9 are Me.

According to one embodiment, in formula (I), X, Y and Z are C and R2 and R3 are each independently selected from the group consisting of C1 -C6 alkyl, and C1 -C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, succinimidyl esters, maleimides, polyethyleneglycol-based substituents.

According to one embodiment of the invention R1 is selected from the group consisting of m(chloro)-Aryl, p(N02)-Aryl, p(OMe)-Aryl, o(OMe)-Aryl, cyclopentyl, C1-C6 alkyl, pyridinil, phenethyl, o(formyl)-Aryl, di(CF3)-Aryl, hydroxypropyl, propanoic acid, o(OCHF2)-Aryl, 1-hydroxyethyl, p(OAr)-Aryl, pentanoic acid, 1-hydroxynonyl, cyclopropyl, aniline, and p(NMe2)-Aryl. According to one embodiment of the invention, X and Y are carbon atoms and R4, R5, R8 and R9 are all Me.

According to a further embodiment of the invention, the compound is selected from the group consisting of:

a derivative and a salt thereof. Preferably the compound is selected from the group consisting of CIR3, CIR4, CIR8, CIR11 , CIR12, CIR13, CIR15, CIR21 , CIR22, CIR25, CIR26, CIR27, CIR29, CIR31 , CIR32, CIR33, CIR34, CIR35, CIR36, CIR38, CIR43, a derivative and a salt thereof.

According to a further embodiment of the invention, the compound is selected from the group consisting of CIR38, CIR38M, CIR38SE, CIR38COOH, and a salt thereof:

According to another embodiment of the invention, the compound is selected from the group consisting of CIR35, CIR35M, CIR35SE and CIR35COOH, and a salt thereof:

According to a further embodiment of the invention, the compound is selected from the group consisting of:

a derivative and a salt thereof, wherein A is selected from the group consisting of C- (CH3)2, N-CH3, O and S, B is either C-(CH3)2 or S, and Z is either C or N. R1 is then preferably selected from the group consisting of m(chloro)-Aryl, p(N02)- Aryl, p(OMe)-Aryl, o(OMe)-Aryl, cyclopentyl, C1 -C6 alkyl, pyridinil, phenethyl, o(formyl)-Aryl, di(CF3)-Aryl, hydroxypropyl, propanoic acid, o(OCHF2)-Aryl, 1 - hydroxyethyl, p(OAr)-Aryl, pentanoic acid, 1 -hydroxynonyl, cyclopropyl, aniline, and p(NMe2)-Aryl.

Preferably, R2 and R3 are each independently selected from the group consisting of C1 -C6 alkyl, and C1 -C6 alkyl substituted with one or more of the following groups: carboxylic acids, substituted amides, succinimidyl esters, maleimides, polyethyleneglycol-based substituents;

According to one embodiment of the invention A is C-(CH3)2.

According to one embodiment of the invention Z is C and R12 is H.

According to another embodiment of the invention Z is N and R12 is tosyl or thiophenyl.

The present invention further provides a process for the preparation of the compound, a derivative or a salt thereof as described above. The method allows isolating the compounds in reasonable yield and high purity. Said process comprises the steps of: a) Performing a nucleophilic substitution with sodium azide on a heptamethine scaffold in a solvent,

b) Extracting the solvent, and

c) Performing a cycloaddition reaction.

When Z in formula (I) is C then in step c) the cycloaddition is performed with a substituted alkyne and is catalysed.

Generally, the compounds of the invention may be prepared by adding sodium azide to a solution of heptamethine in organic solvent (e.g. DMF). The resulting mixture is stirred to form the heptamethine azide. The reaction is then diluted and extracted with organic solvent (e.g. DCM). The crude is then mixed with CuSO ₄ , TBTA, sodium ascorbate and the alkyne (for triazoles) or only with nitrile (for tetrazoles). The resulting mixtures are stirred until reaction completion, extracted with organic solvent and purified by chromatography. Further the present invention provides the compound, a derivative or a salt thereof described above for use in a therapeutic, diagnostic, surgery or analytical method.

The present invention in addition provides diagnostic methods comprising labelling, tracking, and imaging cells in vivo, in vitro or ex vivo.

The present invention also provides a dye-labelled agent, a dye-labelled cell or a dye- labelled molecule comprising of a compound of the invention, a derivative or a salt thereof.

A further embodiment of the invention relates to a diagnostic method comprising administering (e.g. local or systemic administration) to an organism, preferably a mammal dye-labelled agents or dye-labelled cells associated to disease biomarkers (e.g. immune cells, dead cells, cancer cells) comprising a compound of formula (I), and identifying them with an optical readout by an appropriate technique (e.g. optical coherence tomography, fluorescence spectroscopy, fluorimetry, fluorescence microscopy, flow cytometry, fluorescence-assisted cell sorting, fluorescence-guided surgery, fluorescence endomicroscopy, multi-spectral optoacoustic imaging, Raman spectroscopy, Raman imaging or angiography) in a relevant biological sample (e.g. blood, biopsy, tissue, lavage) in order to diagnose a diseased state or stratify patients according to different disease states.

The present invention also provides surgery methods comprising the steps of administration to a mammal a dye-labelled agents or dye-labelled cells (e.g. local or systemic administration), identification of an optical readout by an appropriate imaging modality (e.g. optical coherence tomography, fluorescence spectroscopy, fluorimetry, fluorescence microscopy, flow cytometry, fluorescence-assisted cell sorting, fluorescence-guided surgery, fluorescence endomicroscopy, multi-spectral optoacoustic imaging, Raman spectroscopy, Raman imaging or angiography) and subsequent choice of an appropriate surgical treatment, including fluorescence- guided surgery and ophthalmic surgical procedures.

The present invention further provides therapeutic methods comprising administration to a mammal of a compound of the invention, a derivative or a salt thereof linked to a molecule with therapeutic action (e.g. small molecule, peptide, protein, antibody, nanoparticle) to identify optimal administration routes and dosage by means of optical coherence tomography, fluorescence spectroscopy, fluorimetry, fluorescence microscopy, flow cytometry, fluorescence-assisted cell sorting, fluorescence-guided surgery, fluorescence endomicroscopy, multi-spectral optoacoustic imaging, Raman spectroscopy, Raman imaging or angiography.

The present invention also provides analytical method to characterize dye-labelled molecules by optical coherence tomography, fluorescence spectroscopy, fluorimetry, fluorescence microscopy, flow cytometry, fluorescence-assisted cell sorting, fluorescence-guided surgery, fluorescence endomicroscopy, multi-spectral optoacoustic imaging, Raman spectroscopy or Raman imaging.

According to one embodiment of the invention, the method as defined above is selected from the group consisting of optical coherence tomography, fluorescence spectroscopy, fluorimetry, fluorescence microscopy, fluorescence tomography, whole- body fluorescence imaging, flow cytometry, fluorescence-assisted cell sorting, fluorescence-guided surgery, fluorescence endomicroscopy, multi-spectral optoacoustic imaging, Raman spectroscopy, Raman imaging, fundus camera imaging and angiography.

The method may be a therapeutic method for the diagnosis and/or treatment of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, cancer, macular degeneration, infection, diabetic retinopathy, uveitis, neurodegenerative diseases, dementia, Alzheimer’s disease, multiple sclerosis.

Anyone of the methods described herewith may comprise labelling, tracking, or imaging cells, preferably immune cells, tissues and organs in vivo, in vitro or ex vivo.

According to an embodiment of the invention CIR4, CIR13, CIR21 , CIR26, CIR27, CIR31 , CIR38, a derivative or a salt thereof are for use in fluorescence imaging of cells of post-transferred T cells in a model of immune cell activation.

According to an embodiment of the invention CIR8, CIR12, CIR27, CIR29, CIR32, CIR35, CIR36, a derivative or a salt thereof are for use in optical coherence tomography (OCT) for non-invasive imaging of cells, tissues or blood vessels. According to an embodiment of the invention CIR35, a derivative or a salt thereof is for the determination of retinal barrier integrity using fluorescence or OCT imaging.

According to an embodiment of the invention CIR8, CIR22, CIR25, CIR32, CIR33, CIR34, CIR38, CIR43, a derivative or a salt thereof are for use in Raman spectroscopy or Raman imaging as reporters of metal-based materials (e.g. nanoparticles, biosensors, metal surfaces, chips) conjugated to biomolecules (e.g. antibodies, proteins, peptides, small molecules).

According to an embodiment of the invention CIR3, CIR1 1 , CIR15 a derivative or a salt thereof are for use in multi-spectral optoacoustic imaging (MSOT) on their own or as reporters of biomolecules (e.g. antibodies, proteins, peptides, small molecules).

The present invention further provides a method for imaging of cells in vivo said method comprises the steps of administration of dye-labelled agents or dye-labelled cells (e.g. local or systemic administration) and identification of an optical readout by an appropriate imaging modality (e.g. optical coherence tomography, fluorescence spectroscopy, fluorescence-guided surgery, fluorescence endomicroscopy, multi- spectral optoacoustic imaging, Raman spectroscopy, Raman imaging or angiography).

The present invention further provides a pharmaceutical formulation comprising the compound of formula (I), a derivative or a salt thereof and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier may be for example physiological sterile saline solution, sterile water solution, pyrogen-free water solution, isotonic saline solution, and phosphate buffer solution.

The present invention further provides the pharmaceutical formulation comprising the compound of formula (I), a derivative or a salt thereof and a pharmaceutically acceptable carrier for use in the therapeutic, diagnostic, surgery or analytical method described above. The present invention further provides a kit said kit comprising the compound of the invention, a derivative or a salt thereof as described above as a labelling reagent for biomolecules and/or cells, aqueous buffers and/or solvent for reconstitution, packaging materials and instructions for use thereof.

Definitions

As used herein, the term "derivative" is used to refer to the residue of a chemical compound, such as an amino acid, after it has undergone chemical modification. For instance, these could include derivatives incorporating linkers with reactive groups for bioconjugation (e.g. amines, carboxylic acids, succinimidyl esters, maleimides, azides, alkynes, tetrazines), as well as derivatives of antibodies, proteins, peptides and small molecules.

As used herein, the term "salt" is used to refer to an assembly of cations and anions. These could include sodium, ammonium, quaternary ammonium, calcium, magnesium and potassium as cations or iodine, chloride, bromide, formate, perchlorate, hydrochlorate, sulfate, hydroxide, phosphate and trifluoroacetate as anions. The salt may only include the compound or the derivative of the invention and an anion. The salt may also include additional cations and anions. Preferred cations are of sodium and ammonium. Preferred anions are of iodine, bromide, formate and trifluoroacetate.

As used herein, the term "conjugated" is a system of alternating single and multiple bonds. This can be viewed as an overlapping system of p-orbitals across an intervening o-bonds allowing a derealization of tt-electrons across all the adjacent aligned p-orbitals.

As used herein and in the claims, the term "aromatic ring" is intended to represent ring structures comprising a delocalised conjugated system of electrons forming an aromatic core, which may typically be an arrangement of alternating single and double bonds. An aromatic group may be an aryl group, particularly an aryl group having only carbon atoms in the aromatic core (i.e. no heteroatoms such as O, S and N), such as a C6 aryl group. Alternatively, an aromatic group may be a heteroaryl group, such as a heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C. The aromatic group, including the heteroaryl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms bonded to the aromatic core may be substituted with a substituent independently selected from the group comprising methyl, methoxy, sulfonate, carboxylate, halogen or dialkylamine.

As used herein, the term "alkyl" represents a linear, branched or cyclic alkyl group. The alkyl group may be unsubstituted or substituted.

As used herein, the term "alkenyl" represents a linear, branched or cyclic alkenyl group, The alkenyl group may be unsubstituted or substituted.

As used herein, the term "alkynyl" represents a linear, branched or cyclic alkynyl group. The alkynyl group may be unsubstituted or substituted.

As used herein, the term“polyethyleneglycol” represents a linear, branched or cyclic group containing one or more units of ethylene oxide. The polyethyleneglycol group may be unsubstituted or substituted.

Brief descriptions of the Figures

Figure 1 shows the chemical structures and properties of isosteric tricarbocyanine N- amines (1 ) and /V-triazoles (2);

Figure 2 shows the chemical synthesis of CIR fluorophores, chemical structures of selected CIR fluorophores, absorbance and emission spectra of CIR38 as a representative CIR fluorophore (b), fluorescence NIR intensity of selected CIR fluorophores (c) and time-course analysis of the mean aggregate size of IR780 and CIR38 (d);

Figure 3 shows brightfield, fluorescence and merged microscope images of CD4+ T cells after labeling with CIR38M and CellTracker Green (a); and brightfield, fluorescence and merged microscope images of CD4+ T cells after labeling with IR800CW-SE and CellTracker Green (b);

Figure 4 shows the determination of the limit of detection in suspensions of CIR38M- labeled and DiR-labeled (both at 10 mM) CD4+ T cells by NIR fluorescence imaging.

Figure 5 shows a site-specific accumulation of CIR38M-labeled cells in a model of T cell activation (a) and a flow cytometric ex vivo analysis of inguinal lymph nodes (iLNs) from both left and right hind limbs of mice that had been injected with CIR38M-labeled CD4+ T cells (b).

Figure 6 shows a long-term longitudinal tracking of CIR38M-labeled CD4+ T cells in vivo (day 2, 4 and 7) and Proportion of donor cells retaining NIR fluorescence emission in the inguinal lymph nodes of (il_N) draining the immunization site and the spleen on days 4 and 7.

Figure 7 shows an ex vivo staining of human T cells with CIR38M, a flow cytometric analysis of human CD4+ T cells isolated from peripheral blood after labeling with CIR38M(a); a comparative analysis of human CD4+ T cells labeled with CIR38M or ICG under the same experimental conditions (b); Equal numbers of human CD4 ⁺ T cells were incubated either with CIR38M or PBS and incubated and viable cells were later counted (c) . Human CD4 ⁺ T cells were incubated with CIR38M or PBS and subsequently co-cultured with antigen-presenting cells, concentrations in the supernatant were determined;

Figure 8 shows cell viability of murine macrophages as a readout of CIR dyes toxicity in mammalian cells;

Figure 9 shows spectral centroid shifts derived from OCT measurements for selected CIR dyes (CIR-8, 12, 27, 29, 32, 35, 36) in comparison to ICG;

Figure 10 shows fluorescence intensity of Indocyanine Green (left) and CIR35 (right) in a transwell assay to examine retinal barrier integrity, where Ca2+-depletion (by treatment with EGTA) is used to disrupt retinal cell monolayers. Figure 1 1 shows a bright field image of human A549 cell (a), a pseudo-coloured Raman image of the distribution of the Raman signals after incubation with the cell (b) and a 3D-reconstruction of the Raman image in A549 human cells (c).

Figure 12 shows a flow cytometric analysis of multiple subpopulations of cells and NIR fluorescence emission after labelling with 10 mM CIR38M for 2 min in comparison to unlabelled cells (grey shaded area).

Figure 13 shows Raman intensity of 46 CIR dyes and long-term stability of gold nanoparticles in deionized water after derivatization with CIR22 and a polyethyleneglycol spacer.

Examples

Measurements methods

Thin-layer chromatography was conducted on Merck silica gel 60 F254 sheets and visualised by UV (254 and 365 nm). Silica gel (particle size 35-70 pm) was used for column chromatography.

¹ H and ¹³ C spectra were recorded in a Bruker Avance 500 spectrometer (at 500 and 125 MHz, respectively). Data for ¹ H NMR spectra reported as chemical shift d (ppm), multiplicity, coupling constant (Hz) and integration. Data for ¹³ C NMR spectra reported as chemical shifts relative to the solvent peak. HPLC-MS analysis was performed on a Waters Alliance 2695 separation module connected to a Waters PDA2996 photodiode array detector and a ZQ Micromass mass spectrometer (ESI-MS) with a Phenomenex® column (Cis, 5 pm, 4.6 x 150 mm).

In vitro spectral measurements. Spectroscopic and quantum yield data were recorded on a Synergy HT spectrophotometer (Biotek). Compounds were dissolved at the indicated concentrations and spectra were recorded at r.t. Spectra are represented as means from at least two independent experiments with n=3. Relative quantum yields were calculated by measuring the integrated emission area of the fluorescence spectra and comparing it to the area measured for the standard (e.g. for CIR fluorophores, ICG was used as the reference) (Mendive-Tapia, L; Zhao, C.; Akram, A. R.; Preciado, S.; Albericio, F.; Lee, M.; Serrels, A.; Kielland, N.; Read, N. D.; Lavilla, R.; Vendrell, M. Nat. Commun. 2016, 7, 10940-10948).

Calculations. All quantum chemical calculations were performed with Gaussian 09 (Revision D.01 ) (Frisch, M. J.; Trucks, G. W.; Schelegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Menuucci, B.; Petersson, G. A. et al. Gaussian 09; Gaussian, Inc.: Wallingford CT, 2009). Ground state geometries of compounds 1 and 2 were optimised using the density function theory (DFT) with B3LYP (Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652; and Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785-789), M06-2X, (Zhao, Y.; Truhlar, D. G. Theor. Chem. Acc. 2008, 120, 215-241 ) RBE0 (Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1996, 77, 3865) and wB97xd (Chai, J. D.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2008, 10, 6615-6620) functionals together with the Pople’s basis set 6-31 G ^* . Geometry optimisations were performed both in the gas phase and in EtOH, where the solvent was described by the Polarizable Continuum Model (PCM) (Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669-681 ). The nature of the excitation energies, oscillator strengths and contributions of the different orbitals involved in the electronic transitions were calculated using time-dependent density functional theory (TD-DFT) at the selected levels of theory, both in gas phase and in EtOH.

Particle size analysis. The mean size of aggregates in aqueous media were determined by dynamic light scattering using a PS90 Particle Size Analyser (Brookehaven Instrument Corporation). IR780 and CIR38 were dissolved in PBS (100 mM) and kept at r.t. to measure the size of aggregates every 2 h for a total of 8 h. Data is represented as means ± s.e.m from two independent experiments with n=3.

In vitro labelling and characterisation of CD4+ T cells. Single cell suspensions were made from the spleen after which red blood cells were lysed using NH ₄ CI buffer. CD4+ T cells were purified by magnetic cell sorting as per manufacturer’s instructions (Miltenyi Biotech). CD4+ T cells were re-suspended in PBS and then labelled with the probes for 2 min at the stated concentrations at 37 °C. Cells were washed twice with cell culture medium, after which they were re-suspended in FACS buffer (PBS, 2% FCS, 0.01 % sodium azide) or appropriate cell culture medium. For flow cytometric analysis, cells were then incubated with antibodies for 20 min at 4 °C. The antibodies used were anti-CD4-e450 or anti-CD4-PE, anti-CD45.1 -FITC or anti-CD45.1 -PE, and anti-CD1 1 b-APC (all from eBioscience). Samples were also stained with a fixable viability dye (conjugated with eFluor455, eBioscience) prior to surface staining. Flow cytometry data were collected using an LSR Fortessa (BD Biosciences) and analysed using FlowJo software.

In vitro cell viability and proliferation assays. To study the primary activation of CIR38M-labelled CD4+ T cells, 2 x 10 ⁵ CD4+ Tg4.CD45.1 + T cells were added per well to 2 mg ml. ^-1 anti-CD3 and 2 mg ml. ^-1 anti-CD28 coated, flat-bottomed 96-well plates. After 48 h, cell proliferation was assessed by the addition of [ ³ H]-thymidine at 0.5 pCi per well for the last 18 h of culture. [ ³ H]-thymidine incorporation was measured using a scintillation b-counter (Wallac) as mean counts per minute (c.p.m). The production of cytokines was assessed in culture supernatants by ELISA using Ready- SET-Go ELISA kits according to manufacturer’s instructions after 72 h of culture. In some experiments, CD45.1 + and CD45.2+ CD4+ T cells were co-cultured together to assess the extent of probe transfer between cells. In addition, in some instances, cells were double-labelled with 5 pM CFSE and 10 pM CIR38M for 2 min at 37 °C.

Fluorescence microscopy of CD4+ T cells. Single cell suspensions were made from spleens of C57BL/6 mice and red blood cells lysed using NH ₄ CI buffer. CD4+ T cells were purified by magnetic cell sorting as per manufacturer’s instructions, re- suspended in PBS and then labelled with CellT racker Green (10 pM), MitoT racker Red CMXRos (500 nM) or LysoTracker Red DND-99 (100 nM) at 37 °C, prior to addition of CIR38M or IR800CW-SE (10 pM) and incubation at 37 °C. Cells were washed twice with cell culture medium, twice in PBS, before re-suspension of the cell pellet in 1 % PFA. T cells were mounted and imaged under a fluorescence microscope (Nikon Ti Eclipse) using a 60X oil immersion objective (NA 1 .4) and a Hamamatsu Orca Flash 4.0 V2 camera for NIR detection. Excitation was achieved either by a Ti-sapphire laser (Coherent Mira 900, 740 nm CW) or a 100 W mercury lamp and a NIR epifluorescence filter cube (EX 747/33, DM 776LP, EM 776LP).

Whole-body fluorescence in vivo imaging and ex vivo analysis. C57BL/6 (CD45.1 - CD45.2+) and OT-II (CD45.1 + CD45.2-) mice were bred under specific pathogen-free conditions at the University of Edinburgh. Hair from C57BL/6 mice was removed using hair clippers followed by application of a coat of Nair and subsequent wiping with gauze sponges and water. Hairless albino C57BL/6 (B6.Cg-Tyrc-2J Hrhr/J) mice were purchased from The Jackson Laboratory. The housing facility was compliant with Federation of European Laboratory Animal Science Associations guidelines on screening mice for infectious diseases. All experiments had local ethical approval from the University of Edinburgh’s Animal Welfare and Ethical Review Body and were performed in accordance with UK legislation. OT-II transgenic mice express an l-Ab-restricted T cell receptor, which is reactive toward ovalbumin peptide 323- 339 (Robertson, J. M.; Jensen, P. E.; Evabold, B. D. J. Immunol. 2000, 164, 4706- 4712). The ovalbumin peptide (pOVA) was obtained from Cambridge Research Biochemicals. Tissue culture medium (RPMI 1640 medium) was supplemented with 2 mM L-glutamine, 100 U ml. ^-1 penicillin, 100 mg ml. ^-1 streptomycin, 5 ^c 10 ^-5 M 2-ME (all from Invitrogen Life Technologies) and 10% FCS (Labtech).

Albino hairless female C57BL/6 (CD45.2) mice were intravenously injected with 20 x 10 ⁶ CD45.1 + CD4+ OT-II T cells which had been labelled or not with 10 pM CIR38M for 2 min. On the same day, mice received a 50 pL subcutaneous injection of 10 mg of pOVA peptide emulsified in 2 mg mL ^-1 CFA, or PBS and CFA into each hind leg flank. Whole-body in vivo fluorescence images of

representative mice from both groups (i.e. injected with CIR38M-labelled cells or injected with unlabelled cells) were acquired on a Photonlmager™ (Biospace Lab) on days 2, 4 and 7. Mice were then culled to harvest the spleens and draining inguinal lymph nodes for ex vivo tissue imaging and analysis by flow cytometry as described above.

For CIR38M vs DiR comparative analysis, 10 ⁶ CD4+ Tg4 T cells were transferred into B10.PLxC57BL/6 mice followed by subcutaneous immunisation with 10 mg of MBP Ac1 -9(4Tyr) peptide emulsified in CFA containing 50 mg of heat-killed Mycobacterium tuberculosis H37Ra at a total final volume of 100 pL injected subcutaneously into the hind legs. Whole-body fluorescence images of representative mice from both groups (i.e. injected with CIR38M-labelled cells or DiR-labelled cells) were acquired on a Photonlmager™ (Biospace Lab) 48 h post-injection.

Ex vivo labelling of human CD4+ T cells. Ex vivo experiments with fresh human peripheral blood from healthy donors were approved by the Accredited Medical Regional Ethics Committee (AMREC, reference number 15-HV-013), as previously reported. ⁶⁴ Human CD4+ T cells were purified by magnetic cell sorting as per manufacturer’s instructions (Miltenyi Biotech). CD4+ T cells were re-suspended in PBS and then labelled with CIR38M or ICG (10 pM) at 37 °C. Cells were washed twice with cell culture medium, after which they were re-suspended in FACS buffer or appropriate culture medium. Proliferation studies were undertaken by adding 2 ^c 10 ⁵ CD4+ T cells per well to equal amounts of antigen-presenting cells and 2 mg ml. ^-1 soluble anti-CD3 in flat-bottomed 96-well plates. The production of cytokines was assessed in culture supernatants by ELISA using Ready-SET-Go ELISA kits according to manufacturer’s instructions (eBioscience) after 72 h of culture.

Compounds preparation

Commercially available reagents were used without further purification.

General synthesis of CIR fluorophores. To a solution of IR780 (300 mg, 0.44 mmol, 1 eq.) in DMF (2 mL), was added sodium azide (145 mg, 2.2 mmol, 5 eq.) in H ₂ O (2 mL). The resulting mixture was stirred at 70 °C for 20 min. The reaction was then cooled down, diluted with CH2CI2 (20 mL) and washed with H2O (x1 ). Combined organic layers containing the IR780-azide intermediate (confirmed by HPLC-MS; m/z: 546) were evaporated and used without further purification. IR780-azide was dissolved in 15 mL CH2CI2 and aliquoted to react with different alkynes (typically, in batches of 10 different alkynes). To each aliquot, we added CuSO ₄ (14 mg, 0.08 mmol, 2 eq.), tris[(1 -benzyl-1 H-1 , 2, 3-triazol-4-yl)methyl]amine (TBTA) (42 mg, 0.08 mmol, 2 eq.) and sodium ascorbate (16 mg, 0.08 mmol, 2 eq.) pre-dissolved in DMF:H ₂ O (1 :1 , 0.2 mL), and the alkynes (0.44 mmol, 10 eq.). The resulting mixtures were stirred at r. t. typically for 2 h (longer reaction times depending on the alkynes). The crude reaction mixtures were diluted in CH2CI2 (10-20 mL), and the organic phases were washed with H2O (3 x 10 mL). The organic extracts were dried over MgSO ₄ , filtered and evaporated under reduced pressure. The resulting crudes were then purified by semi-preparative HPLC to yield CIR fluorophores.

Compounds were purified using a Waters semi-preparative HPLC system using a Phenomenex® column (C18 Axial, 10 pm, 21 .2 x 150 mm) and UV detection. HRMS (ESI positive) were obtained in a LTQ-FT Ultra (Thermo Scientific) mass spectrometer.

The schematic preparation of CIR38 derivatives is shown below: The schematic preparation of IR780-maleimide (IR780M) is shown below:

IR780-COOH

IR780-COOH

To a solution of sodium acetate (258 mg, 3.15 mmol) in AcOH (20 ml.) was added 2- chloro-3-(hydroxymethylene)-1 -cyclohexene-1 -carboxaldehyde (86 mg, 0.5 mmol) and 3H-indolium,1 -(5-carboxypentyl)-2,3,3-trimethylbromide (214 mg, 0.6 mmol). The resulting solution was stirred at 130 °C under reflux for 2 h. To the reaction was then added 3H-indolium,2,3,3-trimethyl-1 -propyl iodide (148 mg, 0.5 mmol) and the mixture was stirred overnight at 130 °C. The reaction was then cooled to r.t. and solvents were evaporated under reduced pressure. The resulting crude was then purified by column chromatography (CH2CI2: MeOH, 98:2) to isolate IR780-COOH as a green solid (120 mg, 32% yield).

¹ H NMR (500 MHz, MeOD) d 8.45 (dd, J = 14.1 , 5.0 Hz, 2H), 7.57 - 7.51 (m, 2H), 7.44 (ddt, J = 8.4, 7.5, 1.1 Hz, 2H), 7.38 - 7.33 (m, 2H), 7.30 (tdd, J = 7.4, 3.0, 0.9 Hz, 2H), 6.31 (dd, J = 14.1 , 9.5 Hz, 2H), 4.19 (dt, J = 9.9, 7.5 Hz, 4H), 2.75 (t, J = 6.3 Hz, 3H), 2.32 (t, J = 7.3 Hz, 2H), 1 .99 (s, 3H), 1 .94 - 1.86 (m, 4H), 1.80 (d, J = 2.8 Hz, 1 H),

1 .74 (s, 10H), 1 .73 - 1.67 (m, 2H), 1.63 (s, 1 H), 1.56 - 1.48 (m, 2H), 1.07 (t, J = 7.4 Hz, 3H). ¹³ C NMR (126 MHz, MeOD) 6 176.3, 174.2, 173.1 , 172.8, 149.7, 144.2, 144.0, 142.3, 142.2, 141 .2, 141 .2, 132.8, 132.8, 129.8, 129.1 , 128.5, 128.5, 126.6, 125.2, 125.1 , 123.3, 122.1 , 1 15.2, 1 1 1.0, 1 10.9, 101.1 , 100.9, 53.4, 45.4, 43.7, 33.6, 27.0, 26.0, 24.4, 21 .5, 20.7, 20.5, 19.6, 10.3.

To a solution of IR780-COOH (57 mg, 0.08 mmol) in DMF (1 ml.) was added sodium azide (25 mg, 0.4 mmol) in H2O (1 ml_). The resulting mixture was stirred at r. t. for 20 min. To the reaction was then added 2-ethynylpyridine (79 mg, 0.8 mmol), CuSO ₄ (25 mg, 0.16 mmol), TBTA (85 mg, 0.16 mmol) and sodium ascorbate (32 mg, 0.16 mmol) and the mixture was stirred at r. t. for 2 h. The reaction crude was diluted in CH2CI2 (20 ml_), and the organic phase was washed with H2O (3 x 10 ml_). The organic extracts were dried over MgSO ₄ , filtered and evaporated under reduced pressure. The crude was then purified by normal-phase chromatography (CH2CI2 _: MeOH, 95:5) to yield CIR38-COOH as a green solid (14 mg, 20% yield).

¹ H NMR (500 MHz, MeOD) d 8.87 (s, 1 H), 8.70 (s, 1 H), 8.32 (d, J = 7.9 Hz, 1 H), 8.06 (t, J = 7.8 Hz, 1 H), 7.53 - 7.48 (m, 1 H), 7.45 - 7.36 (m, 4H), 7.34 (d, J = 2.5 Hz, 2H), 7.27 - 7.21 (m, 2H), 6.92 (dd, J = 14.1 , 3.0 Hz, 2H), 6.37 (dd, J = 14.1 , 6.2 Hz, 2H), 4.22 - 4.13 (m, 4H), 2.89 (s, 4H), 2.31 (t, J = 7.3 Hz, 3H), 2.22 - 2.1 1 (m, 2H), 1.91 -

1 .82 (m, 4H), 1.75 - 1.67 (m, 3H), 1 .35 (s, 6H), 1.28 (s, 6H), 1.05 (t, J = 7.4 Hz, 3H). 1 ³ C NMR (126 MHz, MeOD) d 173.1 , 172.9, 149.5, 148.9, 147.3, 142.1 , 142.0, 141.4, 141 .4, 141.1 , 137.7, 128.6, 128.5, 128.5, 126.5, 126.5, 125.4, 125.4, 122.1 , 1 1 1 .2, 1 1 1 .1 , 101 .6, 101 .5, 49.1 , 49.1 , 45.4, 43.8, 26.8, 26.6, 26.0, 24.6, 24.1 , 24.1 , 20.6, 20.4, 10.2.

HRMS: m/z [M ⁺ ] calcd. for C ₄₆ H ₅₃ 0 ₂ N ₆ : 721.4225; found: 721.4206.

CIR38-COOH (9 mg, 0.01 mmol), /V-hydroxysuccinimide (4 mg, 0.03 mmol), N,N'- dicyclohexylcarbodiimide (10 mg, 0.04 mmol) and DIPEA (4 mI_, 0.04 mmol) were dissolved in CHCI ₃ (1.5 ml.) with a catalytic amount of DMAP. The reaction was stirred at r.t. for 2 h. The reaction crude was purified by preparative TLC (CH2CI2 _: MeOH, 95:5) to yield CIR38SE as a green solid (7 mg, 81 % yield).

¹ H NMR (500 MHz, MeOD) d 8.86 (s, 1 H), 8.67 (d, J = 4.6 Hz, 1 H), 8.31 (d, J = 8.0 Hz, 1 H), 8.06 (td, J = 7.8, 1 .8 Hz, 1 H), 7.52 - 7.48 (m, 1 H), 7.43 - 7.37 (m, 4H), 7.33 (d, J = 10.6 Hz, 2H), 7.24 (t, J = 7.9 Hz, 2H), 6.92 (d, J = 14.2 Hz, 2H), 6.37 (dd, J =

14.1 , 4.1 Hz, 2H), 4.23 - 4.1 1 (m, 4H), 2.90 - 2.85 (m, 4H) 2.86 (s, 4H), 1.92 - 1.81 (m, 5H), 1 .64 - 1 .53 (m, 2H), 1.35 (s, 6H), 1 .27 (s, 6H), 1.05 (t, J = 7.4 Hz, 3H), 0.95 - 0.84 (m, 5H). ¹³ C NMR (126 MHz, MeOD) d 173.6, 173.0, 171 .3, 170.4, 149.6, 148.9, 148.0, 147.3, 145.5, 141 .9, 141 .1 , 137.8, 129.4, 128.5, 126.5, 125.4, 123.7, 122.1 , 120.4, 1 14.5, 1 1 1.1 , 1 1 1.1 , 107.4, 104.7, 101.6, 100.6, 96.1 , 53.6, 45.4, 45.3, 43.9,

43.8, 31.7, 30.5, 30.0, 29.3, 26.6, 25.5, 25.1 , 24.9, 24.1 , 23.9, 22.3, 20.6, 20.4, 13.0,

10.2.

HRMS: m/z [M ⁺ ] calcd. for C ₅ oH ₅₆ N ₇ O ₄ : 818.4388; found: 818.4453.

To a solution of 3H-indolium,1-(5-carboxypentyl)-2,3,3-trimethylbromide (50 mg, 0.14 mmol) and 1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylaminomorpholino- carbeniumhexafluorophosphate (COMU) (90 mg, 0.21 mmol) dissolved in ACN (0.4 ml_), DIPEA (37mI_, 0.21 mmol) was added. The mixture was stirred at r.t. for 10 min. Next, 2-maleimidoethylamine (36 mg, 0.21 mmol) and DIPEA (3 7mI_, 0.21 mmol) were added in ACN (0.2 ml.) and the mixture was stirred at r.t. for 2 h. Then, H2O (20 ml.) was added to the reaction mixture which was extracted with CH2CI2 (3 ^c 20 ml_). The organic extracts were dried over MgSO ₄ , filtered and evaporated under reduced pressure. The resulting crude was used in the next step without any purification. To a solution of AcONa (65 mg, 0.80 mmol) in AcOH (5 ml_), 2-chloro-3- (hydroxymethylene)-l -cyclohexene-1 -carboxaldehyde (21 mg, 0.14mmol) and 3 H- indolium,2,3,3-trimethyl-1-propyl iodide (37 mg, 0.14 mmol) were added. The resulting solution was stirred at 130°C under reflux for 2 h. Next, 1-(6-((2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1-yl)ethyl)amino)-6-oxohexyl)-2,3,3-trimethyl-3H-in dol-1-ium bromide (56 mg, 0.14 mmol) was added to the reaction mixture, which was stirred for 2 h at 130 °C. The reaction was then cooled to r.t. and solvents were evaporated under reduced pressure. The resulting crude was then purified by semi-preparative HPLC to isolate IR780-M as a green solid (2 mg). ¹ H NMR (500 MHz, MeOD) d 8.50 - 8.43 (m, 1 H), 7.55 (ddd, J = 7.6, 3.7, 1.1 Hz, 2H), 7.46 (td, J = 7.7, 1 .2 Hz, 2H), 7.41 - 7.34 (m, 2H), 7.32 (tdd, J = 7.5, 3.0, 0.9 Hz, 2H), 6.81 (s, 2H), 6.32 (d, J = 14.1 Hz, 1 H), 5.51 (s, 2H), 4.25 - 4.13 (m, 2H), 4.12 - 4.03 (m, 2H), 3.89 - 3.79 (m, 4H), 3.60 - 3.54 (m, 4H), 2.80 - 2.69 (m, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1 .76 (s, 6H), 1.76 (s, 6H), 1 .69 - 1.59 (m, 2H), 1 .38 - 1.31 (m, 5H), 1 .08 (t, J = 7.4 Hz, 3H), 0.98 - 0.88 (m, 2H).

HRMS: m/z [M ⁺ ] calcd. for C ₄₅ H ₅₄ CIN ₄ O ₃ : 733.3879; found: 733.3849.

CIR6

¹ H NMR (500 MHz, MeOD) d 8.81 (s, 1 H), 7.89 (d, J = 8.1 Hz, 2H), 7.45 - 7.34 (m, 5H), 7.36 - 7.29 (m, 2H), 7.28 - 7.20 (m, 2H), 6.94 (d, J = 14.2 Hz, 3H), 6.36 (d, J = 14.2 Hz, 2H), 4.16 (t, J = 7.3 Hz, 2H), 2.91 - 2.83 (m, 2H), 2.45 (s, 3H), 1 .87 (q, J = 7.4 Hz, 4H), 1 .35 (s, 6H), 1 .30 (s, 6H), 1 .25 - 1.12 (m, 4H), 1.04 (t, J = 7.4 Hz, 6H), 1 .01 - 0.93 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1 , 148.2, 147.7, 142.1 , 141.6,

141 .1 , 138.9, 129.5, 128.5, 126.6, 126.4, 125.5, 125.4, 124.5, 122.1 , 1 1 1 .1 , 101 .4, 49.1 , 45.4, 26.6, 24.1 , 20.6, 20.0, 10.2. CIR7

¹ H NMR (500 MHz, MeOD) d 9.01 (s, 1 H), 8.55 (s, 1 H), 8.12 (dd, J = 8.5, 1.7 Hz, 1 H), 8.08 - 8.03 (m, 1 H), 8.04 - 7.99 (m, 1 H), 7.96 (dd, J = 7.8, 1.9 Hz, 1 H), 7.63 - 7.55 (m, 2H), 7.44 - 7.35 (m, 4H), 7.32 (dt, J = 8.0, 0.8 Hz, 2H), 7.21 (td, J = 7.5, 1.0 Hz,

2H), 6.99 (d, J = 14.1 Hz, 2H), 6.38 (d, J = 14.1 Hz, 2H), 4.16 (t, J = 7.4 Hz, 2H), 2.89 (q, J = 6.7 Hz, 2H), 1.88 (q, J=7.4 Hz, 4H), 1.37 (s, 6H), 1.30 (s, 6H), 1.24- 1.15 (m, 4H), 1.05 (t, J= 7.4 Hz, 6H), 1.03-0.94 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1, 148.1, 147.7, 142.1, 141.6, 141.1, 133.7, 133.6, 128.8, 128.4, 127.9, 127.5, 126.8, 126.5, 126.4, 125.4, 125.1, 124.3, 123.2, 122.2, 111.1, 101.4, 49.1, 45.4, 26.6, 24.1,

20.6, 10.2.

CIR8

1H NMR (500 MHz, MeOD) 68.19 (s, 1H), 7.50 - 7.42 (m, 3H), 7.44 -7.38 (m, 4H), 7.40 - 7.31 (m, 4H), 7.32 - 7.25 (m, 2H), 6.80 (d, J = 14.1 Hz, 2H), 6.33 (d, J = 14.2 Hz, 2H), 4.15 (t, J= 7.4 Hz, 2H), 3.32-3.25 (m, 2H), 3.22-3.16 (m, 2H), 2.89-2.78 (m, 2H), 1.87 (q, J = 7.4 Hz, 4H), 1.34 (s, 6H), 1.22 (s, 6H), 1.05 (t, J = 7.4 Hz, 6H), 1.02-0.85 (m, 6H). ¹³ C NMR(126 MHz, MeOD) d 173.0, 148.0, 142.1, 141.6, 141.1, 140.8, 128.5, 128.3, 128.3, 128.0, 128.0, 126.5, 126.1, 125.4, 123.9, 122.1, 111.1, 101.3, 49.1, 45.3, 35.3, 26.6, 24.0, 20.5, 10.2.

CIR12

¹ H NMR (500 MHz, MeOD) d 10.57 (s, 1H), 8.90 (s, 1H), 8.66 (s, 1H), 8.21 -8.10 (m, 2H), 7.96 - 7.83 (m, 2H), 7.73 (ddd, J = 7.8, 1.4, 0.7 Hz, 1 H), 7.47 - 7.38 (m, 2H), 7.34 (dt, J = 7.9, 0.8 Hz, 2H), 7.27 (td, J = 7.5, 0.9 Hz, 2H), 6.94 (d, J = 14.2 Hz, 2H), 6.39 (d, J = 14.1 Hz, 2H), 4.26 - 4.09 (m, 2H), 2.98 - 2.83 (m, 2H), 1.89 (q, J = 7.4 Hz, 4H), 1.40 (s, 6H), 1.34 (s, 6H), 1.26 - 1.14 (m, 4H), 1.05 (t, J = 7.4 Hz, 6H), 1.03 - 0.88 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 191.8, 173.0, 142.1, 141.4, 141.1, 134.0, 130.1, 129.4, 129.2, 128.5, 127.9, 126.6, 125.5, 122.1, 111.2, 101.6, 49.2, 45.4, 26.7, 24.1, 20.6, 10.2.

CIR17

¹ H NMR (500 MHz, MeOD) d 8.90 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.46 - 7.37 (m, 5H), 7.36 - 7.30 (m, 2H), 7.26 (dd, J = 7.4, 0.9 Hz, 2H), 6.92 (d, J= 14.2 Hz, 2H), 6.36 (d, J= 14.1 Hz, 2H), 4.16 (t, J= 7.3 Hz, 2H), 2.93-2.82 (m,

2H), 1.87 (q, J = 7.4 Hz, 4H), 1.35 (s, 6H), 1.29 (s, 6H), 1.23-1.12 (m, 4H), 1.04 (t, J = 7.4 Hz, 6H), 1.00-0.86 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1, 147.5, 147.0, 142.1, 141.5, 141.1, 132.2, 128.5, 127.2, 126.4, 125.4, 122.2, 111.1 , 101.4, 49.1 , 45.4, 26.6, 24.1, 20.6, 10.2.

CIR22

¹ H NMR (500 MHz, MeOD) d 8.95 (s, 1H), 7.91 - 7.80 (m, 2H), 7.59 (t, J = 8.0 Hz, 1 H), 7.43 - 7.38 (m, 3H), 7.38 - 7.31 (m, 2H), 7.29 - 7.22 (m, 3H), 7.13 (s, 1H), 6.98 (s, 1H), 6.92 (d, J= 14.1 Hz, 2H), 6.37 (d, J = 14.1 Hz, 2H), 4.16 (t, J = 7.4 Hz, 2H),

2.92 - 2.84 (m, 2H), 1.87 (q, J = 7.4 Hz, 4H), 1.35 (s, 6H), 1.30 (s, 6H), 1.21 - 1.10 (m, 4H), 1.05 (t, J = 7.4 Hz, 6H), 0.99 - 0.94 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d

173.1, 152.2, 147.5, 147.0, 141.5, 131.5, 130.6, 128.5, 126.4, 125.4, 125.4, 122.3,

122.1, 119.1, 116.3, 116.2, 111.2, 101.5,49.1,45.4,26.6,26.6,24.1,20.6,20.5, 10.2. CIR26

¹ H NMR (500 MHz, MeOD) d 8.94 (s, 1 H), 8.56 (s, 1 H), 8.08 (t, J = 1.8 Hz, 1 H), 8.01 - 7.93 (m, 2H), 7.88 (d, J = 8.0 Hz, 1 H), 7.59 - 7.52 (m, 1 H), 7.49 (ddd, J = 8.1 , 2.1 , 1.1 Hz, 2H), 7.45 - 7.36 (m, 2H), 7.36 - 7.23 (m, 2H), 6.92 (d, J = 14.3 Hz, 2H), 6.36 (d, J= 14.1 Hz, 2H), 4.16 (t, J= 7.3 Hz, 2H), 2.87 (q, J= 7.5, 7.0 Hz, 2H), 1.87 (q, J =

7.4 Hz, 4H), 1.36 (s, 6H), 1.30 (s, 6H), 1.26- 1.12 (m, 4H), 1.05 (t, J = 7.4 Hz, 6H), 1.02-0.91 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1, 142.1, 141.5, 141.1, 130.6, 128.5, 126.4, 125.5, 125.3, 123.7, 111.1, 101.4, 49.1, 45.4, 26.6, 24.1, 20.6, 10.2.

CIR32

¹ H NMR (500 MHz, MeOD) d 9.19 (s, 1H), 8.72 -8.62 (m, 2H), 8.10 (dt, J= 1.9, 0.9 Hz, 1 H), 7.40 (dd, J = 7.4, 1.3 Hz, 4H), 7.39 - 7.31 (m, 2H), 7.25 (td, J = 7.4, 0.9 Hz, 2H), 6.92 (d, J= 14.1 Hz, 2H), 6.38 (d, J= 14.1 Hz, 2H), 4.17 (t, J = 7.3 Hz, 2H), 2.96 - 2.82 (m, 2H), 1.88 (q, J = 7.4 Hz, 4H), 1.36 (s,6H), 1.29 (s,6H), 1.21 - 1.18 (m, 4H),

1.05 (t, J = 7.4 Hz, 6H), 0.99 - 0.95 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1, 147.1, 145.1, 142.1, 141.3, 141.1, 132.4, 128.5, 126.4, 125.5, 122.2, 111.2, 101.6, 49.1, 45.4, 26.6, 26.4, 24.1, 20.6, 10.2. CIR35

¹ H NMR (500 MHz, MeOD) d 8.21 (s, 1 H), 7.47 (dd, J = 7.6, 1.1 Hz, 2H), 7.46 - 7.39 (m, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.28 (td, J = 7.5, 0.9 Hz, 2H), 6.80 (d, J = 14.1 Hz, 2H), 6.33 (d, J = 14.1 Hz, 2H), 4.15 (t, J = 7.3 Hz, 2H), 3.23 (t, J = 7.1 Hz, 2H), 2.89 - 2.78 (m,4H), 2.10-2.06 (m,4H), 1.87 (q, J = 7.4 Hz, 4H), 1.37 (s,6H), 1.35 (s,6H),

1.05 (t, J = 7.4 Hz, 6H), 1.04 - 0.99 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.0, 147.8, 147.6, 142.1, 141.6, 141.2, 128.5, 126.5, 126.0, 125.4, 122.1, 111.1, 101.3, 49.1, 45.3, 33.8, 26.9, 26.6, 24.0, 20.6, 20.6, 20.4, 10.2.

CIR36

¹ H NMR (500 MHz, MeOD) d 8.20 (s, 1 H), 7.91 (s, 1 H), 7.44 (ddd, J = 20.8, 7.6, 1.2 Hz, 3H), 7.34 (d, J = 8.0 Hz, 2H), 7.28 (td, J = 7.5, 0.9 Hz, 2H), 6.82 (d, J = 17.5 Hz, 2H), 6.34 (d, J = 14.1 Hz, 2H), 4.29 - 4.18 (m, 1H), 4.15 (t, J = 7.3 Hz, 2H), 3.05 (d, J = 6.2 Hz, 2H), 2.83 (q, J = 6.5, 5.4 Hz, 4H), 1.88 (q, J = 7.4 Hz, 4H), 1.39 (s, 6H), 1.37 (s, 6H), 1.29 - 1.21 (m, 3H), 1.20 - 1.13 (m, 4H) 1.05 (t, J = 7.4 Hz, 6H), 1.02-0.84

(m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.0, 147.8, 145.6, 142.1 , 141.6, 141.2, 128.5, 126.7, 126.6, 125.4, 122.1, 111.1, 101.4, 66.5, 49.1 , 48.1 , 45.3, 34.4, 26.9, 26.6, 24.1, 22.3, 20.6, 10.2. CIR39

¹ H NMR (500 MHz, MeOD) d 9.04 (s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 8.49 - 8.46 (m, 1 H), 7.66 (dd, J = 8.0, 4.8 Hz, 1 H), 7.45 - 7.38 (m, 4H), 7.36 - 7.32 (m, 2H), 7.25 (td, J = 7.5, 0.9 Hz, 2H), 6.91 (d, J = 14.1 Hz, 3H), 6.37 (d, J = 14.1 Hz, 2H), 4.16 (t, J = 7.3 Hz, 2H), 2.92 - 2.84 (m, 2H), 1 .88 (q, J = 7.4 Hz, 4H), 1 .36 (s, 6H), 1.29 (s, 6H), 1 .24 - 1.13 (m, 4H), 1 .05 (t, J = 7.4 Hz, 6H), 0.98 - 0.81 (m, 2H). ¹³ C NMR (126 MHz, MeOD) d 173.1 , 149.0, 147.3, 146.0, 144.7, 142.1 , 141 .4, 141 .1 , 133.7, 128.5, 126.4, 125.6, 125.5, 122.1 , 1 1 1.2, 101 .5, 49.1 , 45.4, 26.6, 24.1 , 20.6, 10.2.

Spectral Properties

Figure 1 shows absorbance pictograms of compounds 1 and 2 (10 mM) and a photostability analysis of compounds 1 and 2 (both at 50 pM in PBS) under continuous light irradiation. Solid lines correspond to one phase exponential decay regressions for both sets of values.

As shown in Figure 1 , compounds of the invention (2) display superior spectral properties when compared to their isosteric tricarbocyanine N- amines (1 ). In addition to red-shifted excitation and emission wavelengths, tricarbocyanine /V-triazoles exhibit higher extinction coefficients and quantum yields (i.e. 3% for 1 , 10% for 2) with 30-fold increase in brightness and remarkably enhanced photostability. To analyse the differential behaviour of isosteric amine (1 ) and triazole (2) fluorophores, their electron density distributions and transitions was determined with Gaussian 09.

Photostable NIR fluorophore for labelling CD4+ T cells.

A collection of CIR fluorophores were prepared by modifying IR780 with 46 structurally diverse alkynes (Figure 2a). CIR fluorophores were isolated by semi-preparative HPLC in very high purities (> 95%), and, unlike amino-derivatised tricarbocyanines, all CIR fluorophores showed excitation and emission wavelengths in the NIR window (Aexc.~ 780-800 nm, A _em ~ 805-840 nm) (Figure 2b and Tables 1 and 2). Retention time and UV-based purity were determined under the HPLC conditions described above. Wavelengths and fluorescence quantum yields were determined in DMSO using ICG (in DMSO, QY: 12%) as the reference standard.

Fluorescence NIR intensity of selected CIR fluorophores was determined upon incubation with CD4+ T cells (5 ^c 10 ⁵ cells, 10 mM PBS, A _exc .: 790 nm; A _em. : 820 nm. Values are represented as means ± s.e.m (n=3). Time-course analysis of the mean aggregate size of IR780 and CIR38 in aqueous media (100 pM PBS) determined by dynamic light scattering at r.t. for up to 8 h. Values are represented as means ± s.e.m (n=3).

Table 1. Chemical and spectral characterisation data for the CIR fluorophores.

Table 2. HRMS characterisation of the CIR library.

Table 3. Characterisation of the CIR library in terms of cell permeability and aggregation in aqueous media.

The fluorescence emission of the 46 CIR fluorophores upon incubation with murine T cells was evaluated. CIR fluorophores were uptaken by CD4+ T cells (Table 3) and CIR38 showed the brightest fluorescence emission (Figure 2c).

Water solutions of CIR38 and the generic heptamethine structure IR780 were compared by dynamic light scattering, and it was observed that CIR38 formed less insoluble aggregates in water (Figure 2d), highlighting the pyridinyl-triazole moiety as an effective chemical group to enhance the solubility of heptamethine dyes. Also, most CIR fluorophores show only small aggregates in water (<1000 nm) (Table 3). Furthermore, CIR38 showed excellent photostability, retaining full chemical integrity even after 12 h of continuous UV light irradiation.

Maleimide (CIR38M) and succinimidyl ester (CIR38SE) analogues were then prepared to increase its intracellular retention. The derivatisation of CIR38 with reactive groups did not affect the NIR spectral properties nor the uptake in murine CD4+ T cells.

In vitro characterisation of CIR38M for murine T cell imaging.

First, the staining of CD4+ T cells following rapid 2 min incubation with different concentrations of CIR38M was examined, and it was observed that the incubation of CD4+ T cells with 10 mM CIR38M provided very bright fluorescence signals, close to saturation levels. Next, the intensity of CIR38M after several cell divisions by stimulating CD4+ T cells with plate-bound anti-CD3/CD28 antibodies and measuring the NIR fluorescence emission for several days was assessed. A smaller decline in fluorescence emission was observed after 3 days of in vitro stimulation in CIR38M- labelled CD4+ T cells when compared to CFSE, a standard fluorescent marker of cell proliferation. CIR38M therefore demonstrated the utility to label T cells for multiple rounds of proliferation without a major loss of fluorescence intensity.

The stability of CIR38M labelling was analysed and it was evaluated whether the probe leaked to neighbouring cells, which would lead to the misinterpretation of in vivo imaging data. CIR38M was employed to label CD4+ T cells expressing the CD45.2 cell marker (i.e. CD45.2+ CD4+ T cells), and co-cultured them with the same number of unlabelled CD4+ T cells expressing the CD45.1 cell marker (i.e. CD45.1 + CD4+ T cells). The expression of two different CD45 congenic markers allowed us to distinguish between CIR38M-labelled and unlabelled cells in the co-culture. Flow cytometry analysis after 3 days under stimulatory conditions confirmed the intracellular retention of CIR38M, as labelled cells expressed the CD45.2 marker but not the CD45.1 marker. The fluorescence characterisation of CIR38M-labelled CD4+ T cell lysates also showed that CIR38M was retained inside cells by forming covalent bonds with intracellular proteins, preventing leakage to neighbouring cells. In addition, no significant differences were observed in the viability of CD4+ T cells after CIR38M treatment as well as no reduction in proliferation after culture for 3 days in stimulatory conditions. The non-invasive character of CIR38M was also corroborated by measuring the production of key pro-inflammatory cytokines (e.g. TNF-a, GM-CSF) of stimulated labelled CD4+ T cells, which remained unaltered after treatment with CIR38M.

Fluorescence microscopy of murine CD4+ T cells.

Given the features of CIR38M as a NIR fluorophore for non-invasive labelling of CD4+ T cells, in vitro fluorescence microscopy experiments were performed to compare its imaging capabilities to other fluorophores emitting in the far NIR region (i.e. 800-900 nm). The fluorescence staining of CIR38M was compared to the commercially available IR800CW-SE, a gold standard in NIR fluorescence labelling, by treating CD4+ T cells with both fluorophores under the same conditions and visualising them under a fluorescence microscope. Counter-staining with the green fluorophore CellTracker Green was used to confirm CD4+ T cell labelling.

Figure 3 shows Brightfield, fluorescence and merged microscope images of CD4+ T cells after labeling with CIR38M and CellTracker Green; and Brightfield, fluorescence and merged microscope images of CD4+ T cells after labeling with IR800CW-SE and CellTracker Green. Scale bar: 10 pm. Enhanced labeling of CIR38M over IR800CW- SE (top) when staining murine T cells and ICG (bottom) when staining human T cells.

As shown in Figure 3, the lack of charges and enhanced permeability of CIR38M led to much brighter staining of CD4+ T cells than IR800CW-SE. Direct comparison of CIR38M to IR780M, the maleimide analogue of IR780, showed similar intensity but more photostable NIR signals with the former, highlighting the importance of the N- triazole group within the CIR38M structure. Notably, the strong fluorescence emission of CIR38M enabled imaging of labelled CD4+ T cells using both high and low-power excitation sources (i.e. Ti-Sapphire laser and mercury lamps, respectively) as well as low magnification objectives, expanding the scope of applications for CIR fluorophores to various optical imaging modalities.

In both ICG and IR800CW structures, the potential aggregation of the heptamethine cyanine scaffold is minimised by the incorporation of negatively-charged chemical groups (i.e. sulfonates) which preclude uptake and longitudinal cell tracking. In the CIR dyes, these limitations are addressed by introducing triazole groups directly to the heptamethine core to produce bright, water-soluble and cell-permeable fluorophores. CIR fluorophores show higher sensitivity than than IR800CW-SE and ICG for labelling murine and human cells.

In vivo long-term tracking of labelled T cells.

Next, the properties of CIR38M for tracking CD4+ T cells in vivo were examined using whole-body NIR fluorescence imaging. The limit of detection in suspensions of CIR38M-labeled and DiR-labeled (both at 10 mM) CD4+ T cells was determined by NIR fluorescence imaging.

To assess the capabilities of CIR38M for detecting small populations of therapeutic T cells, CD4+ T cells were labelled with CIR38M and sequential dilutions were prepared before being directly imaged using whole-body imaging acquisition settings. The limit of detection of CIR38M was around 4,000 cells, 3-fold lower than the conventional NIR cell tracer DiR (1 ,T-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine iodide), highlighting the utility of CIR38M for monitoring small numbers of T cells that could not be detected by other methods (Figure 4). Images were acquired in a Photonlmager™ using whole-body NIR fluorescence acquisition settings (A _exc. ~ 760 nm; A _em. ~ 800-900 nm).

CIR38M was then applied in a model of T cell activation, whereby an antigen was injected subcutaneously together with complete Freund’s adjuvant (CFA), to monitor the dynamics of T cell activation in vivo. The co-administration of autoantigens and CFA is one of the most used approaches to induce experimental disease and to examine T cell activation in vivo. With these experiments, it was examined whether:

1 ) CIR38M could be used to image the antigen-specific accumulation of T cells in vivo, and 2) CIR38M could be used to track T cells longitudinally during the whole process of in vivo activation.

To address the first question, CIR38M-labelled CD45.1 + CD4+ OT-II T cells, which express a T cell receptor that responds to ovalbumin peptide (pOVA), was transferred into CD45.2+ CD45.1 - host mice. Host mice were administered a subcutaneous injection of CFA and pOVA antigen on the left hind leg to recruit the labelled OT-II T cells into the antigen-containing site, and CFA and PBS on the right hind leg as a negative control. Right hind legs were injected with PBS emulsified in CFA whereas left hind legs were injected with 10 micrograms pOVA emulsified in CFA. b) Flow cytometric ex vivo analysis of inguinal lymph nodes (iLNs) from both left and right hind limbs of mice that had been injected with CIR38M-labeled CD4+ T cells. Percentages indicate the proportion of CD45.1 + donor cells within the CD4+ T cell compartment.

Whole-body fluorescence images of the mice were acquired 48 h post-injection and it was observed that CIR38M-labelled CD4+ T cells preferentially accumulated in the lymph nodes close to where the pOVA antigen had been injected (Figure 5a: CIR38M-labeled left mouse , unlabeled CD45.1 + CD4+ OT- II T cells right mouse). The CD45.1 + expression on OT-II cells allowed the transferred cells to be identified ex vivo by flow cytometry analysis, and it was confirmed over 2- fold higher CIR38M-labelled cells in the lymph nodes draining the side immunised with CFA and pOVA when compared to the lymph nodes draining the side immunised with CFA and PBS (Figure 5b). Percentages indicate the proportion of CD45.1 + donor cells within the CD4+ T cell compartment.

Ex vivo analysis of the lymph nodes confirmed that CIR38M was only present in CD45.1 + donor cells and had not transferred onto host CD45.1 - cells, confirming that no leakage of CIR38M to other subpopulations of T cells occurred in vivo. These results corroborate that the fluorescence emission of CIR38M directly correlated to the antigen-specific accumulation of CD4+ T cells in mice.

To address the second question, CIR38M was examined for long-term imaging of T cell activation in vivo. CIR38M-labelled CD45.1 + CD4+ OT-II T or unlabeled pOVA- reactive CD45.1 + CD4+ OT-II T cells were transferred into C57BL/6 (BB.Cg-Tyr^ Hr ^h 7J) hairless mice, followed by immunisation with CFA and pOVA at both hind limbs. Whole-body fluorescence images (A _exc. ~ 760 nm; A _em. ~ 800-900 nm) were acquired at 2, 4 and 7 days after immunisation prior to organ harvest and flow cytometric analysis. As shown in Figures 6a-c, CIR38M-labelled T cells were clearly visible in the draining lymph nodes for the entirety of the in vivo activation process. Despite the highly proliferative environment which occurs when T cells interact with antigen in draining lymph nodes, quantitative analysis confirmed that - 40% of the transferred T cells still retained CIR38M at day 4 and over 15% at day 7 (Figure 6d). Values are represented as means ± s.e.m (n=3 per group).

CIR38M-stained T cells were detected in the spleen ex vivo, but were difficult to image in whole bodies due to its deep location in vivo. The lack of NIR fluorescence emission in other immune cells, most importantly CD11 b+ phagocytic myeloid cells, corroborated the applicability of CIR38M to faithfully identify the originally labelled T cells. Furthermore, we confirmed the presence of CIR38M-labelled cells by ex vivo analysis using flow cytometry and NIR tissue imaging.

Furthermore, the sensitivity of CIR38M and DiR for detecting post-transferred cells in mice was compared. Small numbers of CIR38M or DiR-labelled Tg4 CD4+ T cells, which express a transgenic T cell receptor that responds to myelin basic protein (MBP), were injected into mice that were immunised with MBP peptide, and their accumulation in antigen-treated lymph nodes was monitored by whole-body imaging. Brighter signals were detected for CIR38M-labelled cells, confirming its suitability for NIR imaging of activated CD4+ T cells even when low numbers of cells are recruited. Finally, to assess any potential effects of CIR38M-labelled cells on T cell physiology upon in vivo activation, the recruitment of T cells was compared to the lymph nodes in mice that had either received labelled or unlabelled CD45.1 + CD4+ OT-II T cells. No significant differences were found between the absolute numbers nor the proportions of CD45.1 + CD4+ OT-II T cells at any of the time points, which confirms the non- perturbative character of CIR38M for non-invasive in vivo imaging of cellular localisation and proliferation. Potential systemic toxicity derived from the injection of CIR38M-labelled CD4+ T cells was examined. The biochemical profile of serum from mice that had been immunised with pOVA and CFA and injected the same amounts of either CIR38M-labelled or non-labelled CD45.1 + CD4+ OT-II T cells was analysed. Serum biochemistry was performed from mice that had been administered either CIR38M-labelled or unlabelled cells, and no significant differences were observed between the two groups in any of the analytes measured. Furthermore, H&E staining of formalin-fixed livers and kidney sections revealed no tissue pathology after administration of CIR38M-labelled cells. Ex vivo staining of human CD4+ T cells.

Figure 7 shows a flow cytometric analysis of human CD4+ T cells isolated from peripheral blood after labeling with CIR38M and culture with equal amounts of antigen- presenting cells and 2 mg ml. ^-1 soluble anti-CD3 (a); NIR fluorescence emission on day 0 (solid line), day 1 (dashed line) and day 3 (dotted line) in comparison to unlabeled cells (grey shaded area); a comparative analysis of human CD4+ T cells labeled with CIR38M or ICG under the same experimental conditions (b); equal numbers of human CD4 ⁺ T cells were incubated either with CIR38M or PBS and incubated in culture media overnight at 37 °C, and viable cells were counted 24 h later (c), values are represented as means ± s.e.m (n=3), p > 0.05 for n.s.; and human CD4 ⁺ T cells were incubated with CIR38M or PBS and subsequently co-cultured with antigen-presenting cells in the presence of 2 mg ml. ^-1 soluble anti-CD3 (d). GM-CSF concentrations in the supernatant were determined by ELISA 72 h later. Values are represented as means ± s.e.m (n=3), p > 0.05 for n.s.

With the aim of assessing the translational potential of CIR38M in adoptive T cell transfer clinical studies, human CD4+ T cells from the peripheral blood of healthy volunteers were labelled. It was observed that 2 min treatments with 10 mM CIR38M effectively stained human CD4+ T cells, with the cells remaining clearly labelled after 3 days of stimulation (Figure 7a).

The fluorescence staining of human CD4+ T cells was compared with CIR38M to the FDA-approved ICG, and it was observed that CIR38M-labelled cells were over an order of magnitude brighter than ICG-labelled cells under the same conditions (Figure 7b). Subsequent culture and analysis of the CIR38M-labelled cells showed that the probe did not induce significant cytotoxicity in resting human CD4+ T cells after 24 h (Figure 7c) or affected the cytokine expression (e.g. GM-CSF) of activated T cells cultured for 3 days under stimulatory conditions (Figure 7d). The high stability and neutral character of CIR38M asserts its utility for longitudinal imaging studies of murine and human T cell mobility in both preclinical imaging as well as clinical research.

Cell viability of all CIR dyes Cell viability of murine macrophages was used as a readout of toxicity in mammalian cells by incubating CIR dyes at 10 uM for 2 h. DMSO:water (1 :1 ) was used as a positive control for toxicity and media only as a negative control for lack of toxicity. No significant toxicity was detected for CIR dyes as shown in Figure 8.

Comparative readout of CIR dyes vs Indocyanine Green (ICG) under optical coherence tomography (OCT)

In Figure 9, 7 CIR dyes (CIR-8,12, 27, 29, 32, 35, 36) were compared to ICG and the spectral centroid shift was calculated for each dye as in Xu et al.; Opt Lett 2004. The spectral centroid was calculated over 100 Ascans and averaged for each picture for the dye and the no dye control (solid support with no dye). An average spectral shift was calculated on each picture and three pictures were analysed for each dye. Legend of dyes in Figure 9: 1 is CIR-8, 2 is CIR-12, 3 is CIR-27, 4 is CIR-29, 5 is CIR-32, 6 is CIR-35, 7 is CIR-36, 8 is ICG.

These results show that CIR-12, 29, 32, 35, 36 produced a significantly larger spectral centroid shift than ICG, opening the possibility to use these dyes for OCT imaging with enhanced sensitivity than ICG.

Use of CIR dyes to assess retinal barrier integrity

Retinal barrier integrity is one of the hallmarks of acute macular degeneration, which currently cannot be measured in the clinic due to the lack of diagnostic tools that can detect such changes in retinal cells with high sensitivity. The ability of CIR-35 to ICG (only dye used now for imaging ocular changes with OCT and/or angiography) to cross damaged barriers of retinal cells was compared.

ARPE-19 retinal cells were grown in transwell assays under reported conditions to form monolayers for several days. Permeability of both ICG and CIR-35 were assessed in intact monolayers (cell culture medium) as well as damaged monolayers (by treatment with EGTA for 20 min). Dyes were incubated with monolayers and their permeability was assessed by measuring NIR fluorescence in the transwell after 1 h. CIR-35 showed around significantly higher permeability than ICG (5-fold higher). At 1 h CIR-35 also showed significant differences between non-damaged and damaged layers, as shown in Figure 10. CIR-35 also showed stronger OCT contrast than ICG. This opens the possibility of using OCT for the detection of loss of retinal barrier integrity. Other optical modalities: Raman spectroscopy.

Having excitation around 790 nm makes CIR fluorophores fully compatible for surface- enhanced Raman spectroscopy (SERS), where the most common excitation laser line is 785 nm. SERS intensities of the whole CIR library were compared. SERS spectra were measured in a compact Raman scanner with excitation at 785 nm (Figure 13).

Selected CIR dyes (CIR-22 and CIR-25) were then conjugated to Au nanoparticles and full SERS spectra were recorded. Long-term stability for CIR-22 Au nanoparticles in water was assessed (Figure 13)

CIR22-derivatized AuNPs were then further conjugated to anti-EGFR for specific cancer cell detection using Raman imaging. Raman Image of antiEGFR-coated- CIR22-AuNP in A549 human lung cancer epithelial cells. Figure 1 1 (a) shows a bright field image of human A549 cell, Figure 1 1 b a pseudo-coloured Raman image of the distribution of the Raman signals after incubation with the cell. The normalised Raman intensity indicates that bright signals (i.e. yellow) were observed inside the cells with very low Raman intensity in the extracellular space. Figure 1 1 c shows a 3D- reconstruction of the Raman image in A549 human cells.

Other optical modalities: optoacoustic imaging.

The capabilities of some CIR dyes (CIR-3, 1 1 , 15) for optoacoustic imaging were also assessed. CIR-3 showed the brightest optoacoustic (MSOT) readout among the 3, with high sensitivity at low micromolar concentrations.

A flow cytometric analysis of multiple subpopulations of cells and NIR fluorescence emission after labelling with 10 uM CIR38M for 2 min (solid red line) in comparison to unlabelled cells (grey shaded area) is shown in Figure 12.

CIR38M can label different subpopulations of cells presenting different CD surface markers.