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Fluorescence lifetime of injected indocyanine green as a universal marker of solid tumours in patients


The surgical resection of solid tumours can be enhanced by fluorescence-guided imaging. However, variable tumour uptake and incomplete clearance of fluorescent dyes reduces the accuracy of distinguishing tumour from normal tissue via conventional fluorescence intensity-based imaging. Here we show that, after systemic injection of the near-infrared dye indocyanine green in patients with various types of solid tumour, the fluorescence lifetime (FLT) of tumour tissue is longer than the FLT of non-cancerous tissue. This tumour-specific shift in FLT can be used to distinguish tumours from normal tissue with an accuracy of over 97% across tumour types, and can be visualized at the cellular level using microscopy and in larger specimens through wide-field imaging. Unlike fluorescence intensity, which depends on imaging-system parameters, tissue depth and the amount of dye taken up by tumours, FLT is a photophysical property that is largely independent of these factors. FLT imaging with indocyanine green may improve the accuracy of cancer surgeries.

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Fig. 1: FLT enhancement and microscopic cancer specificity in primary and metastatic liver cancers.
Fig. 2: FLT enhancement and microscopic cancer specificity in HN cancers.
Fig. 3: FLT enhancement in primary and metastatic bone and soft tissue sarcomas.
Fig. 4: FLT enhancement in high-grade GBM.
Fig. 5: FLT imaging of tumour infiltrating LNs.
Fig. 6: Mechanistic studies of FLT enhancement in tumour cell lines.

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Data availability

The main data supporting the results in this study, including source data for the figures, are available within the paper and its Supplementary Information. The raw data generated in this study are too large to be publicly shared yet are available for research purposes on reasonable request from the corresponding author. The raw patient data are available from the corresponding author, subject to approval from the IRBs of the respective universities. Source data are provided with this paper.

Code availability

The custom MATLAB codes applied to analyse the TD fluorescence data are available from the corresponding author on reasonable request.


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We acknowledge P. Isermann and D. Tom from Leica Microsystems, and the Harvard Medical School (Boston, MA) MicRoN for access to the Leica STELLARIS 8 used in this work, and for the extensive assistance in imaging. This work was supported in part by the NIH grants R01-CA211084, R01-CA260857 and 1P01CA240239 and the MGH-Executive Committee on Research Interim Support Fund.

Author information

Authors and Affiliations



R.P. designed and performed experiments and data analyses, in addition to co-writing the manuscript. T.M.L. assisted in clinical data acquisition, patient recruitment and project design. C.D.C. designed and performed experiments and analysed the data. T.H.D., H.R.C. and S.A.C. assisted in patient recruitment and IRB application; M.K., H.R.C. and S.S. performed experiments and data analysis, A.L.K. provided nursing services for the clinical study. S.A.C. and M.K. assisted with the imaging system design. M.P.N., A.P., M.S.M., Y.P.H., M.M.-L., L.Z., P.M.S., A.R.S. and W.C.F. provided pathology specimens and assisted in data interpretation. B.V.N., A.L.F., K.S.E., J.Y.K.L., K.S.R., S.L.-C., M.A.V., J.S.D.M, A.L.V., K.R. and K.K.T. provided surgical specimens and guidance in project design and data interpretation. A.T.N.K. conceived the idea for the project, designed and performed the experiments and data analysis, supervised the clinical studies and wrote the manuscript.

Corresponding author

Correspondence to Anand T. N. Kumar.

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Competing interests

A.T.N.K., M.K. and R.P. are co-inventors of a pending US Patent application issued to The General Hospital Corporation. The patent might be the subject of a licensing agreement in the future. Y.P.H. received royalties from Elsevier publishing company unrelated to this study. K.S.R. received remuneration for lecturing on a Stryker Fluorescence Guided Surgery Conference. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Jie Tian, Georg Widhalm and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Measurements of endogenous tissue autofluorescence in the NIR.

Photograph (a), fluorescence intensity (b), and FLT map (c) of a freshly resected metastatic colorectal cancer (mCRC) specimen without ICG injection. T, Tumor; N, Normal liver parenchyma. White dashed lines in a-c indicate the clinically defined tumor boundary. In the absence of ICG, the FLT of tumor autofluorescence (AF) was 0.22 ± 0.01 ns. Histology (d), confocal fluorescence intensity (e), and FLIM (f) images of a 10 µm OCT embedded tissue section obtained from a region of interest (a, yellow dotted outline). The mean tumor AF FLT measured via confocal FLIM was 0.37 ± 0.05 ns. g, Representative wide-field time domain (TD) fluorescence decay curves obtained from an mCRC specimen without ICG (green) and a specimen with ICG (red). The peak of the TD data (represented by the horizontal dashed lines) is representative of the fluorescence intensity in each mCRC tumor. Comparing the AF with tumor ICG intensity, we observed a nearly 20-times stronger signal in the ICG-injected specimen. h, Normalized plots of the TD data from (g), showing a significantly longer FLT of tumor-ICG (red = 0.61 ns) compared to the AF FLT (green = 0.22 ns), thus confirming that the tumor FLT observed with pre-surgery ICG administration arises from the tumor uptake of ICG.

Source data

Extended Data Fig. 2 FLT enhancement of a representative mCRC specimen from a clinical trial conducted at Leiden University.

Histology (a), fluorescence intensity (b), and FLIM (c) images of an mCRC specimen from a patient injected with 0.1 mg/kg ICG 24 h prior to surgery. Imaging was performed in 10 µm thick sections obtained from an FFPE tissue block. Histologically confirmed tumor boundary is shown in the histology (yellow line) and intensity (red line) images. The fluorescence intensity image (b) indicated ICG accumulation primarily around the tumor boundary (‘rim-fluorescence’). Despite the apparent absence of ICG, the FLT in the tumor core (0.74 ± 0.02 ns) was significantly longer (p < 0.001) than the surrounding normal liver parenchyma (0.61 ± 0.04 ns), indicating the presence of ICG in the tumor. Violin plots show the distribution of FLT, d, and intensity, e, across 18 ROIs obtained from the histologically determined tumor (11 ROIs), inflamed tumor stroma (3 ROIs), and normal hepatocyte regions (3 ROIs). f, ROC curves for tumor (tumor + inflamed tumor stroma) (14 ROIs) vs. normal tissue (15 ROIs) classification for this specimen yielded an accuracy (AUC) of 94% using fluorescence lifetime (red) and only 4% for intensity (green) based classification. The dashed lines in d and e represent the threshold FLT (0.68 ns) and intensity (42.7 AU), respectively, corresponding to the highest accuracy as calculated from the ROC curves.

Source data

Extended Data Fig. 3 FLT enhancement of a representative tongue SCC specimen.

Histology (a) and FLT (b) images of a tongue SCC specimen from a patient with ICG injection 20 h prior to surgery. Long FLT of ICG was observed to originate from the microscopic tumor nests (arrows) with short FLT components in the surrounding tissue. One such tumor cell nest (dotted rectangles in a and b) is shown with higher magnification in, c (histology) and d (FLT). c, the tumor nest (dashed outline) can be identified at the center of the histology image within a matrix of desmoplastic stroma and lymphocytes (dotted arrow). d, Long FLT was observed only in the tumor nest while the desmoplastic stroma and lymphocytes showed short FLT (dotted arrow). e, Histology, and f, FLT images of a buccal SCC specimen from another patient injected with ICG 20 h prior to surgery. The boundary of the tumor in the buccal epithelium can be identified by dashed lines in (e) and (f). The cancer cells clearly showed a longer FLT compared to the underlying muscle (solid arrows in (e) and (f)). The dotted arrows in (e) and (f) indicate tumor-infiltrating lymphocytes (TILs) with FLTs comparable to the tumor cells. g, Histology and h, FLT images of a normal oral epithelium showing low ICG uptake and short FLT across the epithelium (solid arrow) and the stroma (dashed arrow).

Extended Data Fig. 4 FLT enhancement of HN cancer specimens from a clinical trial conducted at the University of Pennsylvania.

FLT and intensity contrast in specimens from patients with HN cancer (n = 6) enrolled at the University of Pennsylvania with a high dose of ICG (5 mg/kg) are presented. Histology (a), fluorescence intensity (b), and FLT (c) images of a representative SCC of the tonsil with ICG injection 24 h prior to surgery. The dashed lines indicate histologically identified tumor-normal boundary. The patient-wise (n = 6) mean fluorescence intensity, d, and FLT, e, of tumor (red) and normal tissue (green) are shown as the mean of multiple ROIs ( > 30) of histologically identified tumor and normal tissue in each patient. Statistical significance was calculated using a t-test (two tailed, two sample of unequal variance): n.s. p > 0.05, *** p < 0.001. The average tumor FLTs for all 6 specimens ranged between 0.81–0.87 ns, which was within the FLT range observed with low dose ICG (0.74–0.92 ns) shown in Fig. 2j. The average normal tissue FLTs ranged between 0.72–0.78 ns, which was higher than the normal tissue FLT for the 0.5 mg/kg ICG dose (Fig. 2j; 0.45–0.68 ns) used at MGH, indicating a high non-specific ICG retention in normal tissue presumably due to a 5 mg/kg dose of ICG used in the trial at University of Pennsylvania. Violin plots showing the distribution of fluorescence intensity (f), and FLT (g) in HN tumors and normal tissue types across all 6 patients with high dose ICG. SG: salivary glands; NE: normal epithelium; NS: normal stroma. h, ROC plots of sensitivity vs. false positive rate (1 - specificity) across all patients studied (n = 6) resulted in an area under the curve (AUC) of 0.84 and 0.46 for FLT-based and intensity-based tumor vs normal classification, respectively. The grey-shaded areas represent 95% confidence interval.

Source data

Extended Data Fig. 5 FLT enhancement of breast cancer metastasis to the bone and osteosarcoma.

a, Histology, and b, confocal FLIM images of a specimen obtained from a patient with bone metastasis of breast cancer who was administered a flat dose of 50 mg ICG at the induction of anesthesia. c, Histology, and d, confocal FLIM images of a specimen collected from a patient with osteosarcoma systemically injected with 75 mg ICG one day before surgery. For both tumor types, the cancer cell nests are indicated by solid arrows (a and c) that correspond to long ICG FLT (b and d). Normal bone is indicated by dashed arrows in (a) that did not show a detectable ICG uptake (b). In the osteosarcoma specimen (c), large hemorrhagic pools of red blood cells (dashed arrows) were observed that displayed higher ICG fluorescence, but shorter FLT compared to the nearby cancer cell nests.

Extended Data Fig. 6 Normal tissue adjacent to sarcoma specimens show short ICG FLT.

Histology images of normal tissues, a, Skeletal muscle, b, epidermis with connective tissue, and c, adipose tissue obtained from a patient with myxofibrosarcoma ~2 h after systemic administration of ICG (0.5 mg/kg). Fresh normal tissue specimens away from the primary tumor were collected immediately following surgery and inking. Tissues were frozen in OCT overnight and sectioned (10 μm thickness) for confocal FLIM and H&E staining. A significant amount of connective tissue (red arrows) can be seen along with the epidermis (green arrows) in (b). Confocal FLIM images of the same sections of d, skeletal muscle, e, epidermis with connective tissue, and f, adipose tissues are shown. ICG FLTs in skeletal muscle, skin, connective tissue, and adipose tissue were 0.42 ns ± 0.04 ns, 0.41 ns ± 0.02 ns, 0.45 ns ± 0.01 ns, and 0.44 ± 0.02 ns, respectively. The long FLT component indicated by the ‘while arrowhead’ in (e) was considered an image artifact since it was not associated with the tissue (black arrowhead in (b)).

Extended Data Fig. 7 Wide-field FLT imaging of fresh human resection specimen from a patient with liposarcoma.

a, Photograph of a fresh resection specimen from a patient (S1 in Supplementary Table 13) with liposarcoma, 3 h after systemic ICG injection, showing clinically identified viable tumor (dashed red) and normal muscle (dashed green). Necrosis (solid arrow) and scar tissue (dotted arrow), formed due to extensive neo-adjuvant radiation therapy, are also visible in the FOV. b, Fluorescence intensity was highly heterogeneous within the tumor and significantly overlapped with the intensity of adjacent normal muscle, making it difficult to distinguish tumor from muscle. c, FLT maps clearly distinguished the tumor mass from normal muscle tissue. d, Representative wide-field TD fluorescence decay profiles of liposarcoma (red solid, 0.72 ± 0.07 ns), myxofibrosarcoma (red dashed, 0.73 ± 0.07 ns), and normal muscle (blue, 0.44 ± 0.04 ns) outside the clinically identified tumor margin. e, Fluorescence intensity, and f, FLT histograms of tumor (red) and normal muscle tissue (green) identified from the photograph in (a) are shown. A high degree of overlap of fluorescence intensities between the tumor and normal tissue was observed, while the tumor FLTs were distinctly longer than the normal tissue FLTs with minimal overlap.

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Supplementary dataset 1

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Supplementary dataset 2

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Supplementary dataset 3

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Supplementary dataset 4

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Pal, R., Lwin, T.M., Krishnamoorthy, M. et al. Fluorescence lifetime of injected indocyanine green as a universal marker of solid tumours in patients. Nat. Biomed. Eng 7, 1649–1666 (2023).

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