In Vivo Precision Evaluation of Lymphatic Function by SWIR Luminescence Imaging with PbS Quantum Dots

Abstract

Advancements in lymphography technology are essential for comprehensive investigation of the lymphatic system and its function. Here, a shortwave infrared (SWIR) luminescence imaging of lymphatic vessels is proposed in both normal and lymphatic dysfunction in rat models with PbS quantum dots (PbS Qdots). The lymphography with PbS Qdots can clearly and rapidly demonstrate the normal lymphatic morphology in both the tail and hind limb. More importantly, compared to ICG, SWIR luminescence imaging with PbS Qdots can easily identify the dominant lymphatic vessel and node with higher luminescence signal in rats. Moreover, lymphatic pump is identified as segment contracting sections with a size of ≈1 cm in rat by in vivo SWIR lymphograhy, which propose a direct feature for precise evaluation of lymphatic function. Notably, in vivo SWIR luminescence imaging with PbS Qdots also clearly deciphers the in vivo pattern of morphological and function recovery from lymphatic system in rat model. In summary, SWIR luminescence imaging with PbS Qdots can improve the lymphography and thus deepen the understanding of the morphology and structure of the lymphatic system as well as lymphatic function such as lymphatic pump, which will facilitate the diagnosis of lymphatic dysfunction in the future.

Keywords: lymphatic function; lymphatic system; lymphography; shortwave infrared (SWIR).

Scheme 1

In vivo SWIR luminescence imaging of lymphatic system and lymphedema. a) Schematic route of synthesis and lymphatic uptake of PbS Qdots, respectively; In vivo SWIR luminescence imaging b) Normal lymphatic system and c) tail lymphedema in rat model.

Figure 1

Characterization of PbS Qdots tracer. a) TEM images, b) HRTEM image of the PbS Qdots. c) XRD pattern and d) Zeta‐potential measurement of the PbS Qdots e) FTIR spectrum of the PbS Qdots. f) UV–vis and g) photoluminescence (PL) spectra of the PbS Qdots

Figure 2

SWIR luminescence imaging of normal lymphatic vessel of rat tail. a): Scheme of anatomical position of lymphatic vessels of the SD rat in the cross‐sectional view. V: vein, A: artery, CV: caudal vertebra. The lymphatic vessels are indicated with black arrows. b) The Masson trichrome staining indicating the anatomical position of lymphatic vessels. c) Picture of tail lymphatic vessels on one side of the tail after the excision of circumferential full‐thickness skin and the injection of methylene blue. The lymphatic vessels were labeled with black arrows. d) d1: Schematic illustration and d2: White light pictures showing the injection point (orange cross) and the direction of lymphatic drainage (black arrow, scale bar: 1 cm). e) and f) SWIR luminescence imaging of normal lymphatic vessels on one side of the tail at 1, 2, 3, and 4 min after the injection of PbS Qdots and ICG, respectively. Lymphatic vessels were labeled with arrows in the zoomed image. Scale bar: 1 cm. g) Quantitative analysis of luminescence intensity in (f) with PbS Qdots and ICG at 4 min, respectively. Quantitative analysis of luminescence intensity of h) the cross‐sectional level and (i) the longitudinal axis of rat tail lymphatic vessels of PbS Qdots and ICG. j) The infusion velocity within 4 min post‐injection k) and l) the images acquired 4 min and 1, 3, 6 d after injection and quantitative analysis. Scale bar: 1 cm. (n = 3) Data are represented as the mean ± SD, with ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 3

In vivo SWIR luminescence imaging of normal lymphatic vessel of rat hind limb. Scheme of the lymphatic vessels on (a1) the dorsal and (b1) the ventral side of the rat. a2 – 3 and b2 – 3 Picture of corresponding lymphatic vessels after removal of the skin and the injection of methylene blue. The lymphatic vessels were labeled with black arrows. c) White light picture showing the injection point (white point pointed with the white arrow) and the direction of lymphatic drainage (black arrow). d,e) SWIR luminescence imaging of normal lymphatic vessels on one side of the hind limb 15 min after the injection of PbS Qdots and ICG. On the ventral side, lymphatic vessels were found along the great saphenous vein and then penetrated deeper near the beginning of the thigh. On the dorsal side of the hind limb, a pair of lymphatic vessels was found along the short saphenous vein, penetrating the biceps femoralis and leading to the popliteal lymph node underneath. Scale bar: 1 cm. f) Quantitative analysis of images acquired with PbS Qdots and ICG on dorsal and ventral side, respectively. g) Quantitative analysis of luminescence intensity along the cross‐sectional level of the rat tail lymphatic vessels of PbS Qdots and ICG. h) The distribution of signal intensity manifesting the lymphatic pump along the longitudinal axis of lymphatic vessels with PbS Qdots and ICG on the ventral side. i,j) the images acquired after 1, 3, and 6 d after injection and the quantitative analysis of the luminescence intensity. Scale bar: 1 cm. (n = 3) Data are represented as the mean ± SD, with ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 4

In vivo SWIR luminescence imaging with PbS Qdots of lymphatic dysfunction (lymphedema). a) Schematic illustration of rat tail lymphedema model. b1) Photo of normal rat tail b2) photo of lymphedema rat tail. c) In vivo PbS Qdots imaging of lymphedema rat tail and normal rat tail. Yellow arrow: dermal backflow, white arrow: lymphatic vessels. Scale bar: 1 cm. d) The cross‐sectional luminescence intensity of the swollen rat tail. e) The cross‐sectional luminescence intensity of the rat tail lymphatic vessels by PbS Qdots with or without lymphedema. f) The quantitative analyses of the images acquired 0, 1, 3, 6, and 9 d after injection. (n = 3) Data are represented as the mean ± SD, with ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 5

The biosafety assessment of PbS Qdots for lymphography. a) Ex vivo photo of major organs harvested from the rat treated with PbS Qdots. b,c) Ex vivo SWIR imaging of major organs harvested from the rat treated with PbS Qdots and the quantitative analysis. d) Representative histological H&E staining of major organ tissues harvested from the rat treated with sterile saline (normal) and PbS Qdots, Scale bar: 400 µm. (n = 3) Data are represented as the mean ± SD, with ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.