High-throughput depth-resolved fluorescence imaging inside thick tissues has numerous applications in biomedicine, ranging from fields as diverse as hepatology, neuroscience and oncology. Unfortunately, fast imaging speeds and deep imaging depths are often mutually incompatible, mainly due to autofluorescence of the surrounding tissue, absorption, and scattering of the excitation and emission by impeding biological tissue. Working with the Jain laboratory, we aim to utilize short-wavelength infrared (SWIR) emitting quantum dots (QDs) for whole-body animal imaging based on single-photon epifluorescence microscopy, down to a depth of several hundred microns. We also seek to improve the background rejection and axial resolution of this approach. More precisely, we aim to develop two- or three-photon SWIR temporal focusing (TF) excitation method, utilizing high multiphoton cross section QDs (TRD4) and compressive sensing, to image dynamic events several hundreds of microns inside a solid tumor. This aim has direct impact on our collaboration on cancer biology (CP1) Jain Lab.
There are a number of benefits of short-wavelength infrared (SWIR; 1,000 - 2,000 nm) band over the visible and near-infrared bands. These include (1) minimal autofluorescence of biological tissue that leads to increased sensitivity, and (2) significantly reduced light attenuation from scattering and absorption by the blood and other tissue structures, which enables imaging with high spatiotemporal resolution and increased penetration depth. Consequently, large organisms, such as a whole mouse, may be rendered translucent when imaged using SWIR fluorescence [1]. Through our work, we have shown the capabilities of SWIR imaging through not only quantitative imaging of blood with high-spatial resolution in vivo, but also larger scale real-time whole-body imaging in mice models.
Next-generation in vivo optical imaging with SWIR quantum dots
Figure 1 illustrates multi-pass particle image velocimetry (PIV) in a mouse model using SWIR quantum dots (QDs). Specifically, a tumor-bearing mouse was injected with SWIR QD composite particles (SWIR-emitting InAs-based core-shell (CS) and core-shell-shell (CSS) quantum dots). By imaging the dynamics of the flow in healthy tissue (Fig. 1b) and in the tumor margin of a glioblastoma (Fig. 1a), we visualize the in vivo blood flow dynamics in the glioblastoma margin which exhibits irregular blood flow, including oscillatory 'pendular' flow. On the other hand, the healthy tissue in the contralateral brain hemisphere showed a normal vessel network with regular blood flow. We also exploited the unprecedented high-spatiotemporal resolution of our data to yield z-sectioned measurements of the blood flow through the vasculature at 5 µm increments.
Shortwave infrared fluorescence imaging using clinically approved near-infrared dye indocyanine green
From the microscopic scale, we venture outward to the macroscope scale for whole-body imaging, through skin imaging of the murine mammals for assessing organ functionality, and blood and lymphatic flow in real time, to establish a method for researchers to potentially assess phenotypic manifestations in mice or potentially evaluate the effects of therapeutics in a non-invasive manner [2]. Using indocyanine green (ICG, a FDA approved dye that we have shown to be surprisingly bright and functional in the SWIR) as the contrast agent (fluorescent probe), we demonstrate whole body imaging. Furthermore, we demonstrate resolution and dynamics with real-time, non-invasive SWIR imaging of the lymphatic clearance of ICG (Fig. 2). We injected an aqueous solution of ICG s.c. in the hind feet and the tail and then imaged the flow of lymphatic clearance through intact skin at 9.17 frames per second using 808 nm excitation light and an InGaAs SWIR camera. In more specific applications, ICG can be functionalized for through skin imaging of targeted sites, specifically due to ICG's brightness coupled with the low scattering in the SWIR.
The advantages of SWIR imaging over NIR techniques, such as increased sensitivity, contrast, and resolution of fine anatomical structures, are more readily available for increased adoption in preclinical and clinical imaging systems simply by switching the detection from conventional silicon-based NIR cameras to emerging, high-performance InGaAs SWIR cameras. While no FDA-approved fluorophores with peak emission in the SWIR yet exist, we have shown that detecting the off-peak fluorescence of clinically accessible NIR dyes on SWIR detectors bears the potential for rapid translation of SWIR fluorescence imaging to humans in clinical applications [2].