Intraoperative guidance using targeted near-infrared (NIR) fluorescent tracers can provide surgeons with real-time feedback on the presence of residual tumour tissue. To overcome the still limited depth penetration of NIR light, and limit potentially missing residual occult or deeper lying lesions, the combination of fluorescence with nuclear imaging is proposed. We describe the design and preclinical validation of the anti-HER2 nanobody 2Rs15d, conjugated with a ‘multifunctional single attachment point’ (MSAP), which integrates a Cy5 fluorophore and diethylenetriaminepentaacetic acid (DTPA) chelator into a single label. After random conjugation to primary amines in the nanobody, functionality of the tracer and stability after 111In labelling were evaluated in vitro. Using SKOV3 (HER2+) and MDA-MB-435S (HER2-) xenografted mice, the in vivo biodistribution of 2Rs15d-MSAP.111In was determined by SPECT/CT (1h post-injection) and fluorescence imaging (1h30 post-injection). Ensuing, the ex vivo biodistribution was determined 2h (both xenograft models) and 24h post-injection (SKOV3 only). The tracer retained its affinity after conjugation of the MSAP and remained stable over 24h in both PBS and human serum after 111In labelling. The in vivo SPECT/CT and fluorescence images corresponded well, showing the expected biodistribution pattern for nanobody tracers, meaning low background except for high renal uptake due to clearance, and specific tumour uptake in HER2-overexpressing tumours. Ex vivo biodistribution data revealed a SKOV3 tumour-specific uptake of 7.0 ± 2.5 %ID/g after 2h, significantly higher than 1.1 ± 1.2 %ID/g for control tumours. The tumour-to-blood ratio was 47.6± 25.4, tumour-to-muscle ratio 23.2 ± 11.6, and tumour-to-liver ratio 6.9 ± 3.7. After 24h SKOV3 tumour uptake was 5.6 ± 1.9 %ID/g, tumour-to-blood ratio 229.1 ± 85.1, tumour-to-muscle ratio 16.8 ± 8.0, and tumour-to-liver ratio 5.1 ± 1.9. In conclusion, functional bimodal nuclear/fluorescent nanobody-tracers can be conveniently generated by conjugation of a single-molecule MSAP-reagent carrying both fluorophore and a chelator.
Fluorescence imaging using near-infrared (NIR) fluorescent contrast agents is increasingly being investigated as intraoperative tool to visualize, in real-time, tissues of interest such as tumors, lymph nodes or nerve bundles. Generally, spectral imaging systems are used that measure the intensity of fluorescent signals. However, to aid in a more specific detection of these fluorescent signals, fluorescence lifetime can be added to the image. The lifetime is independent of the intensity and in addition, multiple tracers emitting around the same wavelengths can still be distinguished based on their difference in lifetime. Imaging lifetimes, however, requires a much more advanced imaging system. None of the currently approved fluorescence guidance systems support fluorescence lifetime and today’s available lifetime imaging technology (TCSPC, ICCD) does not allow imaging the sub-nanosecond lifetimes of NIR dyes with the efficiency needed to reach video frame rates. In this paper, we present a 32×32-pixel proof-of-concept camera based on our novel CAPS-pixel based gated image sensor. This camera is specifically targeted at imaging fluorescence lifetimes at NIR wavelengths with high efficiency for the use in fluorescence-guided surgery and is a first step towards a full camera with video resolution at video frame rates. We describe the camera system and how it is used to image fluorescence lifetimes. Next, we show the lifetime imaging capability by imaging the lifetimes of two different nanosecond visible dyes (fluorescein and acridine orange) in cuvette and two more challenging (sub-)nanosecond NIR dyes (ICG and IRDye800CW). Lastly, we validate the camera by imaging NIR fluorescence phantoms in a mouse.
Coronary artery disease (CAD) contributes to millions of deaths each year. The identification of vulnerable plaques is essential to the diagnosis of CAD but is challenging. Molecular probes can improve the detection of these plaques using intravascular imaging methods. Fluorescence lifetime sensing is a safe and robust method to image these molecular probes. We present two variations of an optical system for intravascular near-infrared (NIR) fluorescence lifetime sensing through a multimode fiber. Both systems are built around a recently developed fast and efficient CMOS detector, the current-assisted photonic sampler (CAPS) that is optimized for sub-nanosecond NIR fluorescence lifetime sensing. One system mimics the optical setup of an epifluorescence microscope while the other uses a practical fiber optic coupler to separate fluorescence excitation and emission. We test both systems by measuring the lifetime of several NIR dyes in DMSO solutions and we show that these systems are capable of detecting lifetimes of solutions with concentrations down to 370 nM and this with short acquisition times. These results are compared with time-correlated single photon counting (TCSPC) measurements for reference.