Fluorescence molecular imaging (FMI) is becoming a powerful tool to improve surgical precision and diagnostics. Although significant advances have been made in imaging technology and contrast agents, debate continues regarding suitable standards for FMI. Standardizing all FMI aspects is crucial for adoption in clinical procedures and routine practice. Currently, the lack of clear-cut guidelines on FMI study reporting complicates comparison between studies and impedes standardization. This work presents community-driven REFLECT guidelines for FMI study reporting for fluorescence-guided surgery and interventions, covering preclinical, translational, and clinical studies. It aims to improve quality of reporting, enable comparison between studies, and enhance interpretation of results. The resulting structured checklist encompasses all essential details for reporting on the contrast agent, imaging device, imaging protocol, and image processing and analysis methods. Adoption of this framework is encouraged to enhance reproducibility and establish alignment with standardization efforts in the field - thus fostering advancements in FMI.

Background: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys⁰(IRDye800CW)-Tyr³-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. Results: Binding of 800CW-TATE could be blocked with DOTA⁰-Tyr³-octreotate (DOTA-TATE) on cultured SSTR₂-positive U2OS cells and was absent in SSTR₂ negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR₂ negative cells. No SSTR₂-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. Conclusion: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.

A 64 × 64-pixel current-assisted photonic sampler (CAPS) image sensor is designed for real-time fluorescence lifetime (FLT) imaging. Current-assistance is used for swift detection and time-gating of the photogenerated carriers within the whole substrate. An additional bias node is used to decrease the dynamic power consumption and a photogate is applied to the sensor to reduce the carrier transfer time. The sensor is fabricated in a commercial 350nm CMOS process on a 15μm epi-layer, with 30μm pixels and a 61% fill factor. The pixel features gating widths below 1 ns subject to a 17 ps standard deviation on the gate width uniformity, and a 31 ps standard deviation on its temporal position. The pixel has a quantum efficiency (QE) above 40% for near-infrared (NIR) wavelengths, whereas the peak QE is 61% at 675 nm. The gate’s intrinsic decay is observed to be 320 ps, which allows for the FLT imaging of sub-nanosecond lifetime dyes. The real-time FLT imaging capabilities are demonstrated on commercial fluorescence phantoms and in a preclinical experiment.

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [⁶⁸Ga]Ga-DOTA-PEG₄-IRDye800CW ([⁶⁸Ga]Ga-1) with a radiochemical purity of >96 (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [⁶⁸Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [⁶⁸Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [⁶⁸Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Purpose: To assess our improved NACA for the detection of tumor necrosis. Methods: We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [¹⁸F]FDG-PET and bioluminescence images for complementary detection of tumor viability. Results: We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [¹¹¹In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [¹¹¹In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [¹⁸F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [¹¹¹In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors. Conclusions: Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.