Simultaneous real-time multicomponent fluorescence and reflectance imaging method for fluorescence-guided surgery.

Fluorescence-guided surgical procedures are employed in an increasing number of applications such as tumor delineation, blood perfusion, and sentinel lymph node detection. A new generation of fluorescent probes is expected to increase the number of applications and improve efficiency. Yet, there are no available imaging methods to take full advantage of the forthcoming targeting technologies. We present a novel concept for imaging multiple agents for fluorescence-guided surgery. The system operates without any moving parts and can resolve images of three different fluorochromes while simultaneously recording conventional reflectance images.

Multimodal molecular imaging of integrin αvß3 for in vivo detection of pancreatic cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Late detection of then nonresectable or metastasized tumors emphasizes the need for novel imaging approaches. Here, we report on so far nonexploited potentials of αvß3 integrin-targeted molecular imaging technologies for detection of PDAC using genetically engineered mouse models. METHODS: Immunohistochemistry and Western blot were used for characterization of αvß3 expression in murine and human PDAC. We applied IntegriSense 680 fluorescence molecular tomography, intraoperative fluorescence imaging, and (68)Ga-NODAGA-RGD PET for αvß3 integrin molecular in vivo imaging of spontaneous PDAC occurring in Ptf1a(+/Cre);Kras(+/LSL-G12D);p53(LoxP/LoxP) mice. (NODAGA is 1,4,7-triazacyclononane-1,4-bis[acetic acid]-7-[2-glutaric acid] and RGD is arginine-glycine-aspartic acid.) RESULTS: αvß3 integrin is expressed in tumor cells of human and murine PDAC. IntegriSense fluorescence molecular tomography and (68)Ga-NODAGA-RGD PET enabled faithful visualization of PDAC. Furthermore, intraoperative optical imaging with IntegriSense 680 allowed good delineation of tumor borders. CONCLUSION: Imaging approaches targeting αvß3 integrin expand the potential of molecular imaging for identification of αvß3-positive PDAC with potential implications in early detection, fluorescence-guided surgery, and therapy monitoring.

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results.

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment.

BACKGROUND AND PURPOSE: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. MATERIALS AND METHODS: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. RESULTS: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 °C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 °C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. CONCLUSIONS: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

Enhancing surgical vision by using real-time imaging of αvß3-integrin targeted near-infrared fluorescent agent.

BACKGROUND: This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon's vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome. METHODS: Mice bearing xenograft human tumors were injected with αvß3-integrin receptor-targeted fluorescent probe and in vivo visualized by using a novel, real-time, multispectral fluorescence imaging system. Confirmatory ex vivo imaging, bioluminescence imaging, and histopathology were used to validate the in vivo findings. RESULTS: Fluorescence images were all in good correspondence with the confirming bioluminescence images in respect to signal colocalization. Fluorescence imaging detected all tumors and successfully guided total tumor excision by effectively detecting small tumor residuals, which occasionally were missed by the surgeon. Tumor tissue exhibited target-to-background ratio of ~4.0, which was significantly higher compared with white-light images representing the visual contrast. Histopathology confirmed the capability of the method to identify tumor negative margins with high specificity and better prediction rate compared with visual inspection. CONCLUSIONS: Real-time multispectral fluorescence imaging using tumor specific molecular probes is a promising modality for tumor excision by offering real time feedback to the surgeon in the operating room.

Imaging the bio-distribution of fluorescent probes using multispectral epi-illumination cryoslicing imaging.

PURPOSE: The increasing availability of fluorescent probes for in vivo optical imaging enables the interrogation of complex biological processes in small animals serving as models for human-like tissue function and disease. However, the validation of probe bio-distribution during their development or the study of different disease models, in support of in vivo imaging studies, is not straightforward. PROCEDURES: The imaging system developed consists of a customized multispectral planar imager that has been adapted on a commercial cryomicrotome and provides a powerful modality for ex vivo imaging of small animals. RESULTS: The ability to capture 3D anatomical (color) and fluorescence volumetric distributions of multiple fluorescent markers in high resolution is showcased. CONCLUSIONS: Serving both as a method for accurately imaging the bio-distribution of multiple fluorescent agents inside organisms and as a modality for the validation of non-invasive methods, multispectral cryoslicing imaging offers useful insights to ex vivo optical imaging of molecular probes.

Intraoperative multispectral fluorescence imaging for the detection of the sentinel lymph node in cervical cancer: a novel concept.

PURPOSE: Real-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer. PROCEDURES: In ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system. RESULTS: Real-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases. CONCLUSIONS: We conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity.

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody.

The major stress-inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1-fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near-infrared fluorescence imaging revealed an enrichment of Cy5.5-conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype-matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane-bound Hsp70, the fluorescence-labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour-selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumours. The anti-tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody-dependent cytotoxicity.

Multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases. Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema. As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor. In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix. The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion. It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used. The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Increased cerebral blood volume and oxygen consumption in neonatal brain injury.

With the increasing interest in treatments for neonatal brain injury, bedside methods for detecting and assessing injury status and evolution are needed. We aimed to determine whether cerebral tissue oxygenation (StO(2)), cerebral blood volume (CBV), and estimates of relative cerebral oxygen consumption (rCMRO(2)) determined by bedside frequency-domain near-infrared spectroscopy (FD-NIRS) have the potential to distinguish neonates with brain injury from those with non-brain issues and healthy controls. We recruited 43 neonates < or =15 days old and >33 weeks gestational age (GA): 14 with imaging evidence of brain injury, 29 without suspicion of brain injury (4 unstable, 6 stable, and 19 healthy). A multivariate analysis of variance with Newman-Keuls post hoc comparisons confirmed group similarity for GA and age at measurement. StO(2) was significantly higher in brain injured compared with unstable neonates, but not statistically different from stable or healthy neonates. Brain-injured neonates were distinguished from all others by significant increases in CBV and rCMRO(2). In conclusion, although NIRS measures of StO(2) alone may be insensitive to evolving brain injury, increased CBV and rCMRO(2) seem to be useful for detecting neonatal brain injury and suggest increased neuronal activity and metabolism occurs acutely in evolving brain injury.

Real-time intraoperative fluorescence imaging system using light-absorption correction.

We present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.

Multispectral imaging using multiple-bandpass filters.

We present a method for multispectral imaging. This method uses color CCD cameras with a multiple-bandpass filter, which modifies the spectral response of the cameras used and enables concurrent acquisition of multiple images at defined spectral bands. We experimentally demonstrate methodological feasibility using two color CCD cameras and a polychroic mirror to simultaneously capture eight spectral bands. We discuss how the method developed is well suited for multispectral applications of transient phenomena or for real-time measurements.

Assessment of infant brain development with frequency-domain near-infrared spectroscopy.

This is the first report to demonstrate quantitative monitoring of infant brain development with frequency-domain near-infrared spectroscopy (FD-NIRS). Regionally specific increases in blood volume and oxygen consumption were measured in healthy infants during their first year. The results agree with prior PET and SPECT reports; but, unlike these methods, FD-NIRS is portable and uses nonionizing radiation. Further, new information includes the relatively constant tissue oxygenation with age and location, suggesting a tight control between local oxygen delivery and consumption in healthy infants during brain development. FD-NIRS could become the preferred clinical tool for quantitatively assessing infant brain development.

Near-infrared spectroscopy measurement of the pulsatile component of cerebral blood flow and volume from arterial oscillations.

We describe a near-infrared spectroscopy (NIRS) method to noninvasively measure relative changes in the pulsate components of cerebral blood flow (pCBF) and volume (pCBV) from the shape of heartbeat oscillations. We present a model that is used and data to show the feasibility of the method. We use a continuous-wave NIRS system to measure the arterial oscillations originating in the brains of piglets. Changes in the animals' CBF are induced by adding CO(2) to the breathing gas. To study the influence of scalp on our measurements, comparative, invasive measurements are performed on one side of the head simultaneously with noninvasive measurements on the other side. We also did comparative measurements of CBF using a laser Doppler system to validate the results of our method. The results indicate that for sufficient source-detector separation, the signal contribution of the scalp is minimal and the measurements are representative of the cerebral hemodynamics. Moreover, good correlation between the results of the laser Doppler system and the NIRS system indicate that the presented method is capable of measuring relative changes in CBF. Preliminary results show the potential of this NIRS method to measure pCBF and pCBV relative changes in neonatal pigs.

A clinical study of optical biopsy of the uterine cervix using a multispectral imaging system.

OBJECTIVE: To present the clinical application of the multispectral imaging colposcopic system (MIS colposcopy). METHODS: MIS colposcopy was performed on 123 enrolled women. After a 3% acetic acid application, sequential images were captured, analyzed, and stored automatically. Directed biopsies were taken from distinct marked acetic acid-responsive tissue areas indicated on the monitor, while a real-time assessment of the curves of intensity of the backscattered light (IBSL) vs. time was performed. Blind biopsies were taken from non-acetowhitening areas. Histological findings were correlated with MIS colposcopy results and compared with conventional colposcopy and Pap test results. RESULTS: Acetic acid-tissue interaction resulted in temporal and spatial alterations to the light scattering properties of the abnormal tissue that was analyzed. The shape of IBSL curve and the "relaxation time" (the time it takes for IBSL to decay to 1/e of its peak value) changed in accordance with the underlying lesion. More severe CIN lesions lead to higher maximum IBSL; longer durations of acetowhitening lead to increasingly delayed exponential decay of IBSL curve. To compare with histological examination, MIS colposcopy had a 1.7% false-diagnostic rate, while PAP test and conventional colposcopy had 24.4% and 22% false-diagnostic rates, respectively. A triple exponential function created a "pseudocolor" image that comprised the grade map of the lesion, and this is frequently representative of the duration/degree of the induced alterations. CONCLUSION: Improved diagnostic information can be gained by recording the optical information in a narrow spectral range with high spatial resolution. MIS colposcopy can be used in the diagnosis of uterine cervix pathological conditions and in the differentiation between CIN lesions.