Breast cancer (BC) is the most common cancer in women in the world in western industrialized countries . BC Tumors are draining from the breast tissue to the axillary lymph nodes. Exploration of the axillary region, therefore is part of the surgical staging in breast cancer . If tumor cells are histopathologically present in the extracted axillary lymph nodes, individual cancer prognosis decreases dramatically. The conventional radical axillary lymph node extraction (ALNE) for surgical staging in breast cancer is associated with high morbidity and significant loss of quality of life (QoL). These adverse e_ects were strongly reduced by introducing the concept of the so called sentinel lymph node biopsy (SLNB). Tracer substances like dyes and radio nuclides are used to mark the sentinel lymph nodes (SLNs) within this SLNB method, and are therefore injected into the breast. After a predefined waiting time, enrichment of the tracer is completed in the SLN which can then be localized intra operative as radioactive hot spots by a mobile gamma hand probe and then extracted surgically .
The use of dyes and radio nuclides together for detection of SLNs increases safety in the diagnosis of lymph node involvement through less false negative results . Before starting the surgical procedure a static lymph scintigraphy is demanded for intra operative correlation of the detectable SNLs . Unfortunately, the procedure with combination of dyes and radio nuclides has a longer learning curve than radioactive procedure alone, because of the additional intra operative use of dye . Moreover, this procedure is time consuming within the surgical procedure, as scanning of the axillary region for SLNs by hand probe for radioactivity and visually scanning for blue traces (lymph vessels to lymph nodes) and blue lymph nodes takes time. Axillary morbidity increases particularly when multiple lymph nodes are labeled by radioactivity and/or dye. Moreover, esthetic problems may occur, because blue dyes can generate permanent tattooing skin effects.
We aim to develop a new sentinel lymph node biopsy concept by the use of superparamagnetic iron oxide nanoparticles (SPIOs) and a three-dimensional imaging and distinct localization of SPIOs by magnetic particle imaging (MPI)  in breast cancer to decrease tissue damage while diagnostic surgery of the axilla and to increase patients QoL and safety.
There are SPIOs approved specifically for MR imaging (e.g. ferucarbotran, Resovist®). After intravenous bolus administration uptake is observed in lymph nodes . Nano-sized particles with superparamagnetic iron oxide, coated by carboxydextran could replace the marker substances previously described above within the new SLN procedure. So far, few local and systemic adverse effects were noted when SPIOs like Resovist® were used within MRI procedures [7, 8].
As we know from i.v. application of SPIOs in magnetic resonance imaging (MRI), macrophages are playing a key role in processing of SPIOs (e.g. in the liver) causing a drop of signal intensity [9–12]. But, knowledge lacks concerning enrichment processes of SPIOs after injection in breast tissue, the adjacent lymphatic tissues and associated cells, especially in breast cancer and metastatic lymph nodes. Therefore, tissue and cell associated processing of SPIOs is of major interest for this SLNB concept. Based on our own preliminary studies in healthy and tumor mouse model  we investigate the processing of SPIOs by and in macrophages for the first time .
To investigate those SPIO associated processes in active macrophages (MPs), life imaging by multiphoton laser microscopy seems to be a feasible method. Due to the development Ti.sapphire fs lasers in the last 10 years, multiphoton-microscopy has become a popular method in life sciences. Its ability to excite a broad variety of intrinsic fluorescent proteins such as FAD [15, 16], NADH  or serotonin  without additional staining and without extensive tissue damage is ideal for in vivo applications. Also the combined use with genetical engineered fluorescent proteins has been shown as well as the applicability to standard dyes as for example DAPI in histology or Fluo 4 for calcium measurements. It was also shown that multiphoton microscopy is able to track quantum dots in the colon to investigate the uptake of nanoparticles by the intestines.
Transmission electron microscopy (TEM) is a methodological standard for imaging of subcellular processes after fixation of biological materials [19, 20]. Therefore, it seems to be feasible for approval of in vivo results of multiphoton laser microscopy.
2 Material and Methods
A commercially available mouse monocyte macrophage cell line J774A.1 (BALB/c) was attached to thermanox™ coverslips to growth, than incubated in culture medium (RPMI 1640 (Roswell Park Memorial Institute), 20% fetal bovine serum (FBS), 1% Penicillin/Streptomycin) and further processed, as described follows.
2.1.1 Preparation of MPs for TEM
Pre-incubatad MPs were washed and further processed over 24 hrs either by 100 μl/mg Resovist-Fe (0.5 mmol/l) in culture medium (RPMI 1640, 20% FBS, 1% Penicillin/ Streptomycin), or culture medium only as control.
After incubation, the cells were washed with phosphate buffered saline (PBS) and fixed in paraformaldehyd (Monti Graziadei fixative: 156 ml natrium-cacodylat-buffer 0.12 M; 25 ml glutaraldehyd 25%; 19 ml paraformaldehyd 10%; 3 ml CaCl2, 3%; pH 7.35 accumulated to 312 ml with Aqua bidest ). Macrophages were than analyzed by TEM and phagocytosis of SPIOs was evaluated.
2.1.2 Preparation of MPs for multiphoton microscopy
Pre-incubatad MPs were washed and further processed in heated 6 well plates with 1 × 105 cells per well for immediate in vivo laser procedure. Cells were analyzed directly after incubation either by 100 μl/mg Resovist-Fe (0.5 mmol/l) in culture medium (RPMI 1640, 20% FBS, 1% Penicillin/Streptomycin), or culture medium only as control with live cell imaging.
2.2 Microscope and Lasers
2.2.1 Transmission electron microscope
For TEM imaging, we used a standard transmission electron microscope: JEOL 1011 TEM, Jeol Germany GmbH, Eching. nominal acceleration voltage 80–120 kV.
2.2.2 Multiphoton laser scanning microscope
The multiphoton microscope used is an upright TriM Scope 2 (LaVision Biotec, Bielefeld Germany), with an adapter for an Olympus XLPLN25XWMP2 objective 25× NA 1.05 (Olympus, Tokyo, Japan). The detection is realised with H7422-40 photo multiplier tubes (Hamamatsu, Hamamatsu, Japan) with GaAsP-photocathodes, implemented for optimum quantum effciency in the spectral range from 435 nm to 560 nm. For the detection of the third harmonic below 435 nm a Hamamatsu PMT with an alkali cathode is used. The collected signal from the sample is separated from the excitation light by a 700nmbeam splitter. Afterwards three different dichroic mirrors divide the signal into 4 spectral channels. Below 435 nm, 435 nm to 495 nm, 495 nm to 560 nm and light above 560 nm.
Excitation is realised with two Mai Tai tuneable Ti:sapphire lasers (SpectraPhysics) with a repetition rate of 80 MHz and pulse lengths of about 100 fs. The MaiTai are combined with a 850 nm beam splitter before entering a beam shaper to provide a pre compensation for the dispersion of the fs pulses in the optics of the microscope.
As a third laser source a Insight Deep See (Spectra Physics) broad bandwidth tuneable laser is used. The Insight DS is coupled onto the same beam path as the MaiTais insight the scan head of the TriM 2 with a 1045 nm dichroic mirror. The DS supplies laser pulses at a rate of 80 MHz with about 100 fs length up to 1300 nm.
Three pockels cells control laser power at the sample. Exact confocality for all lasers is checked with fluorescent poly styrole beads of 50 nm size.
2.2.3 Fluorescence Lifetime Imaging Microscopy
The fluorescence lifetime imaging microscopy (FLIM) setup used in our lab is an simple tau 150 with a HPM-100-40 detector (Becker und Hickl GmbH, Berlin, Germany). The software for data acquisition is the proprietary Becker and Hickl SPC Software. The detector module can be easily combined with the filter box module of the TriM Scope 2. Synchronisation with the excitation lasers is done with the MaiTais sync output or a fast photo diode for the Insight DS respectively.
FLIM is a method that allows distinguishing of different fluorescent species by their fluorescence lifetime. Every excited fluorescent molecules excited state has a characteristic halftime. This time can be modified by ambient conditions like pH or calcium concentration or presence of another excitable molecule. This usually is implemented to measure these conditions. On the other hand FLIM can distinguish fluorescence from coherent processes like second harmonic generation (SHG) or third harmonic generation (THG). The limiting factor for this in time correlated single photon counting is the instrument response function (IRF), which is the response of the electronics to the detection of a photon.
The FLIM data was recorded with 100 frames (at 0.6 frames per second with 1.6 μs pixel dwell time) summed up, to get the necessary threshold of photons for fluorescence lifetime fitting. The whole collected fluorescence was directed to the detectors. A 50/50 beam splitter allowed simultaneous image acquisition with the GaAsP PMT of the TriM Scope 2.
2.3 Data collection and software
The cells are imaged under epi-illumination while, submerged in Dulbeccos modified eagles medium. The microscope objective dips into the medium.
The scanning is done at a speed of 1000 lines per second with a pixel dwell time depending on the chosen pixel size of the images taken. PMT amplification is usually set to 90% for the GaAsP detectors and to 95% for the alkali detector.
The laser power for the Insight DS during measurements is about 50mWat the sample for 1200 nm. For most measurements one MaiTai tuned to 740 nm is used to excited intrinsic fluorescent proteins. Laser power at 740 nm is kept below 10mWat all times to limit photo damage and production of photo toxic by products.
The software to collect the data and control the microscope as well as the MaiTai lasers is the Inspector application (32bit version for Windows XP) provided by LaVision Biotec. The Insight DS is controlled by a separate Laptop with Spectra Physics control software.
We are presenting our first results of processing of SPIOs by MPs, which showed activity in phagocytosis of Resovist after incubation in TEM as well as in multiphoton microscopy.
SPIOs were detectable within intracellular vesicles by TEM. Macrophages were incorporating the SPIOs after incubation with Resovist. First, SPIOs are attaching at the cellular membrane, than a phagocytic vesicle was formed and SPIOs were incorporated actively by the cells subsequently. These intracellular phagocytic vesicles are clearly shown by TEM as phagolysosomes (Figure 1 and 2).
Live cell imaging showed that Resovist had no toxic effects on MPs, even after 24 hrs of incubation within the TEM experiment.
Thus, in vivo analysis of living cells by multiphoton microscopy showed at an excitation of 1200 nm, that Resovist was clearly visualized inside the MPs in the channel below 435 nm. The excitation was changed in a range from 1100 nm to 1300 nm to check out optimal excitation wavelength. It peaked at a wavelength of about 1200 nm with a small bandwidth. Experiments with naive cells for control revealed no detectable signal in the spectral range below 435 nm by multiphoton microscopy (Figure 3).
The incubation medium of the cells was changed to SPIO containing medium immediately before the start of continuous multiphoton microscopy scanning with 7.7 s frame intervall to avoid uptake artefacts.
Recordings were done for 760 nm and 1200 nm excitation separately because the laser pulses from MaiTai and Insight DS are not synchronous. The first signal of Resovist was detected after a minimum of 1.5 minutes after adding the iron nanoparticles containing medium. Figure 4 shows the colour-coded image for 760 nm.Mean fluorescence lifetime was determined to 1.36 ns.
Figure 5 shows the FLIM image of the same cells with 1200 nm excitation. The mean fluorescence lifetime was determined to 0.112 ns.
Axillary lymph node detection and extraction are still part of the standard staging procedure in breast cancer diagnosis. To leave the primary radical axillary lymph node extraction procedure the sentinel concept was established to avoid severe side effects. But, even for conventional SLNB the axilla has to be widely explored to identify the SLN. To avoid further surgically caused damage of the axillary region, an intra operative tracer targeted imaging and surgically extraction of the SNL is needed. This would be a relevant improvement in QoL for the patient and could be achieved by the use of SPIO as tracer and MPI as finder [22, 23]. Our own previous findings showed that SPIO detection within tissue is feasible and may be performed withMPI in future scenario . Through the avoidance of intensive surgical exploration of the axilla, may be by using a MPI hand probe later on , the morbidity could be dramatically reduced. The tracer forMPI is easy to obtain. This would make the method accessible to all patients . Furthermore, the SLNB-MPI concept could be applied in principle in all solid tumors if the method has proved its effectiveness under real life conditions.
Nanoparticles are widely discussed as environmental toxins.Unfortunately, knowledge lacks concerning enrichment processes of SPIOs after injection in breast tissue, the adjacent lymphatic tissues and associated cells, especially in BC and metastatic lymph nodes. We already evaluated the distribution of SPIOs in an in vivo healthy and tumor mouse model . Based on these studies we further investigate the processing of the SPIOs in MP.
To our knowledge this is the first time three photon process was used to image superparamagnetic iron oxide nanoparticles in a bio-medical context. The small excitation spectrum as well as the wavelength range of the signal suggests third harmonic generation at the nanoparticle clusters, which can also be seen in the TEM images. Since single nano-particles do not generate a detectable signal, clustering size seems to be directly connected to the conversion effciency.
More experiments are necessary to confirm THG at the nanoparticles. A strong indication might be the fluorescence lifetime of the signal. A frequency tripling would have no detectable lifetime different from the instrument response function of the lifetime measurement device.
The lifetime of the Resovist signal recorded at 1200nm excitation is as short as the IRF of the setup recorded with SHG measurements with Urea cristals excited with 920 nm excitation. This is a strong indication that Resovist particle clusters are a source of THG. Similarly short lifetimes can be recorded with the fluorescence of gold nanoparticles. In contrast to those the excitation and emission spectra of the SPIOs have a much smaller FWHM.Gold nanoparticles can be excited with 740 nm to 1200 nm laser light and detected in the whole spectrum from 435 nm to above 560 nm. The SPIOs used can only be excited around 1200 nm and the spectrum of the signal can only be detected in the spectral channel below 435 nm.
This project is part of a comprehensive test program to develop a new SLNB technique. This might be less complex and incriminating for the patient and the staff, especially if the new MPI hand probe with unilateral solenoid arrangement will be ready for use in the operating theater [22, 27, 28].
The multiphoton based imaging of the SPIO clusters enables us to detect the path of the nanoparticles in histological slices as well as in vivo with a micro-meter resolution. In combination with the system wide scanning methods asMRT orMPI we are now able not only to identify hot spots of the surface modified particles but also to identify the linkage to subcellular handling and localisation of the iron nano-particles. In combination with the broad spectrum of other molecular biological methods the method of multiphoton based imaging will help to further investigate SPIO processing in cells and in the whole body. Since, SPIOs seems not to be toxic to our cells, we conclude that there will not be unexpected toxic effects in the whole organism. But, we are now encouraged to bring the proof for this hypothesis in further experiments.
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animals use.
This work was supported by the German Federal Ministry of Education and Research (BMBF) under Grant 01EZ0912 and by the University Research Program "Imaging of Disease Processes", University of Lübeck.
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