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Label-free discrimination of immune cells by multiphoton microscopy (#314)
O. - M. Thoma1, 2, B. Carlé3, S. Schürmann3, M. J. Waldner1, 2
1 Department of Medicine 1, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Bavaria, Germany
Multiphoton microscopy has emerged as a valuable microscopic technique for in vivo evaluation of pathological processes. Lately, it has been utilized to investigate cell dynamics, for a better understanding of immune responses in inflammatory diseases or cancer as well. However, most applications require different labeling molecules, such as fluorescent-antibodies, to identify individual types of immune cells. As multiphoton microscopy provides specific information on cell autofluorescence, the identification of immune cell subtypes without additional labelling procedures could be possible.
Our work was focused on discriminating different immune cell subtypes by using label-free multiphoton microscopy. Adaptive (T and B) and innate (macrophages, neutrophils and dendritic) immune cells were isolated from the spleen of healthy mice and then analyzed with multiphoton microscopy at an excitation wavelength of 750 nm. The immune cells were then characterized by investigating NAD(P)H (450-500 nm) and flavin (500-580 nm) fluorescence signals and by calculating the redox ratio (NAD(P)H signal intensity / flavin signal intensity).
Since the immune cells can be differentiated by their specific markers, we expected different fluorescence signals in the NAD(P)H and flavin channels as well. The mean fluorescence intensity of flavins was not significant when comparing different immune cell subtypes. However, the investigation of NAD(P)H signals has revealed that adaptive immune cells have a lower fluorescence intensity than most of the tested innate immune cells. Furthermore, when redox ratio was calculated, significant differences among neutrophils/macrophages and B cells/CD8a+ T cells were noticeable (p<0.05), proposing that a discrimination between adaptive and innate immunity by multiphoton microscopy is possible.
This preliminary study shows the great potential of using label-free multiphoton microscopy in identifying and discriminating between individual immune cell subtypes. Further studies would focus on detecting, analyzing and characterizing specific cells in mixed cell cultures, and later, on mucosal surfaces.
1. Martin Lee, Alan Serrels, Methods Mol. Biol., 2016;1467:105-18
2. David R. Miller, Jeremy W. Jarrett, Ahmed M. Hassan, Andrew K. Dunn, Current Opinion in Biomedical Engineering (2017)
3. Takaharu Okada, Sonoko Takahashi, Azusa Ishida, Harumichi Ishigame, Pflugers Arch - Eur J Physiol (2016) 468:1793–1801
Maximilian J. Waldner and Oana-Maria Thoma gratefully acknowledge funding of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG) in the framework of the German excellence initiative.
Simultaneous detection of multiple immune cell populations in human tonsil tissue sections using Imaging Mass Cytometry (#523)
M. Durand1, E. Segura1, T. Walter1, N. Talaei2, T. Vassilevskaia2, O. Ornatsky2
1 Institut Curie, PSL Research University, INSERM, U932, Paris, France
A novel approach for the detection of multiple immune cell types in a single tonsil section is described. Imaging Mass Cytometry™ (IMC™) is an emerging technology that combines single-cell analysis with the selectivity of mass spectrometry. IMC provides simultaneous detection of more than 40 markers in frozen or formalin-fixed, paraffin-embedded (FFPE) tissue sections at the single-cell level with 1 µm resolution. IMC uses metal-tagged antibodies (mAbs), a pulsed UV laser for tissue ablation, and a stream of inert gas to transfer material generated by the laser to the mass cytometer (1,2,3).
Frozen tonsil tissue sections (thickness 5 µm) were prepared as described (4).Tissue sections were fixed in 4% formaldehyde and stained according to the published IMC protocol using a mixture of antibodies (5). Workflow was similar to conventional immunohistochemistry (IHC) with the use of mAbs. The technique allows identification of immune cells within the tissue and their proximity to each other and stromal elements, and it can be used to measure expressed markers and the relative abundance of different cell types. The samples were inserted into the ablation chamber of the Hyperion™ Imaging System. CyTOF® Software v6.7 and MCD™ Viewer 1.0 were used for co-registering all the markers in high-dimensional space and processing the data into computer-generated three-color images.
A single formalin-fixed frozen human tonsil tissue section was probed with a mixture of 20 mAbs. IMC analysis revealed multiple cell types present in the same region. Tonsil tissue architecture was consistent with previous observations of large B cell follicles and inter-follicular T cell zones. E-cadherin and Alpha Smooth Muscle actin mAbs delineated clearly tonsil crypts and blood vessels. Combination of CD19 and Ki67 markers indicated the B cell follicle with proliferating B cells. Specific immune cell populations were identified. T follicular helper (Tfh) cells were located using CD3, PD-1 and Bcl-6 markers simultaneously. Monocytes/macrophages were identified using a combination of CD11c, CD45, and CD14 markers. Analysis of the distribution of these two types of immune cells in situ revealed that tonsil macrophages are located adjacent to Tfh cells. Demonstration of Tfh cell and macrophage population proximity is relevant for unraveling the process of human Tfh differentiation.
IMC technology is a versatile system for multi-parametric tissue analysis. It allows to simultaneously determine spatial distribution of more than 40 targets in a single tissue section with light microscopy resolution. Panel of 20 mAbs demonstrated the capacity of IMC to visualize multiple immune cell populations. Spatial relation of Tfh cell and macrophage populations was analyzed in a histological context of human non-pathological tonsil.
1. Giesen, C., Wang, H.A., Schapiro, D., Zivanovic, N., Jacobs, A., Hattendorf, B., Schuffler, P.J., Grolimund, D., Buhmann, J.M., Brandt, S. et al. (2014). Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods, 11, 417–422.
2. Chang, Q., Ornatsky, O.I., Siddiqui, I., Straus, R., Baranov, V.I., and Hedley, D.W. (2016). Biodistribution of cisplatin revealed by imaging mass cytometry identifies extensive collagen binding in tumor and normal tissues. Sci Rep, 6, 36641.
3. Chang, Q., Ornatsky, O.I., Siddiqui, I., Loboda, A., Baranov, V.I., and Hedley, D.W. (2017). Imaging Mass Cytometry. Cytometry A, 91, 160–169.
4. Segura, E., Durand, M., Amigorena, S. (2013). Similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ-resident dendritic cells. J Exp Med, 210(5), 1035–1047.
5. Chang, Q., Ornatsky, O., and Hedley, D. (2017). Staining of frozen and formalin-fixed, paraffin-embedded tissues with metal-labeled antibodies for Imaging Mass Cytometry analysis. Curr Protoc Cytom, 82, 12.47.1–12.47.8.
Projection Tomography in the NIR-IIa window: potential advantages of using reduced scattering wavelenghts (#366)
A. Marcos Vidal1, D. Ancora2, Z. Giannis2, J. J. Vaquero1, J. Ripoll1
1 Universidad Carlos III de Madrid, Biomedical Imaging and Instrumentation Group, Leganés, Madrid, Spain
Optical Projection Tomography (OPT)1,2,3is a powerful tool to retrieve 3D structural information using light attenuation as a contrast source. However, scattering limits its resolution because light loses its original directionality within mm4. The Near-Infrared (NIR) II-a window comprises wavelengths between 1300 and 1400 nm5 and has promising advantages in the interaction with biological tissues such as reduced scattering, lower autofluorescence and deeper penetration. This allows to image thicker samples without clearing than visible or I NIR window illumination.
We built a simple NIR OPT scanning system with a 1342 nm and 1064 nm lasers expanded through a lens telescope and directed into the sample. A NIR camera with a 100mm f2.8 camera captures the transmitted light, acquiring attenuation projection images. Both lasers had polarized beams, which allows to remove reflections and background illumination with a filter in the lens.
The laser sources can illuminate alternatively acquiring a projection for each wavelength. The sample, mounted in a rotating stage, is turned until full rotation has been completed. Further, the dataset of projections is preprocessed from detector dark currents and reconstructed using filtered backprojection.
The system is fully controlled by LABVIEW and all the processing was implemented in MATLAB.
We have scanned and reconstructed a small anchovy (50mm length, 10mm diameter) with both wavelengths using the described setup. Reconstructed slices show an increment in the contrast for the images at 1342 nm as expected (Figure 1, 2). In fact, there are some structures invisible to the I NIR window that were revealed in the II
The sample was scanned in air with 360 projections, averaging 6 frames with 50ms of exposure for every image. The reconstructed volume had a voxel size of 100 microns.
The inner structures of the sample in the images show and increment in the contrast, especially in the areas with hemoglobin, muscle, fat or skin where the potential benefits of the reduced scattering properties of the NIR II-a are exploited. However, the size of the sample still implies a large loss of directionality of photons. Using fluorescence or moving to smaller samples would be a very interesting possibility for further studies.
In this work we explored the potential benefits of using wavelengths in the II NIR window for optical imaging. At this frequencies light propagates with much lower scattering than in the visible or I NIR regimes. This allowed to reveal structures in a sample that were invisible at smaller wavelengths. Nevertheless, the diffusive behavior of light still blurs the reconstructions.
Further steps would comprise the study of the behavior of II NIR in fluorescence for 3D imaging and the use of smaller samples in transmission.
 Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sørensen, J., Baldock, R. and Davidson, D., “Optical projection tomography as a tool for 3D microscopy and gene expression studies.,” Science 296(5567), 541–545 (2002).
 Arranz, A., Dong, D., Zhu, S., Rudin, M., Tsatsanis, C., Tian, J. and Ripoll, J., “Helical optical projection tomography.,” Opt. Express 21(22), 25912–25925 (2013).
 Rieckher, M., Birk, U. J., Meyer, H., Ripoll, J. and Tavernarakis, N., “Microscopic optical projection tomography in vivo,” PLoS One 6(4), 2–7 (2011).
 Ntziachristos, V., “Going deeper than microscopy: the optical imaging frontier in biology,” Nat Meth 7(8), 603–614 (2010).
 Smith, A. M., Mancini, M. C. and Nie, S., “Second window for in vivo imaging,” Nat. Nanotechnol. 4(11), 710–711 (2009).
The research leading to these results has received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement n°115337, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution.
Keywords: NIR, OPT, near-infrared, scattering
Intracellular Clustering of Plaque-Targeting Super-Paramagnetic Iron Oxide Nanoparticles in Macrophages Switches on Backscattering and Increases Signal in Optical Coherence Tomography (#454)
E. Schellenberger1, A. Ariza de Schellenberger1, W. Poller2
1 Charité - Universitätsmedizin Berlin, Radiology, Berlin, Berlin, Germany
Recently we have shown that accumulation and clustering of Very small iron oxide particles (VSOP) in phagolysosomes of endothelial cells and macrophages allows high quality imaging of atherosclerotic plaques by MRI in less than an hour (1, 2) whereas sufficient uptake of Ferumoxytol in plaques was demonstrated to take more than two days (3). Optical coherence tomography (OCT) is an intravascular, high resolution imaging technique that can be used to characterize atherosclerotic plaques but, the identification of macrophages as important markers of active inflammation, remains difficult.
RAW264.7 macrophage cells were incubated with VSOP (1 mM Fe) that have been in clinical testing (4, 5) and Ferumoxytol (8.9 mM Fe) that is clinically approved for iron deficiency treatment. The scattering light intensity of control macrophages, nanoparticle-labelled macrophages, and nanoparticle suspensions were measured as wavelength scan in synchronous scan mode using a fluorescence spectrophotometer.
Cell pellets of 8,000,000 non-labelled, VSOP-labelled and ferumoxytol-labelled RAW264.7 cells were imaged by OCT (St. Jude Medical) and analyzed with the manufacturers software.
The uptake of VSOP (8.8 pg Fe / cell) increased the backscattering signal of the macrophage cells about 2.5 fold depending of the wave length and for Ferumoxytol (6.8 pg Fe / cell) about 1.5 fold. In contrast the backscattering of both nanoparticles in similar concentrations in suspension is negligible. This increased backscattering signal together with an increased signal attenuation can be visualized by a conventional clinical OCT system (Fig.) although the OCT is not yet optimized for the particle properties.
We conclude that VSOP and to lesser extend Ferumoxytol can serve as a multimodal imaging probe for MRI and OCT and could help to identify macrophages by OCT imaging in the future.
1. Scharlach C, Kratz H, Wiekhorst F, et al. Synthesis of acid-stabilized iron oxide nanoparticles and comparison for targeting atherosclerotic plaques: evaluation by MRI, quantitative MPS, and TEM alternative to ambiguous Prussian blue iron staining. Nanomedicine. 2015;11(5):1085-95.
2. Scharlach C, Warmuth C, Schellenberger E. Determination of blood circulation times of superparamagnetic iron oxide nanoparticles by T2* relaxometry using ultrashort echo time (UTE) MRI. Magn Reson Imaging. 2015.
3. Herborn CU, Vogt FM, Lauenstein TC, et al. Magnetic resonance imaging of experimental atherosclerotic plaque: comparison of two ultrasmall superparamagnetic particles of iron oxide. J Magn Reson Imaging. 2006;24(2):388-93.
4. Taupitz M, Wagner S, Schnorr J, et al. Phase I Clinical Evaluation of Citrate-coated Monocrystalline Very Small Superparamagnetic Iron Oxide Particles as a New Contrast Medium for Magnetic Resonance Imaging. Invest Radiol. 2004;39(7):394-405.
5. Wagner M, Wagner S, Schnorr J, et al. Coronary MR angiography using citrate-coated very small superparamagnetic iron oxide particles as blood-pool contrast agent: Initial experience in humans. J Magn Reson Imaging. 2011;34(4):816-23.
Keywords: OCT, iron oxide nanoparticles, MRI, macrophages
Simultaneous In Vivo Imaging of Adipose-derived Stromal Cells and Vasculature in Rodent Hindlimbs (#369)
R. Schweizer1, J. A. Plock1, P. Seebeck2, E. Ly3
1 University Hospital Zurich, Division of Plastic Surgery and Hand Surgery, Zurich, Switzerland
Vascularized composite allotransplantation (VCA) is a clinical reality and refers to transplantation of functional units (e.g. a hand, a face) composed of multiple tissues (e.g. skin, muscle, nerve, vessel) for restoration of major tissue defects. Here we aimed at investigating the potential of probe-based confocal laser endomicroscopy for in vivo tracking of adipose-derived stromal cells (ASCs) in rodent hindlimbs, to evaluate its future application in rodent hindlimb transplantation research.
ASCs were isolated from Brown Norway rats and cultured to passage 3, then labelled with CellVue Claret dye and subcutaneously injected into the groin of C57/BL6 mice. FITC-Dextran was injected intravenously. Probe-based confocal laser endomicroscopy (pCLE, Cellvizio Dual Band, Mauna Kea Technologies) was performed on the animal’s hindlimb through a small incision to visualize the cells and hindlimb vasculature.
The injected ASCs (red) could be clearly imaged next to vasculature and the hindlimbs vessels could be visualized differentiating between arteries, arterioles, capillary beds and collecting venules (green)(Figure 1). “Indian piles” of erythrocytes could be identified flowing through the capillaries, depending on the mounted confocal microprobe and its associated spatial resolution.
The findings confirm the possibility to track ASCs and simultaneously assess tissue perfusion or angiogenesis in rodent hindlimbs using pCLE. This tool will be useful for a variety of applications in basic science of VCA. Beside monitoring of homing and proliferation of injected cells and assessment of hindlimb perfusion, additional applications include monitoring of nerve regeneration and quantitative assessment of cellular infiltrates during rejection of VCA, among others.
Keywords: vascularized composite allostransplantation, probe-based confocal laser endomicroscopy, adipose-derived stromal cells
Development of a new fluorescence molecular tomography/magnetic resonance imaging hybrid system: from static mode to dynamic mode (#375)
W. Ren1, H. Chen1, M. Rudin1
1 University and ETH Zurich, Institute of Biomedical Engineering, Zurich, Zürich, Switzerland
A hybrid Fluorescence molecular tomography (FMT)/MRI system has been implemented and feasibility has been demonstrated for biomedical applications. One drawback of the system is the poor temporal resolution. A typical FMT measurement takes 5 minutes, which limits its applicability for studying transient processes such as characterizing the tumor neovasculature. In this project, we explored the method to speed up FMT for studying dynamic processes by simulation and evaluated predictions by experiments using tissue phantoms.
Concentration variation was introduced into the reconstruction algorithm for dynamic FMT. Linear approximation was used in building up the weighting matrix after data acquisition. In the simulation, a bolus of the size of one voxel at the depth of 3 mm below the phantom surface was assumed to move at various speeds ranging from 0.1 to 5 mm/s. Dynamic reconstruction was carried out for different speed settings. In the phantom study, a glass capillary containing the fluorescent dye was inserted into a silicon phantom. The capillary was connected with a micropump (Harvard Apparatus, US) using a plastic tubing. Multiple loops of illumination and data acquisition were triggered at the time when the bolus entered the capillary. Finally, the dataset was compared with the simulation result.
In the current configuration, the simulation showed that a moving bolus with a speed of 2.5 mm/s can be captured by a linear model integrated reconstruction method. In contrast, the conventional reconstruction method can only depict the speed of 0.5 mm/s. First phantom experiments revealed a good correspondence to the simulation result. However, Involving k(r, t) in the reconstruction algorithm also increases computational load by a factor of m+1, in which m equals the number of illumination cycles. In order to draw more general conclusion about the relationship among the field of view, voxel size, illumination duration and bolus speed, more simulations and refined phantom experiments have to be carried out.
Both simulation and phantom studies demonstrated the feasibility to integrate the dynamic FMT mode into the hybrid system. Although the reconstruction algorithm has to be refined and speeded up, the functional extension by adding dynamic measurement will broaden its application to dynamic problems. The hybrid design of the setup will enable validating dynamic FMT by comparison with simultaneous dynamic MRI data acquisition.
 Ren, W., Elmer, A., Buehlmann, D., Augath, M.-A., Vats, D., Ripoll, J., & Rudin, M. (2016). Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging. Molecular Imaging and Biology, 18(2), 191-200. doi:10.1007/s11307-015-0884-y
 Leblond, F., Davis, S. C., Valdes, P. A., & Pogue, B. W. (2010). Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications. Journal of Photochemistry and Photobiology B-Biology, 98(1), 77-94.
 Stuker, F., Hybrid Imaging: Combining Fluorescence Molecular Tomography with Magnetic Resonance. 2011, ETH Zurich: Zurich, Switzerland.
Experimental setup for dynamic FMT
(a) A capillary was inserted into the silicon phantom and connected to plastic tubes on both sides. One side lead to the exit and the other connected to the micro-pump. The velocity of the bolus was determined by the pumping rate of the pump (c). (b) Detector array was located above the phantom. Laser position was controlled by pre-programmed software.
Keywords: Fluorescence molecular tomography, Image reconstruction, MRI, Dynamic imaging
Early diagnosis of triple-negative breast cancer using multiphoton fluorescence imaging, and resonance Raman spectroscopy. (#489)
L. Shi1, 2, E. Bendau1, J. Smith1, E. Ackerstaff4, L. Zhang-Chen1, J. Koutcher4, R. Alfano3
1 City College of New York, Biology and Institute for Ultrafast Spectroscopy and Lasers, New York, New York, United States of America
Triple-negative breast cancer (TNBC) is characterized as estrogen receptor (ER) negative, progesterone receptor (PR) negative, and lacking overexpression of human epidermal growth factor receptor 2 (HER2). TNBC has poor prognosis relative to other breast cancer subtypes and thus early detection is crucial for improved patient outcomes.Therefore, the objective of this study is to use in vivo MPM and ex vivo RRS as novel and non-invasive techniques for early detection of TNBC.
Mice were injected with 4T1 (TNBC) and 67NR (non-malignant) cell lines and tumors were grown to around 150mm. Magnetic resonance imaging was used to gather information on the lactic acid content of the tumors. MPM imaging was performed in vivo. Native fluorescence of three key biomolecules, flavin, NADH, and collagen, were probed by two-photon excitation of the tumor at 800nm, 700nm, and 920nm, respectively. The tumors were then immediately excised for ex vivo analysis by RRS. The samples were excited at 532nm at multiple locations on each tumor to obtain the spectral fingerprint from the Raman frequency shift of the molecules.
MPM images revealed morphological differences between the 4T1 and 67NR tumor cells. Collagen signals from 4T1 tumors were concentrated near the surface of the tumors, with lower fiber density in the inner tumoral volume. 67NR tumors exhibited dense, curly collagen fibers with a higher degree of gross alignment that were not found in 4T1 tumors, in which fibers were less densely packed, more disorganized, and straighter or exhibiting only slight long range curvature. Cell density in 4T1 tumors was much higher than in 67NR. Comparison of peak ratios from RRS measurements between these two tumor types revealed key differences in spectral structure. Ratios of Raman signal intensity to that of the normalization peak at 1004cm-1 was higher than those of 67NR (peaks around 470cm-1), while they were lower for 750cm-1 and a band from around 675cm-1-720cm-1
This study used multiphoton fluorescence microscopy (MPM) and resonance Raman spectroscopy (RRS) as a non-invasive and label-free means of cancer diagnosis by detecting unique spectral fingerprints that are associated with different tissue structures and metabolic activity.
We thank grant support from NIH 5 U54 CA137788-10 and P30 CA008748 (MSKCC Support Grant)
Keywords: Triple Negative breast cancer, resonance Raman scattering, multiphoton fluorescence imaging
In vivo characterisation of joint bleeds and visualisation of blood-induced bone pathology in Haemophilia A mice using fluorescence molecular tomography and micro-CT (#217)
K. K. Vøls1, 2, M. Kjelgaard-Hansen2, C. D. Ley2, A. K. Hansen1, M. Petersen2
1 University of Copenhagen, Department of Animal and Veterinary Sciences, Frederiksberg, Denmark
Haemophilia A is a congenital bleeding disorder caused by lack of coagulation factor eight (FVIII) activity. Patients suffer from spontaneous joint bleeds that over time may lead to haemophilic arthropathy1. We wish to implement in vivo imaging to describe active joint bleeds in haemophilia A mice. Fluorescence molecular tomography (FMT) is an in vivo quantitative optical imaging modality that may prove useful for this purpose. Aim: Establish whether FMT imaging can describe induced joint bleeds in haemophilia A mice. Characterise development of bone pathology with μCT.
FVIII-knockout (F8-KO, n=30) and wildtype (WT, n=7) mice were dosed iv. with the fluorescent blood-pool agent AngioSense750 (Perkin Elmer). The mice were then anaesthetised, dosed with analgesia and received an induced knee bleed (left knee). Analgesia was provided via drinking water throughout the study. At t=6min, 6h, 12h, 24h and 48h the mice were imaged with an FMT-scanner (FMT1500, Perkin Elmer) to characterise the induced knee bleed longitudinally by using the fluorescent signal as a surrogate marker for blood. The relative fluorescence (ΔFMT) in the injured knee was quantified using the contralateral knee as control (Fig.1). At day 14, the mice were euthanized and the knee joints were μCT-scanned ex vivo (Quantum FX, Perkin Elmer). The μCT-scans were scored (0-5) for bone pathology.
The average ΔFMT reached max in F8-KO mice 24h after the induced knee bleed, and 12h after the induced knee bleed in WT-mice. F8-KO-mice had a significantly increased ΔFMT compared to WT-mice at 6h, 12h, 24h, and 48h after the induced knee bleed. These results indicate that we can use FMT-imaging to quantitate how the induced knee bleeds develop over time in individual mice. None of the WT-mice developed μCT-detectable bone pathology after the induced knee bleed, whereas half the F8-KO mice had a positive μCT-score, ranging from 1-5.
We can characterise active joint bleeds using optical in vivo FMT imaging with the fluorescent blood-pool agent AngioSense750. Based on the FMT-response in the knee region after induced knee bleed, we can distinguish between F8-KO and WT-mice. F8-KO mice had a significantly higher incidence of μCT-detectable bone pathology than WT mice.
1Srivastava, A., et al. Guidelines for the management of haemophilia. Haemophilia, 2013. 19(1):p e1-47
This project is sponsored by the LIFE in vivo pharmacology centre, University of Copenhagen and Novo Nordisk A/S.
Fig 1: In vivo fluorescent imaging of active knee bleed and ex vivo micro-CT scan of knee joints
A: FMT-image of the lower body of a haemophilia A mouse 24h after induced joint bleed in the left knee (mark: Injured). The bar indicates the intensity of the fluorescent signal. B: Ex vivo μCT scan of the injured knee two weeks after injury. C: Ex vivo scan of the contralateral knee two weeks after injury.
Keywords: optical imaging, micro-CT, in vivo, haemophilia, rodent model
Multimodal Bismuth Ferrite second harmonic nanoparticles for pulmonary macrophage tracking (#499)
F. Ramos-Gomes1, D. Rytz2, W. Moebius3, L. Bonacina4, F. Alves1, 5, A. M. Markus1
1 Max-Planck Institute for Experimental Medicine, Translational Molecular Imaging Group, Goettingen, Lower Saxony, Germany
Nanoparticles (NPs) increasingly govern the development of novel therapeutics and diagnostic enhancers. More recently, second harmonic generation (SHG) nanomaterials have been generated that can be efficiently employed in the near infrared window (NIR-II) using a multiphoton microscopy and which have several inherent advantages: (i) higher penetration depth; (ii) they do not bleach or blink and (iii) no need of cell labelling to be detected1,2. Our aim was to test the use BFO-NPs for monitoring pulmonary macrophages tracking in a mouse model of allergic airway inflammation (AAI).
BFO-NPs or BFO-loaded macrophages (MH-S) were given intranasally (i.n.) or intratracheally (i.t.) to Balb/c mice with AAI. 24h after NP application the lungs were lavaged and/or harvested, embedded in agarose and thick 50-100 µm sections were prepared. Images were performed with a Two-Photon-Laser Scanning Microscope (2P-LSM).
We show that bright BFO-NP signals were detected by 2P-LSM, from superficial cells as well as from deep within the lung tissue. Using a narrow emission filter, BFO-NPs could be identified in the lung with an excellent signal to noise ratio (SNR) and virtually no background signal. The SHG from the nanocrystals could be distinguished from the endogenous SHG from lung collagen around the blood vessels. We found the BFO-NPs were primarily taken up by alveolar macrophages positive for ECF-L marker, providing information about their spatial distribution and interaction. BFO-NPs could thus provide additional knowledge about the role of macrophages in pulmonary inflammation, which is still controversially discussed.
We show that BFO-loaded macrophages in a model of AAI were easily imaged by their SHG signals emission in the context of the surrounding lung tissue. This method could provide novel information about the interaction of macrophages with cells and extracellular matrix in lung disease.
1 Rogov, A. et al. Simultaneous Multiharmonic Imaging of Nanoparticles in Tissues for Increased Selectivity. ACS Photonics 2, 1416-1422, doi:10.1021/acsphotonics.5b00289 (2015).
2 Bonacina, L. Nonlinear nanomedecine: harmonic nanoparticles toward targeted diagnosis and therapy. Molecular pharmaceutics 10, 783-792, doi:10.1021/mp300523e (2013).
We thank Hanna Puchala, Sarah Garbode and Bärbel Heidrich for excellent technical assistance.
BFO-NPs are phagocytized by endogenous alveolar macrophages in mouse lung tissue.
(A) Shows an overview of the staining for macrophages marker ECF-L (green signal), SHG signal emitted from collagen and from BFO-NPs (blue signal) and nuclear marker DRAQ5 (red signal) at the lung tissue sections. The colocalization of i.n. instilled BFO-NPs (blue signal) with macrophage marker ECF-L (green signal) can be seen at the higher magnified picture on (B).
Keywords: second harmonic generation (SHG), 2-Photon microscopy, deep tissue imaging
Detection of Dysplasia and Early-stage Esophageal Adenocarcinoma Using Optical Molecular Imaging with Fluorescently-Labeled Wheat Germ Agglutinin Ex Vivo (#493)
S. J. de Jongh1, A. Johnson2, J. Baeten2, G. M. van Dam3, W. B. Nagengast1
1 University Medical Center Groningen, Gastroenterology and Hepatology, Groningen, Netherlands
Early detection of dysplastic and T1 adenocarcinoma lesions in Barrett’s Esophagus (BE) is crucial for obtaining long-term survival. Patients with BE currently undergo surveillance endoscopies every 3-5 years with random four-quadrant biopsies every 1-2 centimeters. However, this poses a high risk of sampling-error, resulting in a detection miss-rate of up to 57% . In this study, we evaluated the feasibility of optical molecular imaging using the fluorescently labeled lectin ‘wheat germ agglutinin’ (WGA) as a red flag-technique to detect (pre)cancerous lesions in BE ex vivo.
Patients with BE scheduled for endoscopic mucosal resection (EMR) were included. Acetylcysteine was used as an anti-mucolytic prior to topically applying WGA labelled to AlexaFluor-680 (WGA-AF680, 500 µg/ml) on fresh EMR-specimens directly after endoscopy. Fluorescence imaging was performed prior to and one minute after spraying using the IVIS Spectrum. Fluorescence intensities on EMR-specimens and subsequent bread-loaf slices were correlated with histology ex vivo.
So far, five patients were included which yielded 18 EMR-specimens for fluorescence analysis. In contrast to a previous study, WGA-AF680 binding patterns overall positively correlated with histopathological diagnosis . As the degree of dysplasia progressed, increased mean fluorescence intensities (MFI) were observed. High-grade dysplastic and cancer lesions could very well be discriminated from normal squamous epithelium (Figure 1).
We demonstrate the feasibility of optical molecular imaging for detection of dysplasia and early-stage carcinoma in BE using topically applied WGA-AF680 in an ex vivo setting. However, in vivo results are needed to confirm our findings. Therefore, we plan to perform fluorescence molecular endoscopy using topically applied WGA-AF680 on a freshly resected esophageal adenocarcinoma specimen as a proof-of-concept for the design of a first-in-human in vivo study.
 Vieth M, Ell C, Gossner L, May A, Stolte M. Histological Analysis of Endoscopic Resection Specimens From 326 Patients with Barrett’s Esophagus and Early Neoplasia. Endoscopy. © Georg Thieme Verlag Stuttgart · New York; 2004 Sep;36(9):776–81.
 Bird-Lieberman EL, Neves AEA, Lao-Sirieix P, O'Donovan M, Novelli M, Lovat LB, et al. Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus. Nature Medicine. Nature Publishing Group; 2012 Jan 15;18(2):317–23.
Figure 1. WGA-AF680 binding in two EMR specimens within one patient.
Increased fluorescence intensities were observed in a focal lesion containing high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC, EMR-specimen I), whereas low fluorescence intensities were observed in EMR-specimen II, containing mainly low-grade dysplasia and/or squamous and gastric epithelium. Argon plasma coagulation marks are indicated with an arrow.
Keywords: Barrett's Esophagus, Optical Molecular Imaging, Wheat Germ Agglutinin, WGA-AF680
Real Time Laser Speckle Imaging Monitoring Vascular Targeted Photodynamic Therapy (#321)
R. Goldschmidt1, V. Kalchenko2, A. Scherz1
1 Weizmann Institute of Science, Plant Sciences, Rehovot, Israel
Laser Speckle Imaging is a technique that has been developed for monitoring non-invasively in vivo blood flow dynamics and vascular structure with high spatial and temporal resolution.1 There are almost no reports on Real Time Laser Speckle Imaging (RTLSI) during photodynamic therapy (PDT) with real time processing because of light interferences.2 We have set up a system that can be used to monitor online the photodynamic effect on the tumoral blood flow during vascular targeted PDT (VTP). Online treatment monitoring enables a higher selectivity and control of the blood vessel occlusion.
Our home build real time laser speckle imaging system comprises a CCD camera, standard optical lens (focused on a field of view (FOV)) and optical bandpass filter that filters out the PDT illumination wavelength. We developed a special RTLSI module, based on LABVIEW software for data processing. It enables to acquire raw laser speckle images and calculate the value of the laser temporal statistics of time-integrated speckles (at a real time). The RTLSI module was adjusted to acquire continuously sets of 60 raw laser speckle images, then to convert them into a single processed temporal laser speckle contrast image, and finally produces images for display. We aimed to monitor the blood flow effect of VTP on a mice tumor model in real time using the RTLSI new system.
Monitoring vascular shutdown as a marker of response, in particular, live imaging of blood obstruction, could potentially serve as a means for determining the end of treatment and reduce potential damage to the healthy surrounding tissues, as well as a tool to study the basis of VTP mechanisms.3
Application of RTLSI for on-line VTP monitoring, enabled us to observe the immediate effect of the treatment on the blood flow within and around the tumor. Upon application of a moderate VTP regimen, an immediate reduction in blood flow within the tumor, and further in the main tumor’s arteries and draining veins were observed. By the end of treatment, the blood flow within the tumor stopped completely, while the flow in the surrounding blood vessels recovered to some extent. By 24h post-treatment, the tumor blood vessels were completely arrested. Our data were confirmed by angiography (using a fluorescent contrast agent) and histology.
RTLSI presents a low-cost, non-invasive, on-line and selective means of monitoring VTP. It offers an immediate, qualitative and live record of the tumor blood flow dynamics during treatment. More specifically, it could potentially serve as a tool to predict successful VTP treatment, and to adjust treatment parameters, such as light intensity and timing. In doing so, undesirable damage to healthy tissues can be avoided, while ensuring treatment success. Such features are of highest importance, particularly when combining the modality with adjuvant therapies.
1. Briers, D., et al. (2013). "Laser speckle contrast imaging: theoretical and practical limitations." Journal of Biomedical Optics 18(6)
2. Chen, B., et al. (2003). "Blood flow dynamics after Photodynamic therapy with Verteporfin in the RIF-1 tumor." Radiation Research 160(4)
3. Yu, G., et al. (2005). "Noninvasive Monitoring of Murine Tumor Blood Flow During and After Photodynamic Therapy Provides Early Assessment of Therapeutic Efficacy." Clinical Cancer Research 11(9)
The authors would like to thank Viktoria Sergeyev for preparing the histological slides, and Dr. Natasha Kudinova and Keren Sasson for their help with the animal handling. A. S. is the incumbent of the Robert and Yaddle Sklare Professorial Chair in Biochemistry. This work was supported by a grant from the Wade Thompson Family Foundation.
(Right) Perfusion index map obtained during WST11-VTP of a CT26 tumor model in a CD1-nude mouse ear. Images were taken at t = 0, 1, 3 min of treatment, from left to right. Color bar corresponds to blood perfusion intensity and reciprocal to K - laser speckle contrast (a.u.). (Left) Plot of blood velocity changes over time in tumor vs normal blood vessels.
High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup (#241)
K. Prakash1, 2
1 Carnegie Institution for Science, Department of Embryology, Baltimore, Maryland, United States of America
Superresolution imaging techniques have pushed the natural limit to which two closely spaced objects can be resolved by a light microscope. However, over the years, these superresolution techniques have become more sophisticated, restricting their access and application to a few elite optics groups. A part of the problem comes from expensive microscopes, complicated hardware configurations and advanced technical expertise required to reconstruct and analyse the data.
We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and an incoherent light source. The technique termed as Omnipresent Localisation Microscopy (OLM) is an extension of Single Molecule Localisation Microscopy (SMLM) techniques and allows single molecules to be switched on and off, detected and localised. A short burst of deep blue excitation (350-380 nm, instead of 405 nm laser) can be used for a prolonged reactivation of molecules, once the blinking has slowed or stopped.
A resolution of 90 nm based on Fourier ring correlation is achieved on test specimens (mouse and amphibian meiotic chromosomes). Furthermore, using UV activation, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging medium.
It is hoped that the findings can democratise superresolution imaging and help any scientist to generate high-density superresolution images even with a limited budget. Finally, the new photophysical observations reported here should pave the way for more in-depth investigations of the mechanisms underlying the excitation, photobleaching and photoactivation of a fluorophore.
High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup.
bioRxiv 2017. doi: https://doi.org/10.1101/121061
K.P. acknowledges Joseph G. Gall for the financial support. K.P. further acknowledges Joseph G. Gall, Zehra Nizami, Safia Malki, Yixian Zheng and Mahmud Siddiqi for the biological samples, help with microscopy measurements and useful discussion. K.P. thanks David Fournier, Wioleta Dudka, Miguel Andrade-Navarro, Bassam Hajj, Maxime Dahan, Sandra Ritz, Judith Mine-Hattab and Russ Hodge for proofreading the manuscript.
Proof of on/off switching of single molecules induced by a Hg lamp
On the left side: (A-C) Three selected frames (3318, 6128 and 8467) out of 10000 frames are shown where a subset of molecules are 'on'.
On the right side: intensity profiles for three 7X7 (roughly 450X450 nm2) cross sections (yellow boxes in the images on the left) along the image stack are shown.
Correlative STED and SMLM
Synaptonemal complex (SC) proteins from mouse stained with Alexa 594. (A) The confocal image shows the entire complement of SC proteins. Note that the two halves of the SC that unite the meiotic chromosome pair are not resolved. (B) The same chromosome imaged with STED and (C) OLM. The two halves of the SC could be resolved with STED and OLM.
Keywords: Super-resolution microscopy, single molecule localisation microscopy, non-coherent illumination source, Mercury arc lamp, STED, UV activation, PALM, STORM
Intravital 2-photon microscopy in bone combined with whole body imaging for optimizing immunotherapy approaches (#380)
U. Geisen1, C. Desel1, O. Will1, T. Damm1, M. Peipp2, B. Brandt3, S. Tiwari1, C. - C. Glüer1
1 University Medical Center Schleswig-Holstein, Department of Radiology and Neuroradiology, Molecular Imaging North Competence Center, Kiel, Germany
Patients with bone metastases have poor prognosis in various cancer entities. Targeted antibodies and redirected immune cells are among the most promising therapy options in this field. For this, the interactions of antibodies, different immune cells and the tumor are of great interest. However, the composition of bone makes optical intravital imaging still challenging. In this multimodal approach, we combine 2-photon microscopy with µCT, FMT and bioluminescence detection. In a first approach, we analyze the role of Her2 which is overexpressed in various breast cancer bone metastases.
2-photon microscopy was performed at the proximal tibia, shortly below the growth plate. A minor surgical incision was made to place aside the skin and connective tissue. After applying a clearing agent to enhance fluorescent signal, a water immersion objective was used to obtain a homogenous refractive index between lens and specimen. Collagen structure of the bone was observed using the SHG signal. Breast cancer cells expressing Her2 were labelled with tdTomato or Quantum dots for 2-photon microscopy and were injected intratibially or intracardially. In addition to visualization of metastatic tumor cells in bone, methods were also established for the visualization of biomarkers associated with presence of blood vessels.
In our current study we developed an imaging platform which combines systemic imaging of the whole organism with imaging at a single cell level inside the bone. This allows us to observe specific targeting of Her2 antibody to tumor cells in the bone, while quantifying treatment efficacy at the whole organ or tissue level. Preliminary results show, that Her2 overexpressing breast cancer cells form osteolytic lesions inside the tibia, visualized by µCT 3 weeks after injection. Coregistration of longitudinal CT images show increasing bone loss due to the lesion. Intravital 2-photon time-lapse imaging allows the visualization of tdTomato tumor cells together with loss of collagen structure by SHG and vasculature by dextran-FITC accumulation.
Treating bone metastases still remains a major challenge in cancer therapy. Our approach can provide further understanding in the development and effect of immunotargeted agents to the tumor microenvironment. It provides the opportunity to perform 4D imaging (3D and time) to monitor specificity, kinetics and efficacy of targeting agents in order to optimize therapeutic protocols.
Keywords: 2-Photon Microscopy, Intravital Microscopy, µCT, Cancer, Bone Metastases
Gadolinium deposition in human blood cells assessed by Mass cytometry (#460)
A. Ariza de Schellenberger1, 4, S. Baumgart2, A. Grützkau2, E. Schellenberger3, J. Braun4, I. Sack1
1 Charité - Universitätsmedizin, Radiology /Elastography, Berlin, Berlin, Germany
Concerns on the safety of GBCA were raised a decade ago, after Gd was detected in skin and organs from patients with impaired kidney function that developed nephrogenic systemic fibrosis (NSF)1. More recently, Gd was found in the cerebellum in patients with normal kidney function2,3. These post-mortem studies have alerted authorities to take precaution on GBCA administration4,5. Nevertheless, the causes of Gd accumulation and their toxicological effects in vivo remained to be explained.
We assessed the detection of Gd in cells of human blood samples by mass cytometry (CyTOF v1.5). Blood samples were incubated ex vivo for 1h at 37° with two macrocyclic (Gadoterate meglumine and Gadobutrol) and one linear GBCA (Gadopentate dimeglumine) at clinical relevant concentrations. 158Gd signal was monitored on Leucocyte subsets identified by lineage-specific, metal-tagged antibodies and total-Gd was quantified using CyTOF.
The 158Gd cell associated signal was consistently detected for all three GBCA, with the highest intensity on monocytes followed by NK, neutrophils, T, and B -cells. However, the Gd-cell subset association was independent of the GBCA-chemical structure. The average Gd content is in the femtogram (fg) range for the leucocyte cell-subsets.
This study proves the feasibility of CyTOF® to detect GBCA uptake by human blood cells and highlights the relevance of studying the distinctive affinity for GBCA for each leucocyte cells subset and their toxicological effects.
Mass cytometry technology is an excellent tool for quantitative, longitudinal analysis of blood samples at the single cell level from patients after GBCA administration in clinical routine.
1. Grobner T. Gadolinium – a specific trigger for the development of nephrogenic fibrosing dermopathy and nephrogenic systemic fibrosis? Nephrol Dial Transplant. 2006;21: 1104-08.
2. Kanda T, Oba H, Toyoda K, Kitajima K, Furui S. Brain gadolinium deposition after administration of gadolinium-based contrast agents. Jpn J Radiol. 2015;34: 3-9.
3. McDonald RJ, McDonald JS, Kallmes DF, Jentoft ME, Murray DL, Thielen KR, et al. Intracranial gadolinium deposition after contrast-enhanced MR imaging. Radiology. 2015;275: 772-782.
4. Roberts DR, Welsh CA, LeBel DP, Davis WC. Distribution map of gadolinium deposition within the cerebellum following GBCA administration. Neurology. 2017;88: 1206-08.
5. Runge VM. Critical questions regarding gadolinium deposition in the brain and body after injections of the gadolinium-based contrast agents, safety, and clinical recommendations in consideration of the ema's pharmacovigilance and risk assessment committee recommendation for suspension of the marketing authorizations for 4 linear agents. Invest Radiol. 2017; 52: 317-323.
Keywords: Gadolinium, Mass cytometry, MRI, human blood