Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
Fluorescence imaging to study in vivo accumulation and clearance of drug delivery nanoparticles (#583)
Oya Tagit1, Camilla M. Operti1, Yusuf Dolen1, Carl G. Figdor1
1 Radboudumc, Tumor Immunology, RIMLS, Nijmegen, Netherlands
Poly(lactic-co-glycolic acid) (PLGA)-based drug delivery systems are among the most commonly studied materials due to excellent biocompatibility, tuneable degradation characteristics and long clinical history of PLGA . The size and surface functionality of PLGA particles are the main parameters that dictate their fate in vivo. Fluorescence labeling and imaging prove simple, versatile and commonly available tools to track the PLGA particles in vivo . In this study, we monitor in vivo accumulation and clearance of intravenously-administered PLGA nanoparticles on a mouse model .
PLGA nanoparticles encapsulating VivoTag-S 750 were prepared utilizing a microfluidics system composed of a Y-junction cartridge (NanoAssemblr™, Precision Nanosystems Inc.) connected to syringe pumps (Harvard PHD-2000 infusion 70-200). Particle size was ‘tuned’ via varying the flow and formulation parameters. Colloidal characterization of particles was done using dynamic light scattering (DLS) (Nanotrac Flex, Microtrac) and atomic force microscopy (AFM) (Catalyst BioScope, Bruker). Dye-loaded PLGA nanoparticles were injected intravenously to wild-type BALB/cAnNCrl mice through a lateral tail vein. 0.5, 3, 24, and 48 hours after injections mice were shaved and imaged with an IVIS Lumina II (Perkin Elmer) system. Organs of euthanized mice were dissected and imaged separately at 24 and 48 h.
PLGA particles of varying sizes (<100 nm, ~200 nm and >1000 nm) were formed through nanoprecipitation upon controlled mixing of aqueous and organic phases in microfluidics channels. Increasing the PLGA concentration and flow rate of organic phase resulted in the formation of larger particles. DLS and AFM measurements showed a uniform particle size distribution for all particle types. For in vivo studies, particles with <100 nm and ~200 nm size were used to avoid possible accumulation of larger particles in the lung capillaries. Whole-body imaging revealed both particle types mainly in liver and bladder already after 0.5 h. The liver signal decreased gradually at later time points. The decay of liver signal was faster for ~200 nm particles, indicating a faster clearance. Ex-vivo imaging of isolated liver also showed similar variations in signal intensities, such that ~200 nm particles displayed a more pronounced decrease at 48 h compared to ~100 nm particles.
We demonstrated how PLGA particle size can be tuned using a microfluidics system via modulating the formulation and process parameters. We obtained dye-loaded PLGA particles in different sizes, which remarkably affected the particle characteristics in vivo. Tracking the particles through fluorescence imaging, we showed the direct relationship between the size and their pharmacokinetics behavior.
Authors acknowledge the financial support by EU grant PRECIOUS (686089).
 Operti, M.C., Fecher, D., van Dinther, E.A.W., Grimm, S., Jaber, R., Figdor, C.G., Tagit, O. A comparative assessment of continuous production techniques to generate sub-micron size PLGA particles. International Journal of Pharmaceutics 550 140-148 (2018)
 Swider, E.; Maharjan, S.; Houkens, K.; Srinivas, M.; Figdor, C. Tagit, O. Förster resonance energy transfer-based stability assessment of PLGA nanoparticles in vitro and in vivo. ACS Applied Bio Materials 2 1131-1140 (2019)
 Operti, M.C.; Dölen, Y.; Keulen, J.; van Dinther, E.A.W.; Figdor, C.; Tagit, O. Microfluidics-assisted size tuning and biological evaluation of PLGA particles. Pharmaceutics 11 590 (2019)
Tracking the in vivo clearance of PLGA nanoparticles through fluorescence imaging.
A) Fluorescent whole body images of mice obtained at different time points up to 48 h after i.v. administration of ~200 nm and ~100 nm PLGA particles. B) Variations of liver fluorescence in whole body images. C) Variations of liver fluorescence intensities of ~100 nm (blue) and ~200 nm (green) PLGA particles obtained on isolated liver at 24 h and 48 h after particle administration. Data obtained for untreated mice (negative control) are shown in black.
Keywords: PLGA, in vivo clearance, fluorescence imaging, in vivo imaging, drug delivery particles
Demonstrating multiple transport mean free path imaging capabilities of light sheet microscopy in quantifying fluorescence dynamics (#472)
Matthias Rieckher4, 1, Stylianos Psycharakis2, Daniele Ancora2, Evangelos Liapis2, Athanasios Zacharopoulos2, Jorge Ripoll3, Nektarios Tavernarakis4, Giannis Zacharakis2
1 University Hospital Cologne, Institute for Genome Stability in Ageing and Disease, Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases, Cologne, Germany
We demonstrate how LSFM can outperform conventional methods in two scenarios covering multiple transport-mean-free-path regimes from microscopy to mesoscopy. In the first case, LSFM outperforms confocal microscopy when imaging the GFP-tagged DAF-16/FOXO stress response factor in C. elegans. In the second scenario we demonstrate how the implementation of phase retrieval algorithms in three dimensions can radically improve the performance of LSFM when imaging optically opaque live samples in the form of cancer cell spheroids.
Caenorhabditis elegans young adult animals were maintained as previously described  and imaged with a Lightsheet Z.1 microscope (Carl Zeiss AG, Jena, Germany) and a LSM510 laser scanning microscope (Carl Zeiss AG, Jena, Germany). To image temperature-dependent nuclear recruitment of DAF-16::GFP the sample chamber was heated from RT to 30°C, while continuously imaging every 20 sec.
Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in C. elegans and the distribution of necrotic cells in cancer cell spheroids. On the one hand we have shown that LSFM can outperform conventional CSFM in imaging speed, 3D resolution, reduced photo-toxicity, accurate quantification and linear nuclear localization of the translocation of DAF-16::GFP protein upon stress induction in C. elegans resulting in a relatively higher resolved nuclearization dynamics. Furthermore, we present how 3D PRT can improve the depth-to-resolution-ratio and can extend the imaging abilities of LSFM into the mesoscopic regime when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in an optically opaque cancer cell spheroid (size ~200μm), and produce uniform resolution throughout the scattering volume . With these two studies we manage to cover both the microscopic (~1MFP) and mesoscopic (~1 TMFP) regimes of light transport.
We have studied two microscopic imaging scenarios corresponding to widely different regimes of light propagation and demonstrated how modern LSFM combined with novel computational methods can produce high-quality 3D images with advanced spatio-temporal resolution, accurate 3D quantification and precise characterization of fluorescence dynamics, proving its advance as new gold standard for fluorescence microscopy.
AcknowledgmentThis work was supported by the EU Marie Curie ITN “OILTEBIA” (PITN-GA-2012-317526), the H2020 LASERLAB Europe (EC-GA 654148) and the projects “BIOIMAGING-GR” (MIS5002755) and “HELLAS-CH” (MIS 5002735) funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020). We acknowledge F. Geisler, S. Kant and R. Windoffer from the Uniklinik RWTH Aaachen, Germany for access to the Lightsheet microscope Z.1 and technical assistance.
 S. Brenner, 1974, The genetics of Caenorhabditis elegans, Genetics 77, 71-94
 D. Ancora, et al., 2017 Phase-retrieved tomography enables mesoscopic imaging of opaque tumor spheroids, Scientific Reports 7, 11854
 M. Rieckher, et al, 2018, Demonstrating improved multiple transport‐mean‐free‐path imaging capabilities of light sheet microscopy in the quantification of fluorescence dynamics”, Biotechnology Journal 13, 1700419
Comparing LSFM with CSFM to measure the dynamics of DAF-16::GFP nuclearization in C. elegans. (A) Representative 3D images of transgenic C. elegans expressing DAF-16::GFP derived from LSFM and CSFM data. The animals were continuously imaged while being exposed to heat stress increasing from RT to 30°C. Arrows indicate intestinal nuclei. (B) Quantification of the fluorescent signal in intestinal nuclei (n=5) of the individual shown in 2A.
Keywords: Light Sheet Fluorescence Microscopy, Optical Projection Tomography, Phase Retrieved Tomography, Cancer cell spheroids
High resolution 3D imaging of live primary and secondary tumor spheroids using Light Sheet Fluorescence Microscopy (LSFM) (#394)
Stylianos Psycharakis1, 2, Athanasios Zacharopoulos1, Joseph Papamatheakis4, Evangelos Liapis3, Mariam-Eleni Oraiopoulou5, Vangelis Sakkalis5, Jorge Ripoll6, Giannis Zacharakis1
1 Foundation for Research and Technology - Hellas, Institute of Electronic Structure and Laser, Heraklion, Greece
Light Sheet Fluorescence Microscopy has evolved into a powerful tool in cell and developmental biology for high resolution three dimensional imaging of live or fixed tissues and organisms . In this work primary Glioblastoma and secondary MDA-MB-231 breast cancer cells are formed into spheroids and are treated with chemotherapeutic agents. The Multicellular Tumor Spheroids (MCTS) are then imaged with our custom multiangle multicolor LSF Microscope for the study of pre-clinical drug screening and drug efficiency evaluation.
Tumour spheroids are treated with different concentrations of the chemotherapeutic agents (Temozolomide, Doxorubicin, VS 5584) and stained appropriately in order to image cell viability and death (DRAQ7).
LSFM images reveal information about the penetration of the chemotherapeutic agent inside the tumor spheroids, as well as about its efficiency in a dose-response relationship .
In this work we present the use of our custom built LSFM setup for the study of tumor spheroids, the optimization of imaging protocols and the effect of chemotherapy. To illustrate the utility of LSFM for drug screening, patient-derived GB spheroids and MDA-MB-231 breast cancer spheroids were treated with the commonly used anti-cancer agents and spheroid cell viability was estimated using the cell death nuclear stain DRAQ7.
AcknowledgmentResearch supported by the projects “BIOIMAGING-GR” (MIS5002755) and “HELLAS-CH” (MIS 5002735) both implemented under “Action for Strengthening Research and Innovation Infrastructures”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).
 Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. and Stelzer,E. H. K., 2004 "Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy," Science, pp. 1007-1009
 Pampaloni, F., Ansari, N., and Stelzer, E. 2013, 'High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy', Cell Tissue Res., vol. 352, no. 1, pp. 161-77.
 Rieckher,M., Kyparissidis-Kokkinidis,I., Zacharopoulos, A., Kourmoulakis, G., Tavernarakis, N., Ripoll, J. and Zacharakis,G., 2015, 'A Customized Light Sheet Microscope to Measure Spatio-Temporal Protein Dynamics in Small Model Organisms', PLoS ONE, vol. 10(5),.
 Rieckher, M., Psycharakis, S. E., Ancora, D., Liapis, E., Zacharopoulos, A., Ripoll,J., Tavernarakis, N., and Zacharakis,G. , 2018, Biotechnology Journal, 'Demonstrating Improved Multiple Transport‐Mean‐Free‐Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics', vol. 13, 2018.
 Psycharakis, S.E., Liapis,E, Zacharopoulos, A., Oraiopoulou, M.E., Aivalioti, C., Sakkalis, V., Papamatheakis,J., Ripoll J. and Zacharakis, G., 2019, Proc. SPIE 11076, Advances in Microscopic Imaging, 'High resolution 3D imaging of primary and secondary tumor spheroids using multicolor multi-angle Light Sheet Fluorescence Microscopy'
Keywords: LSFM, MCTS, chemotherapeutics, drug efficiency evaluation, anti-cancer agents
3D registration of Light Sheet Fluorescence Microscopy datasets using decomposition to 2D projections (#319)
Athanasios Zacharopoulos1, Stylianos Psycharakis1, Giannis Zacharakis1
1 Foundation for Research and Technology-Hellas (FORTH), Inst. of Electronic Structure & Laser, Heraklion, Greece
A very popular tomographic microscopy technique is Light Sheet Fluorescence Microscopy (LSFM, SPIM)  where a light-sheet is used to excite a biological sample in sequential slices vertically to a fluorescence imaging system. The resulting stacks of slices render a tomographic representation of the sample, while the method maintains low photo-bleaching, and fast acquisition speeds that allow for in-vivo imaging. Still, the effect of light occlusion and scattering is deteriorating the imaging resolution the farther we are from the light-sheet source.
To increase the resolution in regions affected by occlusion or scattering, scans from different viewing angles (0o, 90o, 180o, 270o), are usually acquired. To utilise the complementary information image registration is necessary to resolve any misalignment issues and transform different sets of data into one common coordinate system. The registration of 3D volumes especially when common information between the volumes is sparse and obstructed is a difficult task, and common 3d registration methods fail. On the other hand the addition of artificial landmarks  increases the complexity of the imaging methodology, and are not always available in the necessary wavelengths.
We propose a method that reduces the dimensionality of the 3D problem of aligning the various viewpoints to the sequential registration of 2D projections (for example, maximum intensity projections) created from the initial volumes. We should note that the individual registration of the projections can be performed using any fast 2D image registration algorithm and the current implementation has the option to choose either Random Sample Consensus (RANSAC) based on automatic SIFT points algorithm or image based methods such as square differences or mutual information. Finally, for difficult datasets, where not much common information exists among views, an easy semi-automatic selection of few artificial 2D landmarks (usually 4-5) can be used. Using the projections registration we managed to create volumetric images of high information for multiangle LSFM, and overcome the problems arising from the high complexity of the 3D algorithms.
Using the projections registration, we managed to create volumetric images of high information for multiangle LSFM, and overcome the problems arising from the high complexity of the 3D algorithms. We achieved fast and accurate results without the need for the introduction of added fluorescence beads. Our implementation can be easily used by an experimentalist and it has shown to work even on samples with minimal common information between angles.
We acknowledge support of this work by the project “BIOIMAGING-GR” (MIS5002755) and “HELLAS-CH” (MIS5002755) which are implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).
 M. Rieckher PLoS ONE 10, (2015)
 S. Preibisch, Nature Methods, 7, (2010)
 S.E. Psycharakis SPIE 11076, (2019)
Steps for a 3D projections Registration of a LSFM 4 angles dataset
To quantify the performance of our algorithm we used four views of the Drosophila melanogaster embryo,
(0 degrees (red), 90 degrees (blue), 180 degrees (green), 270 degrees (yellow)) from the dataset http://fly.mpi-cbg.de/preibisch/nm/HisYFP-SPIM.zip.
Keywords: 3D image registation, LSFM, Tomographic Microscopy, SPIM, Projections
Non-linear microscopy reveals the ectopic fat deposition related to age associated pathology in Caenorhabditis elegans (#385)
Meropi Mari1, Konstantinos Palikaras2, Nektarios Tavernarakis2, 3, George Filippidis1
1 Foundation of Research and Technology-Hellas, Institute of Electronic Structure and Laser, Heraklion, Greece
Second and Third Harmonic Generation(SHG,THG) modalities are employed to visualize the age dependent ectopic lipid accumulation in C. elegans.Two Photon Excitation Fluorescence(TPEF) acts complementarily to non-linear scattering methods and all the three imaging modalities reveal that aging is accompanied by lipid deposition in non-adipose tissues such as muscles but also the nervous system of C. elegans.SHG and THG are powerful tools for elucidating subcellular structures and anatomical changes of various biological samples and monitoring complicated developmental processes in vivo [1-3].
The experimental setup consists of a femtosecond laser oscillator, emitting near infrared pulsed light at a central wavelength of 1.028 nm (200fs,50 MHz,1W).The laser beam is guided to a modified upright microscope.Adjustable neutral density filters are utilized to control the power at the sample plane.A set of galvanometric mirrors is employed to perform the fast raster scanning in the selected xy plane of the sample.The focal plane is adjusted by using a motorized translation stage. Diffraction limited focusing is achieved by using a high numerical aperture objective lens.THG is detected in the forward path, while SHG and TPEF signals are detected in the backscattered direction in distinct sets of measurements. The nonlinear signals are recorded by employing photomultiplier tubes.
Non-linear modalities are combined simultaneously to reveal that lipids expand ectopically in pharyngeal and body wall muscle cells during aging in C. elegans various strains .Simultaneous measurements of SHG and THG were performed on the whole body of wild-type C. elegans to monitor and compare ectopic fat storage in young and old animals. We found that fat deposition increases in body wall muscle cells with age (Figure 1).We also investigate whether fat accumulates in C. elegans nervous system. We studied the lipid deposition on Cephalic sheath glial cells (CEPsh) and CEP dopaminergic neurons in nematodes expressing cytoplasmic Green Fluorescent Protein (GFP)(Figure 2).Caloric restriction delays or prevents several potentially harmful age-associated phenotypes, which are known to be involved in metabolic syndrome progression .To this direction, we observed decreased levels of ectopic fat deposition in caloric-restricted eat-2(ad465) mutants compared with wild-type during aging.
Investigating the interplay between aging and ectopic lipid accumulation will enlighten new avenues for therapeutic interventions to cure metabolic syndrome-associated pathologies. Therefore, the establishment of novel, non-invasive methods for visualizing fat deposition in vivo is a prerequisite to tackle the demand for visualizing the fat distribution. Non-linear techniques are successfully employed for in vivo visualisation of ectopic fat.
AcknowledgmentThis work is supported by the project “BIOIMAGING” (MIS 5002755) under the “Action for Strengthening Research and Innovation Infrastructures”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). MM acknowledges the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), for the financial support under grant agreement No .
 Witte, S, Negrean, A, Lodder, J. C, de Kock, CPJ, Testa Silva, G, Mansvelder, HD, Louise Groot, M 2011, 'Label-Free Live Brain Imaging and Targeted Patching with Third-Harmonic Generation Microscopy', Proc. Natl. Acad. Sci. U. S. A., 108 (15), 5970–5975
 Olivier, N, Luengo-Oroz, MA, Duloquin, L, Faure, E, Savy, T, Veilleux, I, Solinas, X, Débarre, D, Bourgine, P, Santos, A, et al. 2010, ‘Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy’, Science, 329 (5994), 967–971
 Tserevelakis, GJ, Megalou, E V, Filippidis, G, Petanidou, B, Fotakis, C, Tavernarakis, N, 2014, ‘Label-Free Imaging of Lipid Depositions in C. Elegans Using Third-Harmonic Generation Microscopy’, PLoS One, 9 (1)
 Palikaras, K, Mari, M, Petanidou, B, Pasparaki, A, Filippidis, G, Tavernarakis, N, 2017, ‘Ectopic Fat Deposition Contributes to Age-Associated Pathology in Caenorhabditis Elegans’, J. Lipid Res, 58 (1)
 Huffman DM, Barzilai N 2009, ‘Role of visceral adipose tissue in aging’, Biochim Biophys Acta, 1790(10):1117-23
Figure 1: Ectopic fat accumulation on body wall muscles
Non-linear imaging on the vulva of a) 1 day and b) 9 days old wild type (N2). Lipid droplets are depicted through THG (yellow) measurements, while the striated muscles are revealed by SHG (magenda) simultaneously recorded measurements. The droplets that are encircled constitute a part of the ectopic fat.
Figure 2: Lipid droplet accumulation around neuronal cell bodies of CEP dopaminergic neurons.
Gradual lipid droplet accumulation in C. elegans nervous system with age a) 1 day and b) 9 days old. Coupling of TPEF (green) and THG (yellow) imaging reveals lipid droplet accumulation around neuronal cell bodies and along neuronal processes of the GFP fluorescent CEP dopaminergic neurons during aging. The images are z-projections of 18 slices to maximum intensity divided by 2 μm.
Keywords: non-linear imaging, C. elegans, fat deposition, aging, Third Harmonic Generation (THG)
Imaging membrane microviscosity of colorectal cancer cells in response to chemotherapy (#267)
Liubov E. Shimolina1, 2, Marina V. Shirmanova1, Marina K. Kuimova3, Maria M. Lukina1, Nadezhda I. Ignatova1, Elena V. Zagaynova1
1 Privolzhsky Research Medical University, Institute of Experimental Oncology and Biomedical Technologies, Nizhny Novgorod, Russian Federation
Chemotherapy is widely used to treat various forms of cancer. However, not all physiological reactions are described that occur during its action. Recent studies have established a link between the change in the microviscosity of tumor cells and the action of platinum drugs. The microviscosity of membrane of live cells can be determined using fluorescence-based methods, including fluorescent molecular rotors. Previously, we developed methodologies that allows obtaining microscopic viscosity maps from individual cancer cells in vitro to a mouse tumor model in vivo [1, 2].
The study was performed on cultured cancer cells CT26 (mouse colorectal cancer) and Hela Kyoto (human cervical cancer). Viscosity was measured in the plasma membranes of individual cells using the fluorescent molecular rotor BODIPY2 (ex. 800 nm, em. 409–680 nm). Chemotherapy was performed with cisplatin (Teva, Israel) at a dose of 2.57 μM for CT26 cells and 2.3 μM for Hela Kyoto cells. Multiphoton tomograph MPTflex (JenLab, Germany) equipped with a tuneable 80 MHz, 200 fs Ti:Sapphire laser (MaiTai) and a TCSPC-based FLIM module (Becker&Hickl Inc., Germany) was used to detect the fluorescence lifetime of a molecular rotor. Laser scanning microscopy on a LSM 880 microscope (Carl Zeiss, Germany) was used to assess cell viability in the monolayer and spheroids.
First, we developed protocol for imaging plasma membrane viscosity of cancer cells in vitro at the microscopic level. The developed protocols were applied to measure changes in membrane viscosity during chemotherapy in vitro in cell monolayers and 3D spheroids. We showed a significant increase in membrane viscosity in viable mouse colorectal cancer cells CT26 and human cervical cancer cells Hela Kyoto in 24h after cisplatin treatment up to 400 ± 27 cP . Measurement microviscosity during spheroid growth did not show significant differences between the viscosity values for spheroids of different size and layers of a spheroid. These data indicates that microviscosity of membrane is a relatively stable parameter, not affected by the metabolic and proliferative activity of cells and by the heterogeneity of the cellular microenvironment. Similar to cell monolayer, a significant increase in viscosity was detected after treatment with cisplatin up to 425 ± 25 cP.
In summary, our results suggest that microviscosity plays a role in the cytotoxicity of the drug and may provide a powerful tool for investigation of tumor responses to chemotherapy and mechanisms of drug resistance.
AcknowledgmentThis work was supported by the RFBR (project No. 18-29-09054 mk).
 Shirmanova, MV, Shimolina, LE, Lukina, MM, Zagaynova, EV, Kuimova, MK 2017, ‘Live Cell Imaging of Viscosity in 3D Tumour Cell Models’ Multi-Parametric Live Cell Microscopy of 3D Tissue Models, 143-153, USA: Springer International Publishing
 Shimolina, LE, Izquierdo, MA, López-Duarte, I, Bull, JA, Shirmanova, MV, Klapshina, LG, Zagaynova, EV, Kuimova, MK 2017, ‘Imaging tumor microscopic viscosity in vivo using molecular rotors’, Scientific Reports, vol. 7, Article number: 41097, UK: Nature Publishing Group
Keywords: microscopy, optical imaging, microviscosity, molecular rotors, lifetime of fluorescence
Fast light sheet microscopy for in-vivo imaging (#372)
María del Carmen Prieto1, Diego Díaz1, María Revuelta1, Manuel Desco Menéndez1, 2, 3, Roberto Fernández1, 4, Jorge Ripoll1
1 Universidad Carlos III de Madrid, Bioengineering and Aerospace Engineering, Leganés, Spain
The in-vivo study of the dwarf shrimp Neocardina David by using fast light sheet microscopy brings out new lines of research to understand the development of this species from early stages of its life until young adult through non-invasive in-vivo three-dimensional imaging. Likewise, its small size and transparency throughout its whole life make it a suitable invertebrate for the study and improvement of new microscopy techniques. Here, the set-up of a selective plane illumination microscope (SPIM) is presented including the characteristics it requires for fast specimen observation.
Stacks of images for 3-D reconstruction were obtained through a light sheet microscope that allowed a fast acquisition of images along the whole volume of the shrimp. The setup of the system is detailed in Fig.1. Laser light is guided through a combination of mirrors and lenses to produce an illumination plane obtained by fast dynamic focusing. The plane scans the sample, producing fluorescence light that goes through the selected emission filter and an electrically tunable lens system that focuses the image at the CMOS camera, thus enabling the acquisition of approximately 10 volumes per second.
In order to obtain images of a macroscopic object such as a young dwarf shrimp (more than 1cm in length), a 1x objective and an achromatic doublet pair (f1=30.0mm, f2=30.00mm) have been used. Therefore, a magnification of M=2.4 and a resolution of R=15.59µm is obtained in this configuration. Fig.2. shows two pictures of Neocardina davidi obtained in-vivo and without anaesthetics. The picture above presents the shrimp under white light illumination while the picture below is made under green light (524nm) excitation, recording light scattered from the animal (i.e. a SPIM version of a dark-field microscope). Thus, stacks of images of the shrimp were acquired focusing the laser light plane at several depths, recovering the whole volume of the animal.
This system constitutes an optimal selective plane illumination microscope for Neocardina davidi. It adjusts the FOV to the sample size without introducing vignetting, granting the whole shrimp observation. Current speeds allow for approximately 10 volumes per second. Image processing of the stacks and 3D reconstruction of the shrimp will be used to study the development and the anatomy of this species during its first stages until young adult.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801347 SENSITIVE and Spanish Ministry of Economy and Competitiveness (MINECO) Grant FIS2016-77892-R. R.F acknowledges funding from Generalitat Valenciana and European Social Fund through postdoctoral grant APOSTD/2018/A/084. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).
 Andrés-Delgado, L., Peralta, M., Mercader, N., & Ripoll, J. (2016, March). Dynamic focusing in the zebrafish beating heart. In Adaptive Optics and Wavefront Control for Biological Systems II (Vol. 9717, p. 971717). International Society for Optics and Photonics.
 Bassi, A., Fieramonti, L., D'Andrea, C., Valentini, G., & Mione, M. (2011). In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography. Journal of biomedical optics, 16(10), 100502.
 Fieramonti, L., Foglia, E. A., Malavasi, S., D'Andrea, C., Valentini, G., Cotelli, F., & Bassi, A. (2015). Quantitative measurement of blood velocity in zebrafish with optical vector field tomography. Journal of biophotonics, 8(1-2), 52-59.
 Huisken, J., & Stainier, D. Y. (2007). Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Optics letters, 32(17), 2608-2610.
 Mickoleit, M., Schmid, B., Weber, M., Fahrbach, F. O., Hombach, S., Reischauer, S., & Huisken, J. (2014). High-resolution reconstruction of the beating zebrafish heart. Nature methods, 11(9), 919.
The SPIM microscope is composed of a CMOS camera, an objective, a motor that enables the motion of the laser plane and the sample, three laser lines (405nm, 488nm and 514nm) and a wheel of emission filters. The lens system that precedes the camera is composed of an achromatic doublet pair (f1=100.00mm; f2=30.00mm) coupled to a 1x objective (1x / 0.025; ∞ / 0; f=200.00mm), a tunable lens, a tube lens (f=200.00mm) and a second achromatic doublet pair (f1=30.00mm; f2=30.00mm).
Keywords: Light Sheet Microscopy, SPIM, Dwarf Shrimp, Neocardina
A novel in vivo multiplexing method for synchronous monitoring of two different cell population in a deep-seated organ of C57BL/6 mice (#13)
Saimir Luli1, 2, Jack Leslie2, Fiona Oakley2
1 Newcastle University, Preclinical In Vivo Imaging, Newcastle upon Tyne, United Kingdom
Advances in optical imaging technology and NIR probes have allowed for in vivo fluorescence cell tracking. However, such a method has been limited to a single cell type and nude/albino mice. Monitoring multiple cell types requires a large number of mice and doesn’t control for mouse-to-mouse variation. Because standard flow cytometry and microscopy are not compatible with NIR probes, data validation at the cellular level remains challenging. In this study, we aim to overcome these barriers and establish an in vivo fluorescence imaging method to monitor two different cell types in a live mouse.
To establish a liver inflammatory response, WT and Rel−/− recipient mice were injured with carbon tetrachloride (CCl4). 24hr post-liver injury neutrophils were isolated from WT and Rel−/− donor mice. These cells were then labelled with Burgundy or CellVue-NIR780; 2 fluorophores with distinct spectral signatures. Following fluorescence labelling, neutrophils were injected into recipient mice. In vivo cell migration was monitored using an IVIS Spectrum fluorescence imaging system. In vivo scans were acquired using the following filter sets: ex/em 675/720nm for the Burgundy and ex/em 745/800nm for the NIR780 dye. To validate the in vivo data, livers were ex vivo imaged and then further processed to isolate neutrophils for Fluorescence-activated cell sorting (FACS) at single-cell resolution.
In vivo fluorescence imaging is limited to tissue depth and autofluorescence. For this reason, literature reports focus on in vivo fluorescence imaging of a single cell population in a superficially localised area in nude or albino mice.
In vivo multiplexing method established by our laboratory has the potential to be applied to different orthotopic mouse models to uncover multidimensional cellular interactions across many research disciplines. This method not only will improve data quality by minimising mouse-to-mouse variability but will also reduce the number of animals used in research by allowing administration of 2 fluorescent biological/chemical agents in the same animal.
This work was funded by a Medical Research Council and Wellcome Trust.
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 Sirbu D, Luli S, Leslie J, Oakley F, Benniston AC. May 2019. Enhanced in vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57BL/6 Mice Using a Highly Bright Near‐Infrared BODIPY Dye. ChemMedChem.17;14(10):995-999
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 Gieling RG, Elsharkawy AM, Caamã No JH, Cowie DE, Wright MC, Ebrahimkhani MR, et al. 2010. The c-rel subunit of nuclear factor-κb regulates murine liver inflammation, wound-healing, and hepatocyte proliferation. Hepatology; 51(3):922–31
Validation of Dual Fluorescent Imaging
(A) In vitro spectral unmixing of Burgundy (710nm) and NIR780 (780nm) using 675/720 and 745/820 nm Ex/Em filters respectively. (B) Graph showing average radiant efficiency for each dye. (C) FACS plots showing cell viability and (D) labelling efficiency of stained neutrophils. (E) In vivo spectral un-mixing of cells labelled with the 710nm and 780nm dyes injected into the flank of C57Bl/6 mice. Left panel shows IVIS scan acquired at 675/720 nm Ex/Em, middle panel shows IVIS scan at 745/800 nm Ex/Em, the right panel demonstrates in vivo cell separation based on the cell labelling fluorophore.
In vivo multiplexing characterises neutrophil migration in a deep-seated organ of C57Bl/6 mice
(A) In vivo scans of WT and Rel-/- recipient mice injected with a mixture of equal number of WT and Rel-/- neutrophils. Left panel: whole body scans acquired at 745/800 nm Ex/Em. Middle panel: images scanned at 675/720 nm Ex/Em; right panel: in vivo multiplexing based on the cell labelling fluorophore. Graph showing average radiant efficiency from WT and Rel-/- recipient mice injected with 780nm labelled WT neutrophils and 710nm labelled Rel-/- neutrophils. (C) FACS plots of live single cells isolated from injured livers of WT and Rel-/- recipient mice. Graph showing flow cytometry data.
Keywords: In vivo spectral unmixing, In vivo cell tracking, in vivo fluorescence imaging, liver, inflammation
Cerenkov luminescence imaging in pulmonary and hepatic metastasectomy (#197)
Esther Ciarrocchi1, 2, Nicola Belcari1, 2, Francesco Bartoli3, Angela G. Cataldi3, Pinuccia Faviana4, Luca Morelli5, 6, Marco Lucchi7, 8, Claudio Traino9, Sara Vitali3, Paola A. Erba3, 10
1 University of Pisa, Department of Physics, Pisa, Italy
Cerenkov luminescence imaging (CLI) is an optical imaging modality to detect distributions of radiopharmaceuticals. CLI can be used to visualize surgical margins immediately after resection and to refine surgery in a single procedure . We are planning a clinical study to evaluate the impact of CLI during surgery of lung and liver metastasis from various primary tumors with respect to conventional post-operative histology, and we are performing in-vitro simulation measurements to optimize the clinical protocol in terms of patient inclusion criteria, activity to inject, radiation monitoring.
We analyzed PET/CT data of 15 patients performed with [18F]-FDG in pulmonary and hepatic metastases and with 68Ga-DOTATOC in neuroendocrine tumors (NETs) to determine typical injected activities, lesion volumes, uptakes and time delays between injection and imaging. We are collecting data for typical histological margins. Since the Cerenkov signal depends on the spectrum of the beta particles and on the optical properties of the tissue, we prepared phantoms to measure the minimum detectable activity as a function of the type of radiopharmaceutical, the type of tissue and the source depth in tissue. The phantoms were imaged with a LightPath system with acquisition time acceptable for clinical needs. We used two short-pass filters to discriminate the depth of origin of the detected light.
Patient data are summarized in Figure 1. The delay between injection and imaging was ~1 hour. For our clinical study, we expect delays up to 4-5 hours between injection and CLI. The decay-corrected mean uptake values to account for this delay (5-11 kBq/cc) are comparable to the minimum detectable activity level of 8 kBq/cc that we measured for [18F]-FDG . For 68Ga-DOTATOC, the final uptake of 4-7 kBq/cc should be well detectable, because our first tests suggest a 13x signal increase with respect to 18F, but an enhancement up to 22x can be expected . Figure 2a shows a representative phantom image. The attenuation of 68Ga signal in the various animal liver samples is shown in Fig. 2b. Independent data-sets for the same type of tissue suggest good reproducibility. We are finalizing the data analysis to determine the target-to-background ratio in both the patient and phantom data.
Patient data suggests that CLI can be performed with standard clinical activities and 5-minute exposure times. The typical lesion volumes are suitable for LightPath imaging. Phantom data for signal attenuation in biological tissue show good reproducibility. We are collecting additional data for lung phantoms, and we are studying the target-to-background ratio for 18F and 68Ga and a method to extract the source depth from the spectral images.
 M.R. Grootendorst, et al. "Intraoperative assessment of tumor resection margins in breast-conserving surgery using 18F-FDG Cerenkov luminescence imaging: a first-in-human feasibility study." Journal of Nuclear Medicine 58.6 (2017): 891-898.
 E. Ciarrocchi, et al. "Performance evaluation of the LightPath imaging system for intra-operative Cerenkov luminescence imaging." Physica Medica 52 (2018): 122-128.
 J. olde Heuvel, et al. "Performance evaluation of Cerenkov luminescence imaging: a comparison of 68 Ga with 18 F." EJNMMI physics 6.1 (2019): 17.
Figure 2. Results of the in-vitro simulation measurements.
a) Representative CLI image (false color) overlaid on the reference photo (black and white). 68GaCl3 was diluted in 4 ml of distilled water and covered with 4 mm of pork liver. The image was acquired with 300 s exposure, binning 8x8 and no optical filters. b) Attenuation of the CLI signal from 68Ga as a function of the source depth in various liver specimens, for phantoms as shown in a). Data were normalized for the source activity and the exposure time. No optical filters were used in this case.
Keywords: cerenkov luminescence imaging, cancer surgery, surgical margin assessment
Application of optical imaging to localisation of the 90Y labelled PSMA inhibitor (#311)
Urszula Karczmarczyk1, Kamil Tomczyk1, Michal Maurin1, Ewa Laszuk1, Arkadiusz Sikora1, Piotr Garnuszek1
1 National Centre for Nuclear Research, Radioisotope Centre POLATOM, Otwock, Poland
Yttrium-90 (90Y), is a well-known radionuclide with a high-energy β− particle emission, which is widely used in nuclear medicine for cancer therapy. High energy beta particles produces light photons (Cerenkov radiation) which can then be imaged using optical imaging systems. The aim of our work was standardisation of Cerenkov measurement of β- emitting radiotracers (90Y) using OptiImager device. We examined how the geometry, radioactivity and refractive index of the tested system affects the record of CLI and if the obtained images may be compare to ex vivo results.
The standardization tests were carried out using 90YCl3 (ItraPol, Poland) solutions as β-emitting sources in 24-well plates. This solution was dissolved at colourless gelatine (type A) and gelatin with pyrocatechol violet (type B). The studies were performed considering Cerenkov sources of different activity and depth of location. In preclinical studies, BALB/c NUDE mice bearing subcutaneous LNCaP cells were administered with 90Y-DOTA-PSMA-D4. In vivo CLI has been performed at 1h, 4h and 3 days post-injection of 90Y-DOTA-PSMA-D4. Ex vivo pharmacokinetics and biodistribution studies at 2h, 4h and 24h p.i.v. were made. The mice were sacrificed and the excised organs, tumors and blood were collected, weight and uptake of radioactivity were measured by scintillation techniques (%ID/g).
Results shows that both the thickness and type of gelatin as dielectric medium used for standardization are of great importance for efficient counting of the emitted Cerenkov luminescence. We observed the linearity of the Cerenkov counting for 90Y up to 11 MBq resulting r2=0.995 for colorless gelatin and r2=0.999 for gelatin with pyrocatechol violet. Moreover, the effect of the type of gelatin used on the number of photon counting was observed at increasing height of the medium above the photon source. The biodistribution study of 90Y-DOTA-PSMA-D4 showed that complex was characterized by the fastest elimination with urine (96%ID at 2 h p.i.v.) and the very low accumulation in kidneys (2.7±0.2%ID/g, at 2h). The pharmacokinetic study in tumour-bearing mice showed very high accumulation of 90Y-DOTA-PSMA-D4 in tumors (20.5±5.5%ID/g, 16.4±6.9%ID/g and 10.3±3.6%ID/g at 2h, 4h and 24h p.i.v. respectively) and impressive T/M ratios what was confirmed at optical imaging studies.
In vivo Cerenkov optical imaging of the 90Y-DOTA-PSMA-D4 in tumor bearing mice confirmed accumulation of radioactivity in tumour pointed out that it is promising candidate for therapy of the prostate cancer.
Keywords: Cerenkov luminescence imaging (CLI), 90Y-DOTA-PSMA-D4, animal study
Assessing Light Sheet Microscopy in Semi-Transparent Media with a Dedicated Monte Carlo Simulator (#585)
Asier Marcos-Vidal1, Roberto Fernández1, 2, Jorge Ripoll1, 3
1 Universidad Carlos III de Madrid, Department of Bioengineering and Aerospace Engineering, Leganés, Madrid, Spain
The study of light propagation using the Monte Carlo (MC) method is a common approach to improve image quality in microscopy and optical imaging, however, this method cannot account for many of the factors that determine the quality of the images in real systems. In this work, light sheet fluorescence microscopy (LSFM) is simulated using a novel MC software that replicates the entire photon flow of a light sheet microscope. The tool is used to assess the performance of LSFM in several spectral windows in terms of resolution loss, depth of penetration and optical sectioning.
The MC-LSFM simulator integrates a custom modified version of the optimized and validated package Monte Carlo eXtreme (MCX)  as the engine to propagate photons in scattering media(see fig1). Detected photons are focused using with a novel focusing and fluorescence computation alogrithm that mimics the lens system and recreates the optical sectioning.
The MC simulations consist of a two step procedure. Firstly a light sheet is first propagated in the medium and its intensity distribution stored. Then the fluorophore distribution is simulated. Each fluorescent photon detected is propagated through a virtual lens system, contributing to the image plane according to its excitation intensity.
The entire workflow is programmed in CUDA GPU and optimized to run a LSFM acquisition within minutes.
The performance of LSFM for three spectral windows was investigated through the evaluation of the depth of penetration for several media of different scattering coefficients. To assess the degradation of the image caused by scattering, absorption remains low and constant throughout the simulations.
The results in figure 2 show how the depth of penetration decreases with the increase of the scattering coefficient. The degradation of the light sheet can be observed at deep x positions.
This work presents a MC-LSFM simulator capable of generating synthetic LSFM images to evaluate the effects of scattering in the performance of this technique.
The software relies on a modified version of a well validated MC package and a novel algorithm to focus the detected fluorescence and compute the optical sectioning. The results can predict the penetration, image quality loss and light sheet degradation in low scattering media.
AcknowledgmentThis project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801347 SENSITIVE and Spanish Ministry of Economy and Competitiveness (MINECO) Grant FIS2016-77892-R. R.F acknowledges funding from Generalitat Valenciana and European Social Fund through postdoctoral grant APOSTD/2018/A/084.
 Qianqian Fang and David A. Boas, "Monte Carlo Simulation of Photon Migration in 3D Turbid Media Accelerated by Graphics Processing Units," Opt. Express, vol. 17, issue 22, pp. 20178-20190 (2009)
Volume rendering of a RGB composite image of the simulated volumes. Channel red was assigned to μ′s = 0.1cm−1, green to μ′s = 1cm−1 and for μ′s = 2cm−1. The fluorophores with whiter color in the figure represent those which have intensity in all the simulated volumes.
Keywords: light sheet, monte carlo, near infrared, simulation
Fluorescent metabolic imaging of the liver at the cellular level during regeneration (#265)
Svetlana A. Rodimova1, 3, Daria S. Kuznetsova1, Nikolay V. Bobrov1, 2, Dmitry G. Reunov1, Emil R. Kryukov1, Natalia V. Vdovina1, Vladimir E. Zagainov1, 2, Elena V. Zagaynova1
1 Privolzhsky Research Medical University, Institute of Experimental Oncology and Biomedical Technologies, Nizhny Novgorod, Russian Federation
A healthy liver has a high regenerative potential, but with an acute injury or after resection, this potential is significantly reduced. Standard methods for assessing the structural and functional state of the liver do not allow studying the processes occurring in the liver cells in dynamics during the regeneration [1,2]. Modern label-free methods of multiphoton microscopy with FLIM (fluorescence lifetime imaging) and SHG (second harmonic generation) modes expand the possibilities of studying the metabolic state at the cellular level, due to its non-invasiveness and high sensitivity .
Experiments were performed on Wistar rats weighing 400-500 g. Removing the left lobe of the liver is a model of 30% hepatectomy, removing the left and medial lobes of the liver is a model of 70% hepatectomy. Metabolic imaging was performed on 3 and 7 days after resection. A separate analysis of NADH and NADPH was presented to evaluate the overall synthetic activity in liver cells. Resected liver samples were researched as control.
The results of the analysis showed an increase in the overall metabolic activity of hepatocytes with an increase of phosphorylation oxidation processes contribution in hepatocytes in both models - 30% PHx and 70 PHx on 3 and 7 days, which may indicate an increase in the energy requirements of proliferating cells.
The obtained parameters may be useful in determining the criteria for the evaluation of the liver regenerative potential after surgery. The obtained data will significantly expand the capabilities of modern diagnostic methods for liver surgery.
This work was supported by the Russian Science Foundation grant № 19-15-00263.
 Forbes, SJ, Newsome, PN 2016, 'Liver regeneration—mechanisms and models to clinical application', Nature reviews Gastroenterology & hepatology, 13(8), 473, UK.
 Wang, H, Liang, X, Gravot, G, Thorling, CA, Crawford, DH, Xu, ZP, Roberts, MS 2017, 'Visualizing liver anatomy, physiology and pharmacology using multiphoton microscopy', Journal of biophotonics, 10(1), 46-60, Institute for Physical Chemistry Friedrich Schiller University, Germany.
 Blacker, TS, Mann, ZF, Gale, JE, Ziegler, M, Bain, AJ, Szabadkai, G, Duchen, MR 2014, 'Separating NADH and NADPH fluorescence in live cells and tissues using FLIM', Nature communications, 5, 393, UK.
Keywords: liver, metabolic imaging, FLIM, multiphoton microscopy, TOF-SIMS
Preclinical Whole-body Fluorescence Tomography Using Infrared Fluorescent Proteins (#504)
Gultekin Gulsen1, Wesley Moy2, Farouk Nouizi1, Austin Moy2
1 University Of California, Irvine, Radiological Sciences, Irvine, United States of America
Reporter gene-based optical imaging is a powerful tool for modern biomedical research. Although optical imaging can be performed in both fluorescence and bioluminescence mode, the latter has been the method of choice in many labs due to easy encoding of firefly luciferase (Fluc), which catalyzes d-luciferin oxidation to produce photons that can be detected with widely available 2D bioluminescence imaging systems. On the other hand, development of the near infrared fluorescent proteins (iRFP) in the recent years paved the way for 3D fluorescence tomographic imaging (FTI) of small animals [1-3].
Full-view tomographic imaging requires acquisition of images all around the medium under investigation. Tomographic imaging in bioluminescence mode is very challenging due to the challenging source localization inverse problem in addition to the dynamic nature of the signals due to distribution of i.p. injected d-luciferin . iRFPs on the other hand provide time independent fluorescence signals that solely based on the excitation light, and therefore perfect for 3D FTI together with the low absorption of tissue in near-infrared region. In this study, mice inoculated with 4T1 cells labeled with both iRFP and Fluc (Imanis Life Sciences, USA) were imaged in both bioluminescence and fluorescence modes using an ICCD camera as well as a commercial gantry-based 3D FTI system (TriFoil Inc, USA)
In this study, first we compared the sensitivity of optical imaging in bioluminescence and fluorescence modes. Although, there were a number of studies in the literature comparing the sensitivity of these to modes , it should be important to note that the choice of the instrument is critical for a fair comparison. For this purpose, we used an intensified CCD camera together with the laser illumination at 660nm. A 725nm (+/-20nm) bandpass filter was used to eliminate the reflected excitation light. Both bioluminescence and fluorescence modes showed similar sensitivity for the subcutaneous 4T1 tumors. In addition to that, tomographic 3D images were acquired using TriFoil InSyTe system in fluorescence mode. This system also acquired X-ray micro-CT images of the animal in the same setting. The cross-sectional fluorescence images superimposed on the X-ray CT images are presented in Fig 1. Providing time independent signal, iRFP enabled successful tomographic imaging of these tumors.
Bioluminescence imaging has an advantage of dark background while reflected excitation light from normal tissue may leak through the bandpass filter and increases background levels in fluorescence imaging. Therefore, the choice of bandpass filter and excitation light combination is very critical. Providing time independent signal, and eliminating the need for injection of an enzyme, iRFP enabled successful tomographic imaging of these tumors.
 D.M. Chudakov et. al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010 Jul; 90(3):1103-63.
 W. Rice et. al. “In vivo tomographic imaging of deep seated cancer using fluorescence lifetime contrast “ Cancer Res. 2015 Apr 1; 75(7): 1236–1243.
 M. Honda et. al. “A Novel Near-infrared Fluorescent Protein, iRFP720, Facilitates Transcriptional Profiling of Prostate Cancer Bone Metastasis in Mice” Anticancer Research June 2017 vol. 37 no. 6 3009-3013
 G. Wang et. al, “Uniqueness theorems in bioluminescence tomography,” Med. Phys. 2004 31, 2289–2299
 C. Genevois et. al. “In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography” Int. J. Mol. Sci. 2016, 17(11), 1815;
Keywords: in vivo fluorescence imaging, bioluminescence imaging, fluorescence tomography