Technologies

Our development team has more than fifteen years of experience in designing and building advanced imaging hardware and software. We work hand in hand with our customers in precisely identifying the specific requirements of their research applications and develop customized imaging solutions that best suit their needs. Having worked ourselves with commercially available microscopy systems, we fully understand what the term “user-friendly” means and we design image acquisition software that can be rightfully described as such. We provide expert consultation in building modular systems. This means that customers actively participate in selecting the key components of their own systems within a wide variety of qualities and prices. This also implies that our systems are dynamic, they can be rapidly and easily adapted as new technologies, better hardware or more advance software are being developed or to better accommodate to the changing demands of your research projects.

What technologies can we help you implement in your laboratories?

• Selective Plane Illumination Microscopy (SPIM)
• Optical Projection Tomography (OPT)
• Fluorescence Molecular Tomography (FMT)

Selective Plane Illumination Microscopy (SPIM)

SPIM, Ultramicroscopy, or in general Laser Sheet Microscopy techniques enable fast high-resolution imaging of large, optically-cleared, three-dimensional specimens. In SPIM, whole organs, thick tissue samples or organotypic or 3D cellular cultures are illuminated using a thin sheet of light that scans the specimens along one axis while emitted light is collected along a perpendicular optical axis, ensuring that the detection objective focal plane coincides with the sheet of light. In the past decade different modalities of SPIM have been developed that vary depending on the configuration of sources and cameras, and on how the sheet is generated to reduce scattering and increase speed. We develop Laser Sheet Microscopy set-ups in all configurations (single-armed, double-sided illumination, fast-acquisition for in-vivo, multiview, etc.)

Advantages

Low photo-toxicity and photo bleaching, high acquisition speeds, sensitive detection, good light penetration, 3D resolution.

For more information on the technologies check the special Collections issue in Nature Methods Journal exclusively dedicated to Laser Sheet Microscopy

References using SPIM and software of 4D-nature

• Photoswitching-Enabled Contrast Enhancement in Light Sheet Fluorescence Microscopy.
  Vettenburg, T., Corral, A., Rodríguez-Pulido, A., Flors, C., & Ripoll, J. (2017). ACS Photonics, 4(3), 424–428.
• Optimized CUBIC protocol for 3D imaging of chicken embryos at single-cell resolution.
  ​Gómez-Gaviro, M. V., Balaban, E., Bocancea, D., Lorrio, M. T., Pompeiano, M., Desco, M., Ripoll, & J, Vaquero, J. J. (2017). Development,
• Looking inside the heart: a see-through view of the vascular tree. Nehrhoff, I., Ripoll, J., Samaniego, R., Desco, M., & Gómez-Gaviro, M. V. (2017). . Biomedical Optics Express, 8(6), 3110.
• Antigen Availability and DOCK2-Driven Motility Govern CD4+ T Cell Interactions with Dendritic Cells In Vivo
  Ackerknecht, M., Gollmer, K., Germann, P., Ficht, X., Abe, J., Fukui, Y., Swoger, J., Ripoll, J., Sharpe, J. &  Stein, J. V. (2017).. The Journal of Immunology.
• 3D imaging in CUBIC-cleared mouse heart tissue: going deeper. 
  Nehrhoff, I., Bocancea, D., Vaquero, J., Vaquero, J. J., Ripoll, J., Desco, M., & Gómez-Gaviro, M. V. (2016). Biomedical Optics Express, 7(9), 3716.
• Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.
  Abe, J., Ozga, A. J., Swoger, J., Sharpe, J., Ripoll, J., & Stein, J. V. (2016).  Journal of Immunological Methods, 431, 1–10.
• Stripe artifact elimination based on non-subsampled contourlet transform for light sheet fluorescence microscopy.
  Liang, X., Zang, Y., Dong, D., Zhang, L., Fang, M., Yang, X., … Tian, J. (2016).  Journal of Biomedical Optics, 21(10), 106005.
• pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion.
  Ozga, A. J., Moalli, F., Abe, J., Swoger, J., Sharpe, J., Zehn, D., … Stein, J. V. (2016).  Journal of Experimental Medicine, 213(12).
• A Customized Light Sheet Microscope to Measure Spatio-Temporal Protein Dynamics in Small Model Organisms
  M Rieckher, I Kyparissidis-Kokkinidis, A Zacharopoulos, G Kourmoulakis, N.Tavernarakis, J. Ripoll and G. Zacharakis,
  Plos One 10(5): e0127869, 2015
• Vertically scanned laser sheet microscopy
  D Dong, A Arranz, S Zhu, Y Yang, L Shi, J Wang, C Shen, J Tian, J Ripoll
  Journal of biomedical optics 19 (10), 106001-106001, 2014
• Alicia Arranz, Di Dong, Shouping Zhu, Markus Rudin, Christos Tsatsanis, Jie Tian, and Jorge Ripoll, “Helical optical projection tomography,” Opt. Express 21, 25912-25925 (2013). In this paper we combined SPIM with OPT (see below) for imaging large samples.

Optical projection Tomography (OPT)

OPT is a mesoscopic technique (Science 296 (5567): 541–5. April 2002) that is used to acquire panoramic 3D microscopy images of samples considered too big for conventional microscopy techniques (1-10mm). In OPT light is projected through a whole specimen treated with optical clearing agents, and the transmitted light is collected as the object rotates. Using backprojection algorithms, a 3D image of the specimen is reconstructed from the series of images acquired. OPT is rapidly becoming a widely used technique in biomedical research and has been employed so far to visualize fixed specimens such as whole mouse embryos, pancreas and peripheral lymph nodes among other (see for example, Kumar V et al, Front Immunol. 2012;3:282).

Advantages

Imaging of large specimens, isotropic resolution.

References using OPT and software of 4D-nature:

• In-vivo Optical Tomography of Small Scattering Specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster
  A Arranz, D Dong, S Zhu, C Savakis, J Tian, J Ripoll
  Scientific reports 4, 2014
• Alicia Arranz, Di Dong, Shouping Zhu, Markus Rudin, Christos Tsatsanis, Jie Tian, and Jorge Ripoll, “Helical optical projection tomography,” Opt. Express 21, 25912-25925 (2013). In this paper we combined SPIM (see SPIM) with OPT for imaging large samples.
• Rieckher M, Birk UJ, Meyer H, Ripoll J, Tavernarakis N (2011) Microscopic Optical Projection Tomography In Vivo. PLoS ONE 6(4): e18963. doi:10.1371/journal.pone.0018963. Note: In this paper we make use of OPT to image C. elegans in-vivo, being the first time OPT is used on live microscopic specimens.
• Heiko Meyer, Alex Darrell, Athanasios Metaxakis, Charalambos Savakis and Jorge Ripoll, “Optical Projection Tomography for In-Vivo Imaging of Drosophila melanogaster”, Microscopy and Analysis, Sept. (2008).
• D. Dong, S. Zhu, C. Qin, V. Kumar, J. V Stein, S. Oehler, C. Savakis, J. Tian, and J. Ripoll. “Automated Recovery of the Center of Rotation in Optical Projection Tomography in the Presence of Scattering.” IEEE Trans. Inf. Tech. in Biomed (Sept. 2012).
• S. Zhu, D. Dong, U. J. Birk, M. Rieckher, N. Tavernarakis, X. Qu, J. Liang, J. Tian, and J. Ripoll. Automated motion correction for in vivo optical projection tomography.” IEEE Trans Med Imaging 31.7 (July 2012), pp. 1358-71.

Fluorescence Molecular Tomography (FMT)

FMT is a tomographic technique that enables in-vivo 3D quantitative images of near infra-red fluorescent probes and proteins in deep tissues. FMT has emerged as a key non-invasive imaging technology in biomedical and preclinical research, due to the fact that it is high-throughput and it employs very specific activatable fluorescent probes or fluorescent proteins. It is based on modeling light propagation in tissues through the diffusion approximation in order to solve what is termed an “inverse problem” to recover the 3D spatial distribution of flurophore concentration.

Advantages

Non-invasive, in vivo deep tissue imaging, high sensitivity and specificity.

References using FMT and software of 4D-nature

• Abraham Martin, Juan Aguirre , Ana Sarasa, Anikitos Garofalakis, Heiko Meyer, Clio Mamalaki, Jorge Ripoll and Anna M. Planas, “Imaging Changes in Lymphoid Organs In Vivo after Brain Ischemia with Three-Dimensional Fluorescence Molecular Tomography in Transgenic Mice Expressing Green Fluorescent Protein in T Lymphocytes”, Mol. Imag. Jul-Aug;7(4):157-67 (2008). Note: In this paper we make use of FMT to follow the changes of GFP expressing T-Lymphocytes in 3D in-vivo after brain ischemia.
• M. Simantiraki, R. Favicchio, S. Psycharakis, G. Zacharakis and J. Ripoll, “Multispectral unmixing of fluorescence molecular tomography data”, J. of Inn. Opt. Health Sci. Vol. 2(4), 353–364 (2009).
• F. Stuker, C. Baltes, K. Dikaiou, D. Vats, L. Carrara, E. Charbon, J. Ripoll, and M. Rudin. Hybrid small animal imaging system combining magnetic resonance imaging with fluorescence tomography using single photon avalanche diode detectors”. IEEE Trans Med Imaging 30.6 (2011), pp. 1265-1273. Note: In this work FMT was used in combination with MRI, making use of an FMT and software developed by members of the 4D-Nature team.
• Zacharakis, G., Favicchio, R., Simantiraki, M. and Ripoll, J. (2011). Spectroscopic detection improves multi-color quantification in fluorescence tomography. Biomedical Optics Express, 2(3), 431–9. doi:10.1364/BOE.2.000431. Note: In this paper FMT is used for multispectral imaging, making use of volumetric multispectral unmixing techniques.
• R. Favicchio, G. Zacharakis, K. Oikonomaki, A. Zacharopoulos, C. Mamalaki, and J. Ripoll. Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epiuorescence laser scanning”. Journal of Biomedical Optics 17.7 (2012), p. 076013.
• A. Zelmer, P. Carroll, N. Andreu, K. Hagens, J. Mahlo, N. Redinger, B. D.Robertson, S. Wiles, T. H. Ward, T. Parish, J. Ripoll, G. J. Bancroft, and U. E. Schaible. “A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging.” The Journal of antimicrobial chemotherapy 67.8 (Aug. 2012), pp. 1948-60.
• A. Arranz, A. Androulidaki, B. Mol, E. Tsentelierou, E. N. Stathopoulos, C. Tsatsanis, and J. Ripoll. “Intravital spectral imaging as a tool for accurate measurement of vascularization in mice.” Journal of angiogenesis research 2.1 (Jan. 2010), p. 22.
• Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging
  Ren, W., Elmer, A., Buehlmann, D., Augath, M.-A., Vats, D., Ripoll, J., & Rudin, M. (2016). . Molecular Imaging and Biology, 18(2), 191–200.