Breast cancer and Glioblastoma brain cancer are aggressive malignancies with poor prognosis. In this study primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. A custom multi-angle Light Sheet Fluorescence Microscope is employed for high resolution imaging of both cancer cell spheroids. Such a technique is successful in realizing pre-clinical drug screening, while enables the discrimination among physiologic tumor parameters. LSFM technique, parameters and method followed are also presented.
Optical tomography in biomedical imaging is a highly dynamic field in which non-invasive optical and computational techniques are combined to obtain a three dimensional representation of the specimen we are interested to image. Although at optical wavelengths scattering is the main obstacle to reach diffraction limited resolution, recently several studies have shown the possibility to image even objects fully hidden behind a turbid layer exploiting the information contained in the speckle autocorrelation via an iterative phase retrieval algorithm. In this work we explore the possibility of blind three dimensional reconstruction approach based on the Optical Projection Tomography principles, a widely used tool to image almost transparent model organism such as C. Elegans and D. Rerio. By using autocorrelation information rather than projections at each angle we prove, both numerically and experimentally, the possibility to perform exact three dimensional reconstructions via a specifically designed phase retrieval algorithm, extending the capability of the projection-based tomographic methods to image behind scattering curtains. The reconstruction scheme we propose is simple to implement, does not require post-processing data alignment and moreover can be trivially implemented in parallel to fully exploit the computing power offered by modern GPUs, further reducing the need for costly computational resources.
Recently great progress has been made in biological and biomedical imaging by combining non-invasive optical methods, novel adaptive light manipulation and computational techniques for intensity-based phase recovery and three dimensional image reconstruction. In particular and in relation to the work presented here, Optical Projection Tomography (OPT) is a well-established technique for imaging mostly transparent absorbing biological models such as C. Elegans and Danio Rerio. On the contrary, scattering layers like the cocoon surrounding the Drosophila during the pupae stage constitutes a challenge for three dimensional imaging through such a complex structure. However, recent studies enabled image reconstruction through scattering curtains up to few transport mean free paths via phase retrieval iterative algorithms allowing to uncover objects hidden behind complex layers. By combining these two techniques we explore the possibility to perform a three dimensional image reconstruction of fluorescent objects embedded between scattering layers without compromising its structural integrity. Dynamical cross correlation registration was implemented for the registration process due to translational and flipping ambiguity of the phase retrieval problem, in order to provide the correct aligned set of data to perform the back-projection reconstruction. We have thus managed to reconstruct a hidden complex object between static scattering curtains and compared with the effective reconstruction to fully understand the process before the in-vivo biological implementation.
Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.
Research on magnetic nanocrystals attracts wide-spread interest because of their challenging fundamental properties, but it is also driven by problems of practical importance to the society, ranging from electronics (e.g. magnetic recording) to biomedicine. In that respect, iron oxides are model functional materials as they adopt a variety of oxidation states and coordinations that facilitate their use. We show that a promising way to engineer further their technological potential in diagnosis and therapy is the assembly of primary nanocrystals into larger colloidal entities, possibly with increased structural complexity. In this context, elevated-temperature nanochemistry (c.f. based on a polyol approach) permitted us to develop size-tunable, low-cytotoxicity iron-oxide nanoclusters, entailing iso-oriented nanocrystals, with enhanced magnetization. Experimental (magnetometry, electron microscopy, Mössbauer and NMR spectroscopies) results supported by Monte Carlo simulations are reviewed to show that such assemblies of surface-functionalized iron oxide nanocrystals have a strong potential for innovation. The clusters’ optimized magnetic anisotropy (including microscopic surface spin disorder) and weak ferrimagnetism at room temperature, while they do not undermine colloidal stability, endow them a profound advantage as efficient MRI contrast agents and hyperthermic mediators with important biomedical potential.
In vivo optical imaging of biological tissue not only requires the development of new theoretical models and experimental procedures, but also the design and construction of realistic tissue-mimicking phantoms. However, most of the phantoms available currently in literature or the market, have either simple geometrical shapes (cubes, slabs, cylinders) or when realistic in shape they use homogeneous approximations of the tissue or animal under investigation. The goal of this study is to develop a non-homogeneous realistic phantom that matches the anatomical geometry and optical characteristics of the mouse head in the visible and near-infrared spectral range. The fabrication of the phantom consisted of three stages. Initially, anatomical information extracted from either mouse head atlases or structural imaging modalities (MRI, XCT) was used to design a digital phantom comprising of the three main layers of the mouse head; the brain, skull and skin. Based on that, initial prototypes were manufactured by using accurate 3D printing, allowing complex objects to be built layer by layer with sub-millimeter resolution. During the second stage the fabrication of individual molds was performed by embedding the prototypes into a rubber-like silicone mixture. In the final stage the detailed phantom was constructed by loading the molds with epoxy resin of controlled optical properties. The optical properties of the resin were regulated by using appropriate quantities of India ink and intralipid. The final phantom consisted of 3 layers, each one with different absorption and scattering coefficient (μa,μs) to simulate the region of the mouse brain, skull and skin.
KEYWORDS: Luminescence, Tomography, Fluorescence tomography, Green fluorescent protein, Skin, In vivo imaging, Tissues, Absorption, Signal detection, Reconstruction algorithms
Autofluorescence has been a significant disadvantage when dealing with tomographic imaging of biological samples or
tissue phantoms. Consequently, the accurate removal of autofluorescence signal has been a major concern in
fluorescence tomography. Here we present a study on three-dimensional mapping and removal of autofluorescence from
fluorescence molecular tomography (FMT) data, both for phantoms and small animal in vivo. The technique is based on
the recording of tomographic data in multiple spectral regions with different excitation light and on the application of a
linear unmixing algorithm for targeting multiple fluorescent probes. Two types of measurements are taken, one with the
excitation being in the region of the maximum absorption of the targeted fluorophore and one in a region away from the
maximum. The relative strengths of the different spectra are employed to calculate the signal to be removed from the
tomographic reconstruction. Autofluorescence spectra are recorded using identical reflection geometry as during the
FMT acquisitions allowing for the correct mapping of the autofluorescence signal. Results from phantoms exhibiting
different background autofluorescence strengths are presented and discussed. In this work we have also studied in vivo
fluorescent activity in mice, involving both subcutaneously implanted fluorescent phantoms and b10 transgenic mice.
KEYWORDS: Luminescence, Green fluorescent protein, Reconstruction algorithms, Tomography, Tissues, In vivo imaging, Optical filters, Control systems, Cameras, Skin
Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo
in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying
the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different
biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral
unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging
modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity
originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated
emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo.
The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of
organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is
highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental
parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to
measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral
unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for
specific applications.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.