Negative Contrast Imaging Research Articles

One-step visualization of natural cell activities in non-labeled living spheroids

3D cultured cell aggregates, including spheroids, reflect the gene expression patterns of living tissues/organs. Mass preparation of spheroids enables high-throughput drug screening (HTS). However, conventional optical imaging of spheroids makes it difficult to obtain sufficient resolution of individual living cells in the thick cellular stack. Rapid and accurate assessment of cellular responses in spheroids is required for effective drug screening. Here, we show that negative contrast imaging (NCI) of spheroids overcomes this issue. Hydrophilic fluorescent dye added into the culture medium rapidly diffused into the intercellular space of living spheroids within a few minutes. Confocal microscopy showed the NCI of individual cells as dark and detailed contours clearly separated with fluorescence signals in the intercellular space. NCI enables the visualization of the alteration of cell morphology after anti-tumor drug application to living spheroids and the measurement of the fluorescent dye diffusion rate without any complicated pretreatments. Using this system, we found that the antitumor drug doxorubicin reduced the intercellular space of spheroids consisting of the human hepatocyte carcinoma cell line HepG2, through the activation of TGF-β signaling and upregulation of ECM protein expression, implicating a drug resistance mechanism. Collectively, the combination of NCI of spheroids and HTS may enhance the efficiency of drug discovery.

A method for detailed analysis of the structure of mast cell secretory granules by negative contrast imaging.

Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG–SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research.

Negative contrast Cerenkov luminescence imaging of blood vessels in a tumor mouse model using [68Ga]gallium chloride

BackgroundCerenkov luminescence imaging (CLI) is an emerging imaging technique where visible light emitted from injected beta-emitting radionuclides is detected with an optical imaging device. CLI research has mostly been focused on positive contrast imaging for ascertaining the distribution of the radiotracer in a way similar to other nuclear medicine techniques. Rather than using the conventional technique of measuring radiotracer distribution, we present a new approach of negative contrast imaging, where blood vessel attenuation of Cerenkov light emitted by [68Ga]GaCl3 is used to image vasculature.MethodsBALB/c nude mice were injected subcutaneously in the right flank with HT-1080 fibrosarcoma cells 14 to 21 days prior to imaging. On the imaging day, [68Ga]GaCl3 was injected and the mice were imaged from 45 to 90 min after injection using an IVIS Spectrum in vivo imaging system. The mice were imaged one at a time, and manual focus was used to bring the skin into focus. The smallest view with pixel size around 83 μm was used to achieve a sufficiently high image resolution for blood vessel imaging.ResultsThe blood vessels in the tumor were clearly visible, attenuating 7% to 18% of the light. Non-tumor side blood vessels had significantly reduced attenuation of 2% to 4%. The difference between the attenuation of light of tumor vessels (10% ± 4%) and the non-tumor vessels (3% ± 1%) was significant. Moreover, a necrotic core confirmed by histology was clearly visible in one of the tumors with a 21% reduction in radiance.ConclusionsThe negative contrast CLI technique is capable of imaging vasculature using [68Ga]GaCl3. Since blood vessels smaller than 50 μm in diameter could be imaged, CLI is able to image structures that conventional nuclear medicine techniques cannot. Thus, the negative contrast imaging technique shows the feasibility of using CLI to perform angiography on superficial blood vessels, demonstrating an advantage over conventional nuclear medicine techniques.

Comparison of Superparamagnetic and Ultrasmall Superparamagnetic Iron Oxide Cell Labeling for Tracking Green Fluorescent Protein Gene Marker with Negative and Positive Contrast Magnetic Resonance Imaging

The objectives of this study were to investigate the feasibility of imaging green fluorescent protein (GFP)-expressing cells labeled with iron oxide nanoparticles with the fast low-angle positive contrast steady-state free precession (FLAPS) method and to compare them with the traditional negative contrast technique. The GFP-R3230Ac cell line (GFP cell) was incubated for 24 hours using 20 microg Fe/mL concentration of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using positive contrast with FLAPS imaging, and FLAPS images were compared with negative contrast T2*-weighted images. The results demonstrated that SPIO and USPIO labeling of GFP cells had no effect on cell function or GFP expression. Labeled cells were successfully imaged with both positive and negative contrast magnetic resonance imaging (MRI). The labeled cells were observed as a narrow band of signal enhancement surrounding signal voids in FLAPS images and were visible as signal voids in T2*-weighted images. Positive contrast and negative contrast imaging were both valuable for visualizing labeled GFP cells. MRI of labeled cells with GFP expression holds potential promise for monitoring the temporal and spatial migration of gene markers and cells, thereby enhancing the understanding of cell- and gene-based therapeutic strategies.

Positiver Kontrast zum Nachweis magnetisch markierter Zellen in der MRT – in-vitro Experimente / Positive contrast in the detection of magnetically labeled cells by MRI – in vitro experiments

Magnetically labeled cells (MLC) cause local field inhomogenities within the single voxels as well as on a macroscopic scale. The related Larmor frequency shift near MLC was exploited to obtain bright visualization applying spectral selective saturation (SSS). SK-Mel28 cells were labeled with the superparamagnetic iron oxide contrast agent SHU 555A. Low cell concentrations (0, 5, 20, 30, 50, 75, and 150 MLC/microl) and high cell concentrations (10 x 10(3), 30 x 10(3), 60 x 10(3), and 100 x 10(3) MLC/10 microl) were examined at 3 Tesla. Shimming and frequency adjustment to spectrometer reference frequency v0 was performed with the built in routine of the scanner. A 2D spin echo sequence with broadband excitation and refocusing pulses was used (BWex = 1.000 Hz). Prior to each TR, a non-selective saturation sinc pulse centred at v0 was applied. Bandwidth (BWsat) of this pulse was varied from 100 Hz to 800 Hz in logarithmic steps. Without SSS the highest value of Crel (i.e. relative MR contrast between labeled to unlabeled samples) was found for 150 MLC/microl and was given by 10%. Applying SSS led to positive contrast of the complete labeled volumes and to remarkable improvements in Crel. With increasing cell concentrations Crel raised to maximum, that was given by 52% (BWsat=100 Hz) and 28% (BWsat = 200 Hz) found for 75 MLC/microl. For 150 MLC/microl Crel decreased. A contrast clarification could also be detected near cell aggregations despite saturation. Using SSS positive contrast can be achieved for voxels containing MLC and voxels close to cell clusters. Under in vitro conditions positive contrast improved the sensitivity to detect MLC as compared to negative contrast imaging techniques. It seems reasonable, that positive contrast approaches can be applied in vivo as the underlying physical mechanism are comparable.