Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice

We investigated the effect of diet on abdominal autofluorescence detected by in vivo fluorescence imaging (FLI) of living mice. Groups of mice were fed a regular, alfalfa-free, or purified diet, and whole-body FLI was performed without the administration of fluorescent probes. In addition, quantum dots were injected intravenously into mice fed one of the three diets, and FLI was performed 3 and 24 hours later. Intense autofluorescence originating from the animals' intestinal contents was observed in mice fed the regular diet. Intestinal autofluorescence decreased substantially after feeding with the alfalfa-free diet and further after feeding with the purified diet. The decline was rapid and took only 1 to 2 days; however, it may have been affected by an intake of feces. The reticuloendothelial system was clearly delineated using a low dose of quantum dots in mice fed the purified diet. On the other hand, intestinal autofluorescence was visible 24 hours postinjection in mice given the alfalfa-free diet and definitely impaired the image quality in mice fed the regular diet. The use of a low-fluorescence diet, especially a purified diet, rapidly reduces intestinal autofluorescence and is expected to enhance the potential of in vivo FLI.     

Dual-laser, differential fluorescence correction method for reducing cellular background autofluorescence

A method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody-binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well-defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell-by-cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent-labeled antibody-binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.     

Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging

Non-invasive measurement of the optical density of the human macular pigment by the autofluorescence method takes advantage of the fluorescence of lipofuscin in the human retinal pigment epithelium. Measuring the intensity of fluorescence above 550 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, provides a single-pass measurement of the macular pigment optical density. The method is implemented either by fluorometry of lipofuscin to yield the optical density of the macular pigment in a 2 degrees diameter central area, or by autofluorescence imaging to yield a high-resolution map of the macular pigment distribution.     

In situ background estimation in quantitative fluorescence imaging

Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of our method and give example applications in ratiometric calcium imaging of bulk-stained olfactory bulb slices. Converting the fluorescence signals into [Ca2+] gives resting values consistent with earlier single-cell staining results, and odorant-induced [Ca2+] transients can be quantitatively compared in different cells. Using these examples we show that inaccurate background subtraction introduces large errors (easily in the range of 100%) in the assessment of both resting [Ca2+] and [Ca2+] dynamics. The proposed method allows us to avoid such errors.     

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction. CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However, the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module) and scFv-sfGFP-NLS(signal module). We removed the nuclear location sequence(NLS) of the signal module and inserted two copies of EGFP into the signal module. The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio) was decreased by 71%, and the ratio of the signal to the background(S/B ratio) was increased by 1.6 times. The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.     

Strategies to minimize false positives and interpret novel microdeletions based on maternal copy-number variants in 87,000 noninvasive prenatal screens

Background

Noninvasive prenatal screening (NIPS) of common aneuploidies using cell-free DNA from maternal plasma is part of routine prenatal care and is widely used in both high-risk and low-risk patient populations. High specificity is needed for clinically acceptable positive predictive values. Maternal copy-number variants (mCNVs) have been reported as a source of false-positive aneuploidy results that compromises specificity.

Methods

We surveyed the mCNV landscape in 87,255 patients undergoing NIPS. We evaluated both previously reported and novel algorithmic strategies for mitigating the effects of mCNVs on the screen’s specificity. Further, we analyzed the frequency, length, and positional distribution of CNVs in our large dataset to investigate the curation of novel fetal microdeletions, which can be identified by NIPS but are challenging to interpret clinically.

Results

mCNVs are common, with 65% of expecting mothers harboring an autosomal CNV spanning more than 200 kb, underscoring the need for robust NIPS analysis strategies. By analyzing empirical and simulated data, we found that general, outlier-robust strategies reduce the rate of mCNV-caused false positives but not as appreciably as algorithms specifically designed to account for mCNVs. We demonstrate that large-scale tabulation of CNVs identified via routine NIPS could be clinically useful: together with the gene density of a putative microdeletion region, we show that the region’s relative tolerance to duplications versus deletions may aid the interpretation of microdeletion pathogenicity.

Conclusions

Our study thoroughly investigates a common source of NIPS false positives and demonstrates how to bypass its corrupting effects. Our findings offer insight into the interpretation of NIPS results and inform the design of NIPS algorithms suitable for use in screening in the general obstetric population.

     

Bioluminescent imaging of hepatocellular carcinoma in live mice

A non-invasive orthotopic hepatocellular carcinoma (HCC) model was created with human HCC cells (HepG-Luc) constitutively expressing luciferase (Luc) in nude mice. Development of tumor growth and response to anti-tumor therapy combined with 5-fluorouracil and cisplatin was monitored by whole-body bioluminescent imaging (BLI). Luciferase activity in the tumor, determined by BLI, correlated with the tumor volume and weight. The anti-tumor therapy proved effective by BLI monitoring. In conclusion, BLI by luciferase provides a non-invasive method of monitoring tumor activities that can prove useful for therapeutic intervention studies.     

In vivo fluorescence imaging of bacteriogenic cyanide in the lungs of live mice infected with cystic fibrosis pathogens

Background

Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex (Bcc), commonly found in the lungs of cystic fibrosis (CF) patients, often produce cyanide (CN), which inhibits cellular respiration. CN in sputa is a potential biomarker for lung infection by CF pathogens. However, its actual concentration in the infected lungs is unknown.

Methods and Findings

This work reports observation of CN in the lungs of mice infected with cyanogenic PA or Bcc strains using a CN fluorescent chemosensor (4′,5′-fluorescein dicarboxaldehyde) with a whole animal imaging system. When the CN chemosensor was injected into the lungs of mice intratracheally infected with either PA or B. cepacia strains embedded in agar beads, CN was detected in the millimolar range (1.8 to 4 mM) in the infected lungs. CN concentration in PA-infected lungs rapidly increased within 24 hours but gradually decreased over the following days, while CN concentration in B. cepacia-infected lungs slowly increased, reaching a maximum at 5 days. CN concentrations correlated with the bacterial loads in the lungs. In vivo efficacy of antimicrobial treatments was tested in live mice by monitoring bacteriogenic CN in the lungs.

Conclusions

The in vivo imaging method was also found suitable for minimally invasive testing the efficacy of antibiotic compounds as well as for aiding the understanding of bacterial cyanogenesis in CF lungs.     

The EZC-prostate model: noninvasive prostate imaging in living mice

Recently, progress in the development of prostate-specific promoters and high resolution imaging techniques has made real-time monitoring of transgenic expression possible, opening a vista of potentially important in vivo models of prostate disease. Herein, we describe a novel prostate reporter model, called the EZC-prostate model that permits both ex vivo and in vivo imaging of the prostate using a sensitive charge-coupled device. Firefly luciferase and enhanced green fluorescent protein were targeted to the prostate epithelium using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P. In EZC-prostate mice, the ventral and dorsal/lateral prostate lobes were brilliant green under fluorescence microscopy, with expression localized to the secretory epithelium. In contrast, enhanced green fluorescent protein was undetectable in the anterior lobes of prostate, seminal vesicles, testes, liver, lung, and brain. The kinetics of luciferase activity in intact and castrated living mice monitored with the IVIS charge-coupled device-based imaging system confirmed that firefly luciferase expression was largely prostate restricted, increased with age up to 24 wk, and was androgen dependent. Decreases in reporter expression after 24 wk may reflect well known, age-related decreases in androgen signaling with age in humans. Ex vivo imaging of microdissected animals further confirmed that the luminescence detected in living mice emanated predominately from the prostate, with minor signals originating from the testes and cecum. These data demonstrate that the hK2-E3/P promoter directs strong prostate-specific expression in a transgenic mouse model. Multigenic models, generated by crosses with various hyperplastic and neoplastic prostate disease models, could potentially provide powerful new tools in longitudinal monitoring of changes in prostate size, androgen signaling, metastases, or response to novel therapies without sacrificing large cohorts of animals.     

Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.     

Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development

A transgenic mouse line named iUBC-KikGR was generated, which expresses the photoconvertible fluorescent protein Kikume Green-Red (KikGR) under the control of the human Ubiquitin C promoter. KikGR is natively a green fluorophore, which can be converted into a red fluorophore upon exposure to UV light. KikGR is expressed broadly throughout transgenic embryos from the two-cell stage onward and in the adult. Specificity of photoconversion can range from the entire embryo to a region of an organ, to a few individual cells, depending on the needs of the experimenter. Cell movements, tissue reorganization, and migration can then be observed in real time by culturing the tissue of interest as an explant on the microscope stage. The iUBC-KikGR transgenic line represents a singular genetic reagent, which can be used for fate mapping, lineage tracing, and live visualization of cell behaviors and tissue movements in multiple organs at multiple time points.     

High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy

Conventional two-dimensional cell monolayers do not provide the geometrical, biochemical and mechanical cues found in real tissues. Cells in real tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Such a highly interconnected communication network extends along all three dimensions. This architecture is lost in two-dimensional cultures. Therefore, at least in many cases, two-dimensional cell monolayers do not represent a suitable in vitro tool to characterize accurately the biology of real tissues. Many studies performed over the last few years have demonstrated that the differences between three-dimensional and two-dimensional cultured cells are striking at the morphological and molecular levels and that three-dimensional cell cultures can be employed in order to shrink the gap between real tissues and in vitro cell models. End-point and long-term imaging of cellular and sub-cellular processes with fluorescence microscopy provides direct insight into the physiological behavior of three-dimensional cell cultures and their response to chemical or mechanical stimulation. Fluorescence imaging of three-dimensional cell cultures sets new challenges and imposes specific requirements concerning the choice of a suitable microscopy technique. Deep penetration into the specimen, high imaging speed and ultra-low intensity of the excitation light are key requirements. Light-sheet-based fluorescence microscopy (LSFM) offers a favorable combination of these requirements and is therefore currently established as the technique of choice for the study of three-dimensional cell cultures. This review illustrates the benefits of cellular spheroids in the life sciences and suggests that LSFM is essential for investigations of cellular and sub-cellular dynamic processes in three-dimensions over time and space.     

Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research

Recently, there has been tremendous interest in developing techniques such as MRI, micro-CT, micro-PET, and SPECT to image function and processes in small animals. These technologies offer deep tissue penetration and high spatial resolution, but compared with noninvasive small animal optical imaging, these techniques are very costly and time consuming to implement. Optical imaging is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. In vivo optical imaging is the result of a coalescence of technologies from chemistry, physics, and biology. The development of highly sensitive light detection systems has allowed biologists to use imaging in studying physiological processes. Over the last few decades, biochemists have also worked to isolate and further develop optical reporters such as GFP, luciferase, and cyanine dyes. This article reviews the common types of fluorescent and bioluminescent optical imaging, the typical system platforms and configurations, and the applications in the investigation of cancer biology.     

Integrated lymphography using fluorescence imaging and magnetic resonance imaging in intact mice

We assessed lymph drainage in living mice by an integrated imaging method using fluorescence imaging (FLI) and magnetic resonance imaging (MRI). Mice were subcutaneously injected with quantum dots and gadofluorine 8 into the right rear footpad. They were fixed on a transparent flat plate and underwent FLI and MRI successively. Small markers were attached to the mouse surface for spatial coregistration, and image fusion of FLIs and MRIs was performed. Two-dimensional fluorescence reflectance imaging was used for FLI. FLI and MRI provided generally consistent results and demonstrated lymphatic flow to the popliteal, sacral, and iliac lymph nodes in most mice and to the renal, inguinal, and lumbar-aortic lymph nodes in some mice. On the fusion images, the locations of the lymph nodes in the mouse trunk were in good agreement between FLI and MRI, indicating successful spatial registration even for the deep structures. The popliteal node tended to be visualized a little farther caudally in FLI than in MRI, presumably because the overlying tissues were thicker in the cranial portion. Integrated FLI/MRI lymphography with image fusion appears to be a useful tool for analysis of the murine lymphatic system.     

Monomeric CFTR in plasma membranes in live cells revealed by single molecule fluorescence imaging

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel. There is indirect and conflicting evidence about whether CFTR exists in cell membranes as monomers, dimers, or higher order oligomers. We measured fluorescence intensities and photobleaching dynamics of distinct fluorescent spots in cells expressing functional CFTR-green fluorescent protein (GFP) chimeras. Intensity analysis of GFP-labeled CFTR in live cells showed single-component distributions with mean intensity equal to that of purified monomeric GFP, indicating monomeric CFTR in cell membranes. Fluorescent spots showed single-step photobleaching, independently verifying that CFTR is monomeric. Results did not depend on whether GFP was added to the CFTR N terminus or fourth extracellular loop or on whether CFTR chloride conductance was stimulated by cAMP agonists. Control measurements with a CFTR chimera containing two GFPs showed two-step photobleaching and a single-component intensity distribution with mean intensity twice that of monomeric GFP. These results provide direct evidence for monomeric CFTR in live cells.     

High-resolution nonlinear optical imaging of live cells by second harmonic generation

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.     

Smartphone-based multimodal tethered capsule endoscopic platform for white-light,narrow-band,and fluorescence/autofluorescence imaging

Multimodal low-cost endoscopy is highly desirable in poor resource settings such as in developing nations. In this work, we developed a smartphone-based low-cost, reusable tethered capsule endoscopic platform that allows white-light, narrowband, and fluorescence/autofluorescence imaging of the esophagus. The ex-vivo studies of swine esophagus were performed and compared with a commercial endoscope to test the white-light imaging capabilities of the endoscope. The efficacy of the capsule for narrow-band imaging was tested by imaging the vascularization of the tongue. To determine the autofluorescence/fluorescence capability of the endoscope, fluorescein dye with different concentrations was imaged. Furthermore, swine esophagus injected with fluorescein dye was imaged using the fluorescence/autofluorescence and the white-light imaging modules, ex-vivo. The overall cost of the capsules is approximately 12 €, 15 €, and 42 € for the white light imaging, the narrow-band imaging, and the fluorescence/autofluorescence imaging respectively. In addition, the cost of the laser source module required for the narrow-band imaging and the fluorescence/autofluorescence imaging is approximately 218 €. This device will open the possibility of imaging the esophagus in underprivileged areas.     

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs

Background

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells.

Methods and results

polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells.

Conclusions

The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

     

Synthesis of the Cyanine 7 labeled neutrophil-specific agents for noninvasive near infrared fluorescence imaging

A neutrophil-binding peptide, cinnamoyl-F(D)LF(D)LF (cFLFLF), was labeled with the near infrared (NIR) fluorophore, Cyanine 7 (Cy7). This construct was modified with a polyethylene glycol (PEG, M_W 3.4 kDa) moiety in order to increase its solubility and bioavailability to circulating neutrophils. A preliminary noninvasive fluorescence imaging of a mouse model of ear inflammation with the fluorescent probe is presented.     

Simple method for reduction of autofluorescence in fluorescence microscopy.

Autofluorescence of aldehyde-fixed neural tissue often complicates the use of fluorescence microscopy. Background fluorescence can be notably reduced or eliminated by irradiation with light before treatment with fluorescence probes, resulting in a higher contrast without adversely affecting the staining probabilities.