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.     

Pontamine sky blue: a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry

The stain pontamine sky blue (PSB) has been shown to reduce background autofluorescence in catecholamine fluorescence and immunofluorescence histochemical preparations. Using PSB as a counterstain on whole-mount stretch preparations of human mesenteric blood vessels, a medium dense noradrenergic nerve plexus is clearly revealed, which previously had been only partially visible because of background autofluorescence. Image analysis of nerve densities in whole-mount stretch preparations of guinea-pig arteries containing noradrenergic, substance P-, and vasoactive intestinal polypeptide (VIP)-positive nerve plexuses shows that PSB staining does not alter the specific neuronal fluorescence and that it improves image definition.     

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.

     

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3‐D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.

     

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.     

Noninvasive multimodal imaging by integrating optical coherence tomography with autofluorescence imaging for dental applications

We report the development of an integrated multifunctional imaging system capable of providing anatomical (optical coherence tomography, OCT), functional (OCT angiography, OCTA) and molecular imaging (light‐induced autofluorescence, LIAF) for in vivo dental applications. Blue excitation light (405 nm) was used for LIAF imaging, while the OCT was powered by a 1310 nm swept laser source. A red‐green‐blue digital camera, with a 450 nm cut‐on broadband optical filter, was used for LIAF detection. The exciting light source and camera were integrated directly with the OCT scanning probe. The integrated system used two noninvasive imaging modalities to improve the speed of in vivo OCT data collection and to better target the regions of interest. The newly designed system maintained the ability to detect differences between healthy and hypomineralized teeth, identify dental biofilm and visualize the microvasculature of gingival tissue. The development of the integrated OCT‐LIAF system provides an opportunity to conduct clinical studies more efficiently, examining changes in oral conditions over time.     

Fast noninvasive functional diffuse optical tomography for brain imaging

Advances in epilepsy studies have shown that specific changes in hemodynamics precede and accompany seizure onset and propagation. However, it has been challenging to noninvasively detect these changes in real time and in humans, due to the lack of fast functional neuroimaging tools. In this study, we present a functional diffuse optical tomography (DOT) method with the guidance of an anatomical human head atlas for 3‐dimensionally mapping the brain in real time. Central to our DOT system is a human head interface coupled with a technique that can incorporate topological information of the brain surface into the DOT image reconstruction. The performance of the DOT system was tested by imaging motor tasks‐involved brain activities on N = 6 subjects (3 epilepsy patients and 3 healthy controls). We observed diffuse areas of activations from the reconstructed [HbT] images of patients, relative to more focal activations for healthy subjects. Moreover, significant pretask hemodynamic activations were also seen in the motor cortex of patients, which indicated abnormal activities persistent in the brain of an epilepsy patient. This work demonstrates that fast functional DOT is a valuable tool for noninvasive 3‐dimensional mapping of brain hemodynamics.      

Visualization of pulmonary inflammation using noninvasive fluorescence molecular imaging.

The ability to visualize molecular processes and cellular regulators of complex pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), or adult respiratory distress syndrome (ARDS), would aid in the diagnosis, differentiation, therapy assessment and in small animal-based drug-discovery processes. Herein we report the application of normalized transillumination and fluorescence molecular tomography (FMT) for the noninvasive quantitative imaging of the mouse lung in vivo. We demonstrate the ability to visualize and quantitate pulmonary response in a murine model of LPS-induced airway inflammation. Twenty-four hours prior to imaging, BALB/c female mice were injected via tail vein with 2 nmol of a cathepsin-sensitive activatable fluorescent probe (excitation: 750 nm; emission: 780 nm) and 2 nmol of accompanying intravascular agent (excitation: 674 nm; emission: 694 nm). Six hours later, the mice were anesthetized with isoflurane and administered intranasal LPS in sterile 0.9% saline in 25 microl aliquots (one per nostril). Fluorescence molecular imaging revealed the in vivo profile of cysteine protease activation and vascular distribution within the lung typifying the inflammatory response to LPS insult. Results were correlated with standard in vitro laboratory tests (Western blot, bronchoalveolar lavage or BAL analysis, immunohistochemistry) and revealed good correlation with the underlying activity. We demonstrated the capacity of fluorescence tomography to noninvasively and longitudinally characterize physiological, cellular, and subcellular processes associated with inflammatory disease burden in the lung. The data presented herein serve to further evince fluorescence molecular imaging as a technology highly appropriate for the biomedical laboratory.     

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.     

An optical system for noninvasive microscopy of psoriatic mice in vivo

Psoriasis is a chronic inflammatory skin disease involved with both complex morphological changes of skin and immune processes. The clinical diagnostics and research of psoriasis often require invasive biopsy which lacks their real-time dynamics in vivo. Here we report a noninvasive microscopic system developed by combining in vivo fluorescent microscopy, optical clearing, and immunolabeling to enable real-time imaging of immune cells and cytokines in blood flow in psoriatic animal models. The vascular morphology and time-lapse kinetics of interleukin (IL)-23, IL-17, tumor necrosis factor-α, and CD4+ cells in blood are captured at submicron resolution through the thickening epidermis and opaque scales during the development of psoriasis in vivo. Our data suggest IL-23 recruits CD4+ cells to release IL-17 in blood that further leaks out in the psoriatic skin area. This optical system enables noninvasive and real-time assessment of immune molecules and cells in vivo, providing good potential for medical researches on psoriasis.     

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.     

Long-term, noninvasive imaging of regulated gene expression in living mice

We describe here an approach for monitoring regulated gene expression by noninvasive imaging in living mice. We have utilized the tetracycline inducible system to simultaneously coregulate the expression of two genes encoding the firefly luciferase and the Cre recombinase, respectively. Results from our model system demonstrate that luciferase can be used as a noninvasive imaging marker for the regulated expression of a second gene in living mice. The integration of noninvasive imaging and inducible gene expression into current approaches of functional genomics should greatly advance our capabilities of carrying out highly controlled long-term studies of gene function in individual mice.     

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.