Raman ChemLighter: Fiber optic Raman probe imaging in combination with augmented chemical reality

Raman spectroscopy using fiber optic probe combines non‐contacted and label‐free molecular fingerprinting with high mechanical flexibility for biomedical, clinical and industrial applications. Inherently, fiber optic Raman probes provide information from a single point only, and the acquisition of images is not straightforward. For many applications, it is highly crucial to determine the molecular distribution and provide imaging information of the sample. Here, we propose an approach for Raman imaging using a handheld fiber optic probe, which is built around computer vision–based assessment of positional information and simultaneous acquisition of spectroscopic information. By combining this implementation with real‐time data processing and analysis, it is possible to create not only fiber‐based Raman imaging but also an augmented chemical reality image of the molecular distribution of the sample surface in real‐time. We experimentally demonstrated that using our approach, it is possible to determine and to distinguish borders of different bimolecular compounds in a short time. Because the method can be transferred to other optical probes and other spectroscopic techniques, it is expected that the implementation will have a large impact for clinical, biomedical and industrial applications.      

Diffuse and nonlinear imaging of multiscale vascular parameters for in vivo monitoring of preclinical mammary tumors

Diffuse optical imaging (DOI) techniques provide a wide‐field or macro assessment of the functional tumor state and have shown substantial promise for monitoring treatment efficacy in cancer. Conversely, intravital microscopy provides a high‐resolution view of the tumor state and has played a key role in characterizing treatment response in the preclinical setting. There has been little prior work in investigating how the macro and micro spatial scales can be combined to develop a more comprehensive and translational view of treatment response. To address this, a new multiscale preclinical imaging technique called diffuse and nonlinear imaging (DNI) was developed. DNI combines multiphoton microscopy with spatial frequency domain imaging (SFDI) to provide multiscale data sets of tumor microvascular architecture coregistered within wide‐field hemodynamic maps. A novel method was developed to match the imaging depths of both modalities by utilizing informed SFDI spatial frequency selection. An in vivo DNI study of murine mammary tumors revealed multiscale relationships between tumor oxygen saturation and microvessel diameter, and tumor oxygen saturation and microvessel length (|Pearson's ρ| ≥ 0.5, P < 0.05). Going forward, DNI will be uniquely enabling for the investigation of multiscale relationships in tumors during treatment.      

Comparative study of optical coherence tomography angiography algorithms for rodent retinal imaging

Optical coherence tomography angiography (OCTA) is a functional extension of optical coherence tomography for non-invasive in vivo three-dimensional imaging of the microvasculature of biological tissues. Several algorithms have been developed to construct OCTA images from the measured optical coherence tomography signals. In this study, we compared the performance of three OCTA algorithms that are based on the variance of phase, amplitude, and the complex representations of the optical coherence tomography signals for rodent retinal imaging, namely the phase variance, improved speckle contrast, and optical microangiography. The performance of the different algorithms was evaluated by comparing the quality of the OCTA images regarding how well the vasculature network can be resolved. Quantities that are widely used in ophthalmic studies including blood vessel density, vessel diameter index, vessel perimeter index, vessel complexity index were also compared. Results showed that both the improved speckle contrast and optical microangiography algorithms are more robust than phase variance, and they can reveal similar vasculature features while there are statistical differences in the calculated quantities.     

倏逝波光纤免疫传感器的光纤再生

倏逝波光纤免疫传感器作为一种新兴的检测技术,在环境检测、食品卫生检测及生物医学检测等领域具有广泛的应用前景。为了降低检测成本并达到快速检测,光纤再生问题就显得尤为重要。我们在对倏逝波光纤免疫传感器原理和光纤再生原理介绍的基础上,对光纤再生的酸性碱性溶液、高浓度盐溶液、去污剂等方法进行简要综述,探讨了光纤再生存在的问题,并展望了解决光纤重生后倏逝波光纤免疫传感器的应用前景。     

High-resolution imaging of proteins in human teeth by scanning probe microscopy

High-resolution studies of dental tissues are of considerable interest for biomedical engineering and clinical applications. In this paper, we demonstrate the application of piezoresponse force microscopy (PFM) to nanoscale imaging of internal structure of human teeth by monitoring the local mechanical response to an electrical bias applied via a conductive tip. It is shown that PFM is capable of detecting dissimilar components of dental tissues, namely, proteins and calcified matrix, which have resembling morphology but different piezoelectric properties. It is demonstrated that collagen fibrils revealed in chemically treated intertubular dentin exhibit high piezoelectric activity and can be visualized in PFM with spatial resolution of 10 nm. Evidence of the presence of protein inclusions of 100-200 nm wide and several micrometers long in tooth enamel has been obtained. Furthermore, it is found that the peritubular dentin and intertubular dentin exhibit different piezoelectric behavior suggesting different concentration of collagen fibrils. The obtained results demonstrate a high potential of PFM in providing an additional insight into the structure of dental tissues. It is suggested that the PFM approach can be used to study the structure of a wide range of biological materials by monitoring their electromechanical behavior at the nanoscale.     

A novel DAD type and folic acid conjugated fluorescent monomer as a targeting probe for imaging of folate receptor overexpressed cells

We describe a modification and post‐functionalization technique for a donor–acceptor–donor type monomer; 6‐(4,7‐bis(2,3‐dihydrothieno[3,4‐b][1,4]dioxin‐5‐yl)‐2H‐benzo[d][1,2, 3]triazol‐2‐yl)hexan‐1‐amine. Folic acid was attached to the fluorescent structure. The conjugation was confirmed via NMR and Fourier transform infrared analyses. Cytotoxicity was investigated and the comparison of association of targeted monomeric structures in tumor cells was monitored via fluorescence microscopy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:952–959, 2014     

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.     

Miniaturized all fiber probe for optical coherence tomography and pH detection of biological tissue

We present a novel all-fiber probe with 710-μm outside diameter for combined optical coherence tomography and pH detection. In cancer surgery, a significant challenge is how to completely remove the malignant tumor without cutting too much normal tissue. The difference between cancer tissue and normal tissue not only lies in morphology and structure but also in tissue pH, where malignant tissue has a lower pH. This dual-modality probe combined optical coherence tomography and pH detection of biological tissue, is expected to determine whether the tissue is cancerous quickly and accurately. The probe utilizes a typical three-segment structure (double-clad fiber - no-core fiber - graded-index fiber). We obtained a lateral resolution of ~10.6 μm, a working distance of ~506 μm and a pH measurement accuracy of 0.01 pH unit for the probe. The performance of the all-fiber probe was verified through an ex vivo experiment using the porcine brain specimen.     

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.     

Spectral imaging and its applications in live cell microscopy

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.     

AFM as a high-resolution imaging tool and a molecular bond force probe

This focused review summarizes certain aspects of recent developments in the use of atomic force microscope in high-resolution imaging of membrane-bound biological macromolecules and in molecular force probing of the interaction between biological conjugates. We point out the pros and cons in these approaches and critically analyze details involved in experiments.     

Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe

A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8+/-0.3ns but was reduced to 2.4+/-0.4 and 1.9+/-0.2ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5+/-0.5 and 1.7+/-0.4ns at a doxorubicin concentration of 750nM after 2 and 4h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.     

Indocyanine green-enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA-approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG-RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15-fold at 24 h post-injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.     

Cell imaging and manipulation by nonlinear optical microscopy

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second-harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.      

Photoacoustic and ultrasound (PAUS) dermoscope with high sensitivity and penetration depth by using a bimorph transducer

A bimorph transducer was proposed to improve the detection sensitivity and imaging depth of photoacoustic and ultrasound (PAUS) dermoscope. By applying the bimorph transducer, the imaging depth and sensitivity of PAUS dermoscope were enhanced by simultaneously improving excitation efficiency and reception bandwidth. The integrated design of the imaging head of the dermoscope makes it highly convenient for detecting human skin. The PAUS imaging performance was demonstrated via visualizing subcutaneous tumor and depicting full structures of different skin layers from epidermis to subcutaneous tissue. The results confirm that the dermoscope with the bimorph transducer is well suited for PA and US dual‐modality imaging, which can provide multi‐information for skin disease.     

Novel biomedical applications of Cerenkov radiation and radioluminescence imaging

The main goals of this review is to provide an up-to-date account of the different uses of Cerenkov radiation (CR) and radioluminescence imaging for pre-clinical small animal imaging. We will focus on new emerging applications such as the use of Cerenkov imaging for monitoring radionuclide and external radiotherapy in humans. Another novel application that will be described is the monitoring of radiochemical synthesis using microfluidic chips.Several pre-clinical aspects of CR will be discussed such as the development of 3D reconstruction methods for Cerenkov images and the use of CR as excitation source for nanoparticles or for endoscopic imaging.We will also include a discussion on radioluminescence imaging that is a more general method than Cerenkov imaging for the detection using optical methods of alpha and gamma emitters.     

Optimization for continuous-wave terahertz reflection imaging for biological tissues

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuous-wave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application.