Chunqiang Li

Effect of surface coating and organic matter on the uptake of CeO2 NPs by corn plants grown in soil: Insight into the uptake mechanism

Little is known about the fate, transport, and bioavailability of CeO(2) nanoparticles (NPs) in soil. Moreover, there are no reports on the effect of surface coating upon NPs uptake by plants. In this study, Zea mays plants were grown for one month in unenriched and organic soils treated with coated and uncoated CeO(2) NPs. In addition, plants were exposed to fluorescein isothiocyanate (FITC)-stained CeO(2) NPs and analyzed in a confocal microscope. In organic soil, roots from uncoated and coated NPs at 100, 200, 400, and 800mg kg(-1) had 40, 80, 130, and 260% and 10, 70, 90, and 40% more Ce, respectively, compared to roots from unenriched soil. Conversely, shoots of plants from unenriched soil had significantly more Ce compared with shoots from organic soil. Confocal fluorescence images showed FITC-stained CeO(2) NP aggregates in cell walls of epidermis and cortex, suggesting apoplastic pathway. The μXRF results revealed the presence of CeO(2) NP aggregates within vascular tissues. To the authors knowledge this is the first report on the effects of surface coating and organic matter on Ce uptake from CeO(2) NPs and upon the mechanisms of CeO(2) NPs uptake by higher plants.

Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence

We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

Multiphoton Microscopy of Live Tissues With Ultraviolet Autofluorescence

Current research on multiphoton autofluorescence microscopy is primarily focused on imaging the signal from reduced nicotinamide adenine dinucleatide (NADH) in tissue. NADH levels in cells are useful reporters of metabolic information, as well as early indicators in precancer and cancer diagnosis. While NADH is typically imaged in the 400-500 nm spectral window, the amino acid tryptophan is the major source of tissue fluorescence in the Ultraviolet range. Here, we briefly review current progress in multiphoton autofluorescence imaging of live tissues and cells, and report our recent findings of in vivo mouse skin imaging based on multiphoton excited tryptophan autofluorescence. This new method enables noninvasive imaging of skin tissue at video-rate and allows for the visualization and identification of cellular components in the epidermis, dermis, and muscle layers. It is also possible to image through small blood vessels in the mouse skin and observe circulating leukocytes in situ.

Inflammation Modulates Murine Venous Thrombosis Resolution In Vivo Assessment by Multimodal Fluorescence Molecular Imaging

Objective—Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here, we develop and evaluate 2 integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo. Methods and Results—Murine DVT were created with topical 5% FeCl3 application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy. Day 4 analyses showed robust relationships among in vivo thrombus macrophages, matrix metalloproteinase activity, and fluorescein isothiocyanate-dextran deposition (r>0.70; P<0.01). In a serial 2–time point study, mice with DVT underwent intravital microscopy at day 4 and day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (P<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography was used and detected macrophages within jugular DVT (P<0.05 versus sham controls). Conclusion—Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo and can inform the magnitude of thrombus resolution.

High-contrast mapping of basal cell carcinomas

Because of low optical contrast in the visible spectral range, accurate detection of basal cell carcinomas (BCC) remains a challenging problem. In this letter, we experimentally demonstrate that reflectance confocal imaging in the vicinity of 1300 nm can be used for the detection of BCC without exogenous contrast agents. We present high-contrast reflectance confocal images of thick fresh skin tissues with clearly delineated cancer and discuss possible reasons for causing decreased scattering of BCC. Comparison with histopathology confirms that tumors scatter less and exhibit lower pixel values in the images, as compared to benign skin structures. The results demonstrate the feasibility of real-time noninvasive detection of BCC using intrinsic differences in scattering between tumors and normal skin.

Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy

Transition from latency to active tuberculosis requires Mycobacterium tuberculosis (Mtb) to penetrate the phagosomal membrane and translocate to the cytosol of the host macrophage. Quantitative two-photon fluorescence resonance energy transfer (FRET) microscopy is developed to measure cytosolic translocation using Mycobacterium marinum (Mm) as a model organism for Mtb. Macrophages were infected with Mm or non-pathogenic Mycobacterium smegmatis (Ms) as a control, then loaded with a FRET substrate. Once translocation occurs, mycobacterium-bearing β-lactamase cleaves the substrate, resulting in decrease of FRET signal. Quantification of this FRET signal change revealed that Mm, but not Ms, is capable of translocating to the cytosol.

Dual-color multiple-particle tracking at 50-nm localization and over 100-µm range in 3D with temporal focusing two-photon microscopy.

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking.

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs), mast cells, vascular smooth muscle cells (SMCs). Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG) were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs) which participate in the degradation of collagen.

Photolysis of a peptide with n-peptidyl-7-nitroindoline units using two-photon absorption

N-acyl-7-nitroindolines have been used as caged compounds to photorelease active molecules by a one- or two-photon excitation mechanism in biological systems. Here, we report the photolysis of a polypeptide that contains 7-nitroindoline units as linker moieties in its peptide backbone for potential materials engineering applications. Upon two-photon excitation with femtosecond laser light at 710 nm the photoreactive amide bond in N-peptidyl-7-nitroindolines is cleaved rendering short peptide fragments. Thus, this photochemical process changes the molecular composition at the laser focal volume. Gel modifications of this peptide can potentially be used for three-dimensional microstructure fabrication.

Imaging immune response of skin mast cells in vivo with two-photon microscopy

Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.