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1.
J Biomed Opt ; 22(6): 66018, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28665439

ABSTRACT

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.


Subject(s)
Cytological Techniques/methods , Induced Pluripotent Stem Cells/cytology , Optical Imaging , Tomography , Animals , Fibroblasts/cytology , Lasers , Mice , Photons
2.
Methods Mol Biol ; 1453: 39-47, 2016.
Article in English | MEDLINE | ID: mdl-27431245

ABSTRACT

Mouse whiskers containing hair-follicle-associated pluripotent (HAP) stem cells, from nestin-driven green fluorescent protein (ND-GFP) transgenic mice, were placed in 3D histoculture supported by Gelfoam(®). ß-III tubulin-positive fibers, consisting of ND-GFP-expressing HAP stem cells, extended up to 500 mm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F-actin indicating they were growing axons. The growing whisker sensory nerve was highly enriched in ND-GFP HAP stem cells which appeared to play a major role in its elongation and interaction with other nerves placed in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results suggested that a major function of HAP stem cells in the hair follicle is for growth of the hair follicle sensory nerve.


Subject(s)
Hair Follicle/cytology , Nestin/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Vibrissae/metabolism , Animals , Cell Culture Techniques , Ganglia/cytology , Gene Expression , Mice , Mice, Transgenic , Microscopy, Confocal , Nestin/metabolism , Peripheral Nerve Injuries , Sciatic Nerve/cytology
3.
J Biomed Opt ; 21(6): 60501, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27251075

ABSTRACT

We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user's practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation.


Subject(s)
Cell Membrane/radiation effects , Cytological Techniques/instrumentation , Cytological Techniques/methods , Lasers , Optics and Photonics , Cell Membrane/chemistry , Microscopy, Confocal , Titanium , Ytterbium
4.
J Biomed Opt ; 21(3): 36002, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26953661

ABSTRACT

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.


Subject(s)
Cornea/diagnostic imaging , Microscopy, Confocal/methods , Optical Imaging/methods , Animals , Equipment Design , Swine
5.
J Neurooncol ; 127(3): 473-82, 2016 May.
Article in English | MEDLINE | ID: mdl-26830089

ABSTRACT

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.


Subject(s)
Brain Neoplasms/diagnosis , Glioma/diagnosis , Microscopy, Fluorescence, Multiphoton/methods , Optical Imaging/methods , Animals , Brain Neoplasms/surgery , Fluorescence , Glioma/surgery , Humans , Mice , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
J Biophotonics ; 9(9): 942-7, 2016 09.
Article in English | MEDLINE | ID: mdl-26530487

ABSTRACT

Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Microfluidics/methods , Transfection/methods , Cells, Cultured , Humans , Lasers
7.
J Biomed Opt ; 20(11): 115008, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26618522

ABSTRACT

We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.


Subject(s)
Cellular Reprogramming Techniques/instrumentation , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Lasers , Photic Stimulation/instrumentation , Transfection/instrumentation , Animals , Cell Differentiation/physiology , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Humans , Infrared Rays , Mice
8.
Cell Cycle ; 14(21): 3430-3, 2015.
Article in English | MEDLINE | ID: mdl-26397024

ABSTRACT

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.


Subject(s)
Cell Lineage , Cell Tracking/methods , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Hair Follicle/metabolism , Microscopy, Fluorescence, Multiphoton , Pluripotent Stem Cells/metabolism , Vibrissae/metabolism , Animals , Green Fluorescent Proteins/genetics , Hair Follicle/cytology , Mice, Inbred BALB C , Mice, Transgenic , Nestin/genetics , Promoter Regions, Genetic , Vibrissae/cytology
9.
J R Soc Interface ; 12(110): 0609, 2015 Sep 06.
Article in English | MEDLINE | ID: mdl-26333813

ABSTRACT

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54-1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26-2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.


Subject(s)
Bedbugs/metabolism , NADP/metabolism , Spermatozoa/metabolism , Animals , Cell Survival/physiology , Female , Male
10.
Anticancer Res ; 35(10): 5225-9, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26408681

ABSTRACT

BACKGROUND/AIMS: We previously developed a genetically-modified bacterial strain of Salmonella typhimurium, auxotrophic for leucine and arginine, which also expresses green fluorescent protein (GFP), termed S. typhimurium A1-R. S. typhimurium A1-R was found to be effective against metastatic human prostate, breast, pancreatic, cervical and ovarian cancer, as well as osteosarcoma, fibrosarcoma and glioma, in clinically relevant nude mouse models. MATERIALS AND METHODS: To understand the tumor cell-killing mechanism of S. typhimurium A1-R-GFP, we studied the interaction of S. typhimurium A1-R-GFP with three different prostate cancer cell lines in vitro. S. typhimurium-GFP invasion, proliferation, and means of killing in three different human prostate cancer cell lines were visualized by confocal fluorescence microscopy with the Olympus FV1000. RESULTS: We found that S. typhimurium A1-R-induced cancer-cell death had different mechanisms in different prostate cancer cell lines, occurring through apoptosis and necrosis in the PC-3 prostate cancer cell line, and by cell bursting in the LNCaP and DU-145 prostate cancer cell lines. The time required for S. typhimurium A1-R-GFP to kill the majority of cancer cells varied from line to line, ranging from 2 hours to 48 hours. CONCLUSION: Understanding the various mechanisms of cancer-cell killing by S. typhimurium A1-R will be important for its use as a general therapeutic for cancer.


Subject(s)
Biological Therapy , Cell Death , Prostatic Neoplasms/therapy , Salmonella typhimurium , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Male , Prostatic Neoplasms/pathology , Salmonella typhimurium/classification , Salmonella typhimurium/genetics
11.
J Med Imaging (Bellingham) ; 2(1): 016003, 2015 Jan.
Article in English | MEDLINE | ID: mdl-26158089

ABSTRACT

We report on a flexible multipurpose nonlinear microscopic imaging system based on a femtosecond excitation source and a photonic crystal fiber with multiple miniaturized time-correlated single-photon counting detectors. The system provides the simultaneous acquisition of e.g., two-photon autofluorescence, second-harmonic generation, and coherent anti-Stokes Raman scattering images. Its flexible scan head permits ex vivo biological imaging with subcellular resolution such as rapid biopsy examination during surgery as well as imaging on small as well as large animals. Above all, such an arrangement perfectly matches the needs for the clinical investigation of human skin in vivo where knowledge about the distribution of endogenous fluorophores, second-harmonic generation-active collagen as well as nonfluorescent lipids is of high interest.

12.
Sci Rep ; 5: 11185, 2015 Jun 08.
Article in English | MEDLINE | ID: mdl-26053047

ABSTRACT

Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.


Subject(s)
Automation, Laboratory/methods , Cell Membrane/metabolism , Lasers , Transfection/methods , Animals , CHO Cells , Cell Culture Techniques , Cell Line , Cricetulus
13.
Microsc Res Tech ; 77(12): 974-9, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25123087

ABSTRACT

We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in lab-on-a-chip devices.


Subject(s)
Lasers , Transfection/methods , Animals , CHO Cells , Cell Membrane/physiology , Cell Membrane/radiation effects , Cell Membrane Permeability/radiation effects , Cricetinae , Cricetulus , Flow Cytometry/methods
14.
Methods ; 66(2): 230-6, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-23867337

ABSTRACT

An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below -10 °C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20 mW mean powers.


Subject(s)
Cryopreservation , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Kinetics , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods
15.
J Cell Biochem ; 114(7): 1674-84, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23444061

ABSTRACT

We have previously reported that nestin-expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice), and were placed in 3D histoculture supported by Gelfoam®. ß-III tubulin-positive fibers, consisting of ND-GFP-expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F-actin. These findings indicate that ß-III tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND-GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin-expressing stem cells in the hair follicle is for growth of the follicle sensory nerve.


Subject(s)
Hair Follicle/cytology , Hair Follicle/metabolism , Intermediate Filament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Vibrissae/cytology , Vibrissae/metabolism , Animals , Ganglion Cysts/metabolism , Mice , Mice, Transgenic , Microscopy, Confocal , Nestin , Peripheral Nerves/cytology , Peripheral Nerves/metabolism , Sciatic Nerve/cytology , Sciatic Nerve/metabolism , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism , Trigeminal Nerve/cytology , Trigeminal Nerve/metabolism
16.
J Cell Biochem ; 114(1): 99-102, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22886742

ABSTRACT

Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo high resolution SHG multi-photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate.


Subject(s)
Colonic Neoplasms/metabolism , Extracellular Matrix/metabolism , Fibrillar Collagens/analysis , Molecular Imaging/methods , Photons , Tomography/methods , Animals , Cell Adhesion , Cell Line, Tumor , Cell Shape , Colonic Neoplasms/ultrastructure , Extracellular Matrix/ultrastructure , Fibrillar Collagens/metabolism , Genes, Reporter , Green Fluorescent Proteins , Injections, Subcutaneous , Mice , Mice, Nude , Neoplasm Transplantation , Transfection , Tumor Microenvironment
17.
J Biomed Opt ; 17(10): 101502, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23223978

ABSTRACT

ABSTRACT. Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima: 770 nm/830 nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles.


Subject(s)
Chromosomes/radiation effects , Cytological Techniques/methods , Lasers , Microscopy, Confocal/methods , Microsurgery/methods , Nanotechnology/methods , Chromosomes/ultrastructure , Cytological Techniques/instrumentation , Humans , Image Processing, Computer-Assisted , Infrared Rays , Leukocytes, Mononuclear/ultrastructure , Metaphase , Microscopy, Atomic Force/instrumentation , Microscopy, Atomic Force/methods , Microscopy, Confocal/instrumentation , Microsurgery/instrumentation , Nanomedicine , Nanotechnology/instrumentation
18.
Anticancer Res ; 32(10): 4331-7, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23060555

ABSTRACT

Our laboratory has previously developed a bacterial cancer therapy strategy by targeting tumors using engineered Salmonella typhimurium auxotrophs (S. typhimurium A1-R) that were generated to grow in viable as well as necrotic areas of tumors but not in normal tissue. The mechanism by which A1-R kills cancer cells is unknown. In the present report, high-resolution multiphoton tomography was used to investigate the cellular basis of bacteria killing of cancer cells in live mice. Lewis lung cancer cells (LLC) were genetically labeled with red fluorescent protein (RFP) and injected subcutaneously in nude mice. After tumor growth was observed, the mice were treated with A1-R bacteria expressing GFP, via tail-vein injection. Mice without A1-R treatment served as untreated controls. The imaging system was 3D scan head mounted on a flexible mechano-optical articulated arm. A tunable 80 MHz titanium:sapphire femtosecond laser (710-920 nm) was used for the multiphoton tomography. We applied this high-resolution imaging tool to visualize A1-R bacteria targeting the Lewis lung cancer cells growing subcutaneously in nude mice. The tomographic images revealed that bacterially-infected cancer cells greatly expanded and burst and thereby lost viability. Similar results were seen in vitro using confocal microscopy. The bacteria targeted the tumor within minutes of tail-vein injection. Using mice in which the nestin-promoter drives GFP and in which blood vessels are labeled with GFP, the bacteria could be imaged in and out of the blood vessels. Collagen scaffolds within the tumor were imaged by second harmonic generation (SHG). The multiphoton tomographic system described here allows imaging of cancer cell killing by bacteria and can therefore be used to further understand its mechanism and optimization for clinical application.


Subject(s)
Neoplasms/therapy , Organisms, Genetically Modified , Salmonella typhimurium , Animals , Cell Line, Tumor , Female , Genetic Engineering , Green Fluorescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Mice, Nude , Microscopy, Fluorescence, Multiphoton , Tomography , Red Fluorescent Protein
19.
In Vitro Cell Dev Biol Anim ; 48(5): 301-5, 2012 May.
Article in English | MEDLINE | ID: mdl-22580909

ABSTRACT

We have previously demonstrated that nestin-expressing multipotent hair follicle stem cells are located above the hair follicle bulge and can differentiate into neurons and other cell types in vitro. The nestin-expressing hair follicle stem cells promoted the recovery of pre-existing axons when they were transplanted to the severed sciatic nerve or injured spinal cord. We have also previously demonstrated that the whisker hair follicle contains nestin-expressing stem cells in the dermal papilla (DP) as well as in the bulge area (BA), but that their origin is in the BA. In the present study, we established the technique of long-term Gelfoam® histoculture of whiskers isolated from transgenic mice in which nestin drives green fluorescent protein (ND-GFP). Confocal imaging was used to monitor ND-GFP-expressing stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam® forming nerve-like structures. This new method of long-term histoculture of whiskers from ND-GFP mice will enable the extensive study of the behavior of nestin-expressing multipotent stem cells of the hair follicle.


Subject(s)
Hair Follicle/cytology , Multipotent Stem Cells/cytology , Vibrissae/cytology , Animals , Cell Differentiation , Green Fluorescent Proteins , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Mice , Mice, Transgenic , Microscopy, Confocal , Nerve Tissue/cytology , Nerve Tissue/growth & development , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , Organ Culture Techniques , Vibrissae/growth & development
20.
Cell Cycle ; 10(12): 2017-20, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21558804

ABSTRACT

We demonstrate noninvasive, high-resolution multiphoton tomography of nestin-expressing stem cells of hair follicles in living transgenic nude mice. An imaging system comprised of a compact femtosecond laser, 3D scan head mounted on a flexible mechano-optical articulated arm for simultaneous intra-tissue fluorescence and second harmonic detection (SHG) detection was used. This noninvasive method enables long-term in vivo tracking of intra-tissue stem cells in living animals. Multiphoton animal sectioning with subcellular resolution can visualize the real-time behavior of single stem cells in their native tissue microenvironment.


Subject(s)
Skin/cytology , Stem Cells/cytology , Tomography/instrumentation , Animals , Equipment Design , Intermediate Filament Proteins , Lasers , Mice , Mice, Nude , Mice, Transgenic , Nerve Tissue Proteins , Nestin
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