Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source

The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near‐infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two‐photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep‐tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher‐order nonlinearities such as three‐photon absorption that we use to induce spatially localized photodamage in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

用二次谐波成像技术研究经飞秒激光切削后角膜变化

本文用二次谐波成像技术（second harmonic generation SHG）来研究飞秒激光切削后角膜结构的变化.在生物学研究,材料科学等方面都有很广泛应用的SHG成像技术能在不破坏的角膜情况下获得高对比度的角膜层析图像,分辨率为500 nm,实验装置是利用现有的双光子显微镜.本文还根据成像结果评价了飞秒激光在角膜切削中的质量,为飞秒激光微米级的精确切削和临床应用提供了实验支持.     

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.     

Towards 3D modelling and imaging of infection scenarios at the single cell level using holographic optical tweezers and digital holographic microscopy

The analysis of dynamic interactions of microorganisms with a host cell is of utmost importance for understanding infection processes. We present a biophotonic holographic workstation that allows optical manipulation of bacteria by holographic optical tweezers and simultaneously monitoring of dynamic processes with quantitative multi‐focus phase imaging based on self‐interference digital holographic microscopy. Our results show that several bacterial cells, even with non‐spherical shape, can be aligned precisely on the surface of living host cells and localized reproducibly in three dimensions. In this way a new label‐free multipurpose device for modelling and quantitative analysis of infection scenarios at the single cell level is provided. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging

High power femto‐second (fs) laser pulses used for in‐vivo nonlinear optical (NLO) imaging can form cyclobutane pyrimidine dimers (CPD) in DNA, which may lead to carcinogenesis via subsequent mutations. Since UV radiation from routine sun exposure is the primary source of CPD lesions, we evaluated the risk of CPD‐related squamous cell carcinoma (SCC) in human skin due to NLO imaging relative to that from sun exposure. We developed a unique cancer risk model expanding previously published estimation of risk from exposure to continuous wave (CW) laser. This new model showed that the increase in CPD‐related SCC in skin from NLO imaging is negligible above that due to regular sun exposure. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.     

Selective Visualization of Fluorescent Sterols in Caenorhabditis elegans by Bleach‐Rate‐Based Image Segmentation

The nematode Caenorhabditis elegans is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE), is challenged by C. elegans’ high autofluorescence in the same spectral region as emission of DHE. We present a method to detect DHE selectively, based on its rapid bleaching kinetics compared to cellular autofluorescence. Worms were repeatedly imaged on an ultraviolet‐sensitive wide field (UV‐WF) microscope, and bleaching kinetics of DHE were fitted on a pixel‐basis to mathematical models describing the intensity decay. Bleach‐rate constants were determined for DHE in vivo and confirmed in model membranes. Using this method, we could detect enrichment of DHE in specific tissues like the nerve ring, the spermateca and oocytes. We confirm these results in C. elegans gut‐granule‐loss (glo) mutants with reduced autofluorescence and compare our method with three‐photon excitation microscopy of sterol in selected tissues. Bleach‐rate‐based UV‐WF imaging is a useful tool for genetic screening experiments on sterol transport, as exemplified by RNA interference against the rme‐2 gene coding for the yolk receptor and for worm homologues of Niemann‐Pick C disease proteins. Our approach is generally useful for identifying fluorescent probes in the presence of high cellular autofluorescence.     

Quantitative analysis of shape and volume changes in activated thrombocytes in real time by single‐shot spatial light modulator‐based differential interference contrast imaging

We suggest to use a combination of optical tweezers and single‐image quantitative differential interference contrast (DIC) emulated by a spatial light modulator (SLM) to study physiological shape changes in thrombocytes after activation and demonstrate the effectiveness of this system for the given task. A specially designed phase mask displayed at the SLM enables quantitative phase calculation from only a single recording. The optical tweezers stabilize trapped thrombocytes for long‐time monitoring of changes in the optical thickness profile of thrombocytes during activation by adenosine diphosphate (ADP). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Real‐time interactive two‐photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice

The potential usefulness of intravital two‐photon microscopy for fate mapping is limited by its inability to track cells beyond the confines of the imaging volume. Therefore, we have developed and validated a novel method for in vivo photolabelling of spatially‐restricted cells expressing the Kaede optical highlighter by two‐photon excitation. This has allowed us to optically mark a cohort of follicular B cells and track their dissemination from the original imaging volume in the lymph node to the spleen and contralateral lymph node. We also present the first demonstration, to our knowledge, of in vivo photoconversion of a freely moving single cell in a live adult animal. This method of `discontinuous' cell tracking therefore significantly extends the fate mapping capabilities of two‐photon microscopy to delineate the spatiotemporal dynamics of cellular processes that span multiple anatomical sites at the single cell level. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Two‐photon excited fluorescence lifetime measurements through a double‐clad photonic crystal fiber for tissue micro‐endoscopy

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two–photon excitation and time‐correlated single photon counting detection through a double‐clad photonic crystal fiber (DC‐PCF) without pre‐chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one‐photon‐based techniques, may be combined with morphology‐yielding techniques such as photoacoustic and ultrasound imaging. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Artefact reduction for cell migration visualization using spectral domain optical coherence tomography

Visualization of cell migration during chemotaxis using spectral domain optical coherence tomography (OCT) requires non‐standard processing techniques. Stripe artefacts and camera noise floor present in OCT data prevent detailed computer‐assisted reconstruction and quantification of cell locomotion. Furthermore, imaging artefacts lead to unreliable results in automated texture based cell analysis. Here we characterize three pronounced artefacts that become visible when imaging sample structures with high dynamic range, e.g. cultured cells: (i) time‐varying fixed‐pattern noise; (ii) stripe artefacts generated by background estimation using tomogram averaging; (iii) image modulations due to spectral shaping. We evaluate techniques to minimize the above mentioned artefacts using an 800 nm optical coherence microscope. Effect of artefact reduction is shown exemplarily on two cell cultures, i.e. Dictyostelium on nitrocellulose substrate, and retinal ganglion cells (RGC‐5) cultured on a glass coverslip. Retinal imaging also profits from the proposed processing techniques. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (α < eqid1 > β₂) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol {23}: 975–987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio–temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

Simultaneous multi‐site two‐photon photostimulation in three dimensions

We demonstrate simultaneous multi‐site two‐photon photolysis of caged neurotransmitters with close to diffraction‐limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two‐photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)