The use of ultrashort-pulsed lasers for molecule delivery and transfection has proved to be a non-invasive and highly efficient technique for a wide range of mammalian cells. This present study investigates the effectiveness of femtosecond photoporation in plant cells, a hard-to-manipulate yet agriculturally relevant cell type, specifically suspension tobacco BY-2 cells. Both spatial and temporal shaping of the light field is employed to optimise the delivery of membrane impermeable molecules into plant cells using a reconfigurable optical system designed to be able to switch easily between different spatial modes and pulse durations. The use of a propagation invariant Bessel beam was found to increase the number of cells that could be viably optoinjected, when compared to the use of a Gaussian beam. Photoporation with a laser producing sub-12 fs pulses, coupled with a dispersion compensation system to retain the pulse duration at focus, reduced the power required for efficient optical injection by 1.5-1.8 times when compared to a photoporation with a 140 fs laser output.
We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5-5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.
We report the non linear fluorescence real-time detection of methylboldenone, an androgenic anabolic steroid used
illegally as growth promoter based on a resonant sensing chip: a double grating waveguide structure. The limit of
detection of this synthetic steroid is two orders of magnitude lower than the Minimum Required Performance Limit
required by the World Anti-Doping Agency. The immunoreagents have been have been immobilized onto the surface of
the resonant sensor after being activated with phosphonohexanoic acid spacers. The developed immunosensor presents
great potential as a robust sensing device for fast and early detection of illegal dopants and food contaminants.
Specimen-induced aberrations often affect microscopes, particularly when high numerical aperture lenses are used to
image deep into biological specimens. These aberrations cause a reduction in resolution and focal intensity. This is
particularly problematic in multiphoton microscopy, where the
non-linear nature of the signal generation process means
that the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of
adaptive optics to correct aberrations in two-photon fluorescence and harmonic generation microscopes, restoring image
quality. In particular we have used these microscopes for studies in developmental biology and for the imaging of
mammalian embryos.
The image resolution and contrast of microscopes are often detrimentally affected by aberrations that are introduced
when focusing deep into specimens. These aberrations arise from spatial differences in optical properties
of the specimen or refractive index mismatches. This is particularly problematic in multiphoton microscopy,
where short pulsed lasers are used to generate contrast through non-linear optical effects, such as two-photon
fluorescence or third harmonic generation. The non-linear nature of the signal generation process means that
the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of
adaptive optics to measure and correct the aberrations, restoring image quality. In particular, this has been
demonstrated in harmonic generation microscopy of developing mouse embryos. Similar aberration problems
affect the resolution and efficiency of three-dimensional optical fabrication systems, such those used for the manufacture
of photonic crystals or optical waveguides. These systems are based around microscope optics and use
short pulsed laser illumination to induce localized multiphoton effects in a fabrication substrate. In this case,
significant aberrations are introduced when focusing deep into the substrate. We report on the development
of adaptive optics systems for these applications and discuss the specific challenges for wave front sensing and
correction that are presented by these systems.
The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.
Because of its polarization sensitivity, SHG microscopy can provide information about the orientation and degree of
structural organization inside biological samples. To fully exploit the above potential, the state of the polarization at the
sample plane needs to be known. In this work we present starch granules as a reliable probe for the polarization state of
the excitation beam at the sample plane of a high resolution multiphoton microscope. Polarization dependent SHG series
of images demonstrated the radial distribution of SHG active molecules inside starch granules. This allowed the granule
to exhibit symmetrical SHG emission regions. The pattern rotates along with the rotation of a λ/2 waveplate and thus,
can demonstrate the polarization at the sample plane. Maximum signal in the forward detected geometry appears when
imaging starch granules exactly at the hemisphere plane. Symmetric SHG regions rotating with the incoming linear
polarization were also recorded in the backward detected geometry. A portion of the backwards detected SHG signal,
which corresponds to two rotating equator arcs, does not overlap with the forward SHG signal. Importantly, polarization
measurements, performed either in the forward or the backwards directions, have demonstrated the suitability and
flexibility of this technique for both detection schemes. As result, observation of the starch signal allowed us to know the
polarization of our SHG microscope. Furthermore, by coding this information in an angular representation, we corrected
the input values in a theoretical model that predicts the average orientation of SHG active molecules. This has allowed us
to map the mean orientation of SHG active molecules in body walls muscle of Caenorhabditis elegans, with pixel
resolution.
Dorsal closure is a key morphogenic process that occurs at the last stages of Drosophila melanogaster embryogenesis. It involves a well coordinated rearrangement and movement of tissues that resemble epithelial wound healing in mammals. The cell dynamics and intracellular signaling pathways that accompany hole closure are expected to be similar during would healing providing a model system to study epithelial healing. Here we demonstrate the use of
two-photon fluorescence microscope together with femtosecond laser ablation to examine the epithelial wound healing during embryonic dorsal closure. By using tightly focused NIR femtosecond pulses of subnanojoule energy we are able to produce highly confined microsurgery on the epithelial cells of a developing embryo. We observed that drosophila epidermis heals from the laser wounds with increased activity of actin near the wound edges.
KEYWORDS: Second-harmonic generation, Polarization, In vivo imaging, Image resolution, Signal detection, Harmonic generation, Tissues, Collagen, Nonlinear optics, Data modeling
Second harmonic generation (SHG) imaging has emerged in recent years as an important laboratory imaging technique
since it can provide unique structural information with submicron resolution. It enjoys the benefits of non-invasive
interaction establishing this imaging modality as ideal for in vivo investigation of tissue architectures. In this study we
present, polarization dependant high resolution SHG images of Caenorhabditis elegans muscles in vivo. We imaged a
variety of muscular structures such as body walls, pharynx and vulva. By fitting the experimental data into a cylindrical
symmetry spatial model we mapped the corresponding signal distribution of the χ(2) tensor and identified its main axis
orientation for different sarcomeres of the earth worm. The cylindrical symmetry was considered to arise from the thick
filaments architecture of the inside active volume. Moreover, our theoretical analysis allowed calculating the mean
orientation of harmonophores (myosin helical pitch). Ultimately, we recorded and analysed vulvae muscle dynamics,
where SHG signal decreased during in vivo contraction.
It is well known that the efficiency and selectivity of two-photon excited fluorescence (TPEF) process can
depend on various parameters of the ultrashort pulses, such as the pulse intensity and phase, which interact with the
specimen. In order to completely understand this dependence and to obtain optimal TPEF images, techniques like
Collinear Frequency Resolved Optical Gating (CFROG) arrangement can be implemented in a microscope for complete
pulse characterisation at the sample plane. However, this adds complexity that that additional forward collecting optics is
required as well as a suitable frequency doubling crystal.
Here we report a simple way to characterize the pulses within a multiphoton microscope that do not require
forward collecting optics. This is achieved by taking advantage of the fact that backward propagating second harmonic
generation (SHG) signal can be easily generated from starch granules. Since both the fluorescence and SHG signals can
be collected using the same detection scheme the experimental arrangement is considerably simplified. Starch, being a
non- toxic and non-soluble material does not affect living cells allowing the pulse characteristics to be measured in situ,
without the need to move the sample.
We obtained real-time SHG-autocorrelations traces by using a single starch granule that was placed alongside
the living HeLa cells (GFP labeled) being imaged. Furthermore by placing a spectrometer at the output port of the
microscope, a spectrally resolved SHG autocorrelation was acquired allowing complete characterisation of the pulse to
be carried out. The temporal intensity and phase profile were retrieved using CFROG technique. Marginal analysis was
carried out to ensure that the experimental data was successfully acquired.
We demonstrate an ultra sensitive method for Two Photon Fluorescence (TPF) excitation using resonant Grating Waveguide Structures (GWS). In its basic configuration, a GWS consists of a substrate, a waveguide layer and an additional grating layer. When illuminated with laser light under resonant conditions, the GWS reflects all light and leads to very high local surface intensities. This field enhancement can be exploited for TPF spectroscopy, without the need for a highly intense, focused laser light. We present the enhanced TPF signal obtained from a 23 nM drop of tetramethylrhodamine (TMR) on the top of high-finesse resonant polymeric GWS. The resonant behaviour of the GWS was tested for normal incidence with TE polarization illumination. As expected, the transmission spectral profile has a dip at resonant wavelength. The TPF spectra of TMR molecules were observed for different excitation wavelengths. Close to resonance, TPF intensity increases and the maximum signal is obtained when the excitation wavelength coincides with the resonance wavelength of the GWS. These results clearly indicate that the huge field localization at grating surface is responsible for the TPF excitation. We obtained a detection limit down to picomolar concentration of the dye molecules, offering the possibility of a highly sensitive, compact and non-destructive tool for widespread biochemical applications.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.