In minimally destructive SHG biomedical imaging (high resolution optical slicing) is greatly desirable to extract the
maximum of information from the light matter interaction. Here we develop a 3-D biophysical model and a
methodology, which extracts molecular information below the experimental resolution limit. Firstly, it provides the pitch
angle (SHG effective orientation) of the SHG source helix of the sample. This information is used to characterize and
categorize the SHG sources among them. And secondly, it provides the degree of organization of the SHG source
molecules. This can be used as a quantitative imaging biomarker able to characterize the degree of organization
(homeostasis) of the sample. Here we applied the model in dried and hydrated wheat starch granules. Our results show
that the SHG source molecule in starch is amylopectin. We also conclude that under hydration, the amylopectin
molecules are further organized but they do not change structure. This organization is reflected to the width of the pitch
angles pixels' histograms' distributions. The shorter the width is, the more organized the amylopectin molecules in starch
are.
Polarization sensitive second harmonic generation (PSHG) provides additional information in intensity only SHG
imaging. In particular, it offers the SHG effective orientation of the implicated SHG active structures. Assuming that
those structures possess cylindrical symmetry, the supplementary contrast is based on the ratio of two non-vanishing,
independent elements of the χ2 tensor. This ratio is experimentally extracted by fitting pixel by pixel a theoretical model
to the PSHG images and by finding the maximum frequency value or the mean of the consequent pixels' histogram. In
the present study we show that the above χ2 elements' ratio critically depends on the tilted-off the plane SHG active
structures. We performed PSHG in different z-planes of a starch granule (presenting radially oriented amylopectin
molecules, the SHG source in starch) and we found different pick values of the χ2 elements' ratio histogram for each
plane. By assuming a fixed value for the χ2 elements' ratio, we present here a generalized three dimensional (3D) model
that determines the 3D orientation of the SHG active structures.
Starch is one of the major constituents of our everyday diet and it forms granules. Starch granules are basically
consisting of amylose and amylopectin molecules and they are among the brightest nature-made second harmonic
generation (SHG) converters. In this study, we take advantage of that and we perform polarization sensitive SHG
(PSHG) imaging of starch granules. We fit the SHG signal variation of each pixel of the PSHG images into a biophysical
model and we extract molecular information below the experimental resolution limit. Specifically, by assuming that the
SHG source molecule is a helix with cylindrical symmetry along its long axis, the model extracts the helical pitch angle
of the SHG source and the orientation of its supporting filament for every pixel of the image. Pixel by pixel fitting
consequently creates new images which their contrast is based on the values of the fitting to the theoretical model. Then
we chose a region of interest in the image and we create pixels' values histograms. We applied the above in wheat starch
granules and we found a highly peaked pixels' histogram of helical pitch angles at θe = 36.2°. This pitch angle
corresponds to the strand of the parallel double helical structure, called amylopectin (as measured by small angle X-ray
scattering). Thus, using an optical technique we extracted the helical pitch angle of amylopectin in starch. This angle
value can be used as a quantitative biomarker capable of characterizing the quality of starch based structures and
products.
Polarization sensitive second harmonic generation (PSHG) imaging can provide useful information which is unreachable
by intensity SHG imaging. Specifically, it can provide geometrical characteristics of the SHG source molecular
architecture. The information is obtained by rotating the excitation linear polarization and by fitting the SHG intensity
variation to a cylindrical symmetry biophysical model. As a result, the ratios of the non-vanishing χ2 tensor elements,
responsible for the SHG conversion, are retrieved. In the end, by assuming a SHG source with dominant
hyperpolarizability, its molecular orientation can be estimated. Here, we developed and used this approach to retrieve
submicron structural information from cultured neurons and to provide estimation on the effective orientation of the
molecular SHG source in axons. For that purpose, the PSHG images of axons were fitted pixel by pixel using an
algorithm based on the above mentioned model. A coefficient of determination of r2>90% was chosen as a filtering
mechanism. For a selected region of interest we then retrieved the pixels' values histogram of the harmonophores'
effective orientations, θe. The distribution was centred at θe=34.93°, with σ=7.62°. These angle values correspond to the
geometrical characteristics of the tubulin heterodimmers forming the microtubules. Modifications on tubulin dimers may
alter θe, σ thus the PSHG optical technique suggests novel quantitative biomarkers able to characterize neurons'
plasticity as well as disease progression.
We use high resolution polarization second harmonic generation (PSHG) imaging microscopy in cultured neurons, and
we provide estimation on the effective orientation of the SHG source in neuronal processes in vitro. We performed pixel
by pixel analysis and we found a picked distribution of angles with maximum frequency at θe = 34.54°, with Δθe=11.44°.
This angle value is very close to the inclination of the tubulin heterodimmer with respect to the long axis of the
microtubule.
In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain
contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian
tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an
algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For
the whole image we retrieve for every pixel, the effective orientation, θe , of the SHG active structures. As a result a new
image is formed which its contrast depends on the values of θe . Collagen presented in the forward direction for a
predefined region of interest (ROI), peak distribution of angles θe centered in the region of ~45°, while muscle in the
region of ~65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we
show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any
exogenous labeling or any co-localization with fluorescence imaging.
Fetal growth restriction (FGR) has recently shown a strong association with cardiac programming which
predisposes to cardiovascular mortality in adulthood. Polarization Second Harmonic Microscopy can
quantify molecular architecture changes with high sensitivity in cardiac myofibrils. In this work, we use
myosin helical pitch angle as an example to quantify such alterations related to this high risk population.
Importantly, this shows a potential use of the technique as an early diagnostic tool and an alternative
method to understand pathophysiological processes.
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.
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.
Measurable change in the sensory motor machinery of growth cones are induced by non contact femtosecond laser. The
focused laser beam with an average power of 3 mW was positioned at some distance away from the closest fillopodia of
cortical neurons from primary cell cultures (mice E15). By identifying a set of preliminary parameters we were able to
statistically analyze the phenomenological behavior of the fillopodia and classify the effects different conditions of laser
light has on the growth cone. Results show that fillopodia become significantly biased towards the focused femtosecond
laser light. The same experiment performed with continuous wave (CW) produced results which were indistinguishable
from the case where there is no laser light present (placebo condition) indicating no clear effects of the CW laser light on
the fillopodia at a distance. These findings show the potential for ultrashort pulsed light to become a new type of
pathfinding cue for neuronal growth cones.
Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma
skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images
were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The
results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The
developed technique may provide an efficient real-time optical tool for detecting skin pathology.
In this paper we report the use of a starch as a non-linear medium for characterising ultrashort pulses. The starch suspension in water is sandwiched between a slide holder and a cover-slip and placed within the sample plane of the nonlinear microscope. This simple arrangement enables direct measurement of the pulse where they interact with the sample.
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