Alzheimer’s disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.
Higher-order nonlinearity of light–matter interactions, such as second and third harmonic
generation (SHG and THG) and Coherent anti-Stokes Raman Scattering (CARS) can be used for
improving spatial resolution in microscopy as a consequence of the confinement of the nonlinear
polarization to the high-intensity region of the focal volume. However, the resolution is limited to
~300 nm, not sufficient to resolve macromolecules or nanostructures of interest in the bio-, life-and
nano-sciences. In the strive to push the resolution beyond the diffraction limit, allowing for
nanoscale imaging, we have equipped a nonlinear optical microscope with a scanning-probe
setup operated in tapping-mode feedback. A tapered, gold-coated, open-aperture tip with an
aperture diameter of ~150 nm is scanned over the sample, probing the nonlinear nearfield
generated by free-beam excitation. First nonlinear coherent Raman nearfield images of biological
macromolecules and metallic nanostructures are shown. Limitations and future challenges with
nonlinear nearfield microscopy are discussed.
For studies of neuronal cell integration and neurite outgrowth in polymeric scaffold materials as a
future alternative for the treatment of damages in the neuronal system, we have developed a
protocol employing CARS microscopy for imaging of neuronal networks. The benefits of CARS
microscopy come here to their best use; (i) the overall three-dimensional (3D) arrangement of
multiple cells and their neurites can be visualized without the need for chemical preparations or
physical sectioning, potentially affecting the architecture of the soft, fragile scaffolds and (ii)
details on the interaction between single cells and scaffold fibrils can be investigated by close-up
images at sub-micron resolution. The establishment of biologically more relevant 3D neuronal
networks in a soft hydrogel composed of native Extra Cellular Matrix (ECM) components was
compared with conventional two-dimensional networks grown on a stiff substrate. Images of
cells in the hydrogel scaffold reveal significantly different networking characteristics compared
to the 2D networks, raising the question whether the functionality of neurons grown as layers in
conventional cultivation dishes represents that of neurons in the central and peripheral nervous
Microparticles loaded with antigens, proteins, DNA, fungicides, and other functional agents emerge as
ideal vehicles for vaccine, drug delivery, genetic therapy, surface- and crop protection. The
microscopic size of the particles and their collective large specific surface area enables highly active
and localized release of the functional substance. In order to develop designs with release profiles
optimized for the specific application, it is desirable to map the distribution of the active substance
within the particle and how parameters such as size, material and morphology affect release rates at
single particle level. Current imaging techniques are limited in resolution, sensitivity, image
acquisition time, or sample treatment, excluding dynamic studies of active agents in microparticles.
Here, we demonstrate that the combination of CARS and THG microscopy can successfully be used,
by mapping the spatial distribution and release rates of the fungicide and food preservative IPBC from
different designs of PMMA microparticles at single-particle level. By fitting a radial diffusion model
to the experimental data, single particle diffusion coefficients can be determined. We show that release
rates are highly dependent on the size and morphology of the particles. Hence, CARS and THG
microscopy provides adequate sensitivity and spatial resolution for quantitative studies on how singleparticle
properties affect the diffusion of active agents at microscopic level. This will aid the design of
innovative microencapsulating systems for controlled release.
The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH2-groups at 2845 cm−1 permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.
Hallmarks of high-fat Western diet intake, such as excessive lipid accumulation in skeletal muscle and liver as well as liver fibrosis, are investigated in tissues from mice using nonlinear microscopy, second harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS), supported by conventional analysis methods. Two aspects are presented; intake of standard chow versus Western diet, and a comparison between two high-fat Western diets of different polyunsaturated lipid content. CARS microscopy images of intramyocellular lipid droplets in muscle tissue show an increased amount for Western diet compared to standard diet samples. Even stronger diet impact is found for liver samples, where combined CARS and SHG microscopy visualize clear differences in lipid content and collagen fiber development, the latter indicating nonalcoholic fatty liver disease (NAFLD) and steatohepatitis induced at a relatively early stage for Western diet. Characteristic for NAFLD, the fibrous tissue-containing lipids accumulate in larger structures. This is also observed in CARS images of liver samples from two Western-type diets of different polyunsaturated lipid contents. In summary, nonlinear microscopy has strong potential (further promoted by technical advances toward clinical use) for detection and characterization of steatohepatitis already in its early stages.
A compact high-power fiber-based femtosecond laser system is presented for coherent anti-Stokes Raman scattering/second-harmonic generation (CARS/SHG) microscopy, and quantitatively compared with a conventional picosecond optical parametric oscillator (OPO)-based system. While the broad spectral width of the femtosecond pulses results in 2.5 times lower image contrast and limited spectral selectivity, lipid stores, myosin, and collagen filaments in living cells can clearly be identified at 60 times lower excitation powers compared to the picosecond system. Visually the images contain the same information. Together with simple operation, small footprint, and low cost, the capabilities of this high-power all-fiber-based laser system promise a more general use of nonlinear microscopy within the biosciences.
Major efforts are presently made to develop artificial replacement tissues with optimal architectural and
material characteristics, mimicking those of their natural correspondents. Encouraged by the readiness with
which cellulose fibers woven by the bacteria Acetobacter xylinum can be formed into organ-like macroscopic
shapes and with different microscopic textures, it emerges as an interesting material within tissue engineering.
We have developed a protocol employing simultaneous CARS and SHG microscopy for monitoring the
cellulose network characteristics and its impact on the integration of smooth muscle cells (SMCs) for
functionalized artificial tissues. CARS and SHG overlay images of the cells and the cellulose fibers reveal an
immediate interaction irrespective of scaffold morphology and that the SMCs attach to the cellulose fibers
already during the first cultivation day without cell-adhesive coatings. During the subsequent 28 days, SMCs
were found to readily proliferate and differentiate on the cellulose scaffold without the need for exogenous
growth factors. However, the efficiency with which this occurred depended on the topography of the cellulose
constructs, benefited by porous and less compact matrices. This brings forward the need for in-depth studies
on how the microstructure of tissue scaffolds influences and can be optimized for native cell integration and
proliferation, studies where the benefits of multi-modal non-linear microscopy can be fully exploited.
We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the
biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a
product with material properties similar to those of human blood vessels. Grown in a tubular geometry
it could then be used as a natural and biocompatible source of replacement tissue in conjunction with
cardiovascular surgery. The bacteria (<i>Acetobacter xylinum</i>) were selectively visualized based on the
CH<sub>2</sub> vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering
(CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose
fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection
approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of
e.g. different growth conditions on fiber thickness and orientation, their assembling into higher-order
structures and overall network density. The bacterial and fiber distributions were monitored in a
simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation
process of tubular cellulose grafts. The CARS and SHG
co-localization images reveal that highest
bacterial population densities can be observed in the surface regions of the cellulose tissue, where the
primary growth presumably takes place. The cellulose network morphology was also compared with
that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively
homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This
prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and
shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to
contribute to the fundamental knowledge required for optimal production of bioengineered cellulose
tissues, eventually being available for clinical use.
After several years of proof-of-principle measurements and focus on technological development, it is timely to
make full use of the capabilities of CARS microscopy within the biosciences. We have here identified an urgent
biological problem, to which CARS microscopy provides unique insights and consequently may become a
widely accepted experimental procedure. In order to improve present understanding of mechanisms underlying
dysfunctional metabolism regulation reported for many of our most wide-spread diseases (obesity, diabetes,
cardio-vascular diseases etc.), we have monitored genetic and environmental impacts on cellular lipid storage in
the model organism <i>C. elegans</i> in vivo in a full-scale biological study. Important advantages of CARS
microscopy could be demonstrated compared to present technology, i.e. fluorescence microscopy of labelled
lipid stores. The fluorescence signal varies not only with the presence of lipids, but also with the systemic
distribution of the fluorophore and the chemical properties of the surrounding medium. By instead probing high-density
regions of CH bonds naturally occurring in the sample, the CARS process was shown to provide a
consistent representation of the lipid stores. The increased accumulation of lipid stores in mutants with
deficiencies in the insulin and transforming growth factor signalling pathways could hereby be visualized and
quantified. Furthermore, spectral CARS microscopy measurements in the C-H bond region of 2780-2930 cm<sup>-1</sup>
provided the interesting observation that this accumulation comes with a shift in the ordering of the lipids from
gel- to liquid phase. The present study illustrates that CARS microscopy has a strong potential to become an
important instrument for systemic studies of lipid storage mechanisms in living organisms, providing new
insights into the phenomena underlying metabolic disorders.
We present a new Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique for label-free imaging of biomolecules
in living cells; dual-CARS microscopy. The use of three synchronized laser pulses in a dual-pump/dualdetection
configuration enables imaging of two species with different molecular vibrations simultaneously, as well as
acquisition of images free of non-resonant background. We show the power of the method by imaging deuterated
nonadecane slowly diffusing into a suspension of living yeast cells in medium, clearly distinguishing the medium and
the lipid droplets in the cells by probing the CH<sub>2</sub> vibration from the D-nonadecane by probing the CD vibration. In
addition, images of lipid stores in living <i>C. elegans</i> nematodes free of non-resonant background are shown. This results
in a significant enhancement of the image contrast, allowing the visualization of emerging, low-density lipid stores in a
dauer larva, difficult to distinguish in conventional CARS microscopy. The separation of the non-resonant background
is shown to be beneficial also when monitoring molecules with weak vibrational modes. The improved sensitivity
obtained is illustrated by probing the C=C vibration in polyunsaturated lipids extracted from fish. This enables the
monitoring of the degree of unsaturation of lipids, a high value of which is reported in foods known to have positive
effects on human health.
We introduce near-infrared Coherent Anti-Stokes Raman Scattering (CARS) microscopy as a method for the monitoring of fat deposition in a living organism by directly probing the CH<sub>2</sub> vibration of the lipids without the need for staining or labeling. This study nicely brings forward all the advantages of the technique: deep probe depth, low excitation powers, high 3-dimensional resolution, and visualization without the interference of exogenous label molecules, or fixation and staining procedures. Differences in fat deposition during the life cycle of the nematode <i>Caenorhabditis elegans</i> were evaluated quantitatively from the CARS microscopy images, showing that the technique can be used to study mechanisms that regulate lipid storage. Beside the wild type nematode, the feeding-deficient mutant <i>pha-3</i> was studied. It was shown that the embryonal accumulation of energy stores is enough for the development of a full-sized pre-adult larva, being possible also for the mutant. However, the volume density of lipid stores at the fourth and last pre-adult development stage seems to determine its adult body size. Whereas the wild type larva maintains its size when becoming adult, though at the cost of reduced lipid density, the feeding deficient mutant instead has to reduce its body size in order to reach the same volume density of lipid stores. Both strains start off their adult life with a volume fraction of lipid stores corresponding to 6-7%; the wild type with a radius of 24±2 µm and the <i>pha-3</i> mutant with a significantly smaller radius of 16±3 μm.
We report the first successful study of the use of Raman spectroscopy for quantitative, noninvasive ("transcutaneous") measurement of blood analytes, using glucose as an example. As an initial evaluation of the ability of Raman spectroscopy to measure glucose transcutaneously, we studied 17 healthy human subjects whose blood glucose levels were elevated over a period of 2–3 h using a standard glucose tolerance test protocol. During the test, 461 Raman spectra were collected transcutaneously along with glucose reference values provided by standard capillary blood analysis. A partial least squares calibration was created from the data from each subject and validated using leave-one-out cross validation. The mean absolute errors for each subject were 7.8%±1.8% (mean±std) with R2 values of 0.83±0.10. We provide spectral evidence that the glucose spectrum is an important part of the calibrations by analysis of the calibration regression vectors.
Coherent Anti-Stokes Raman Scattering (CARS)-microscopy has in recent years developed as a promising microscopical
technique for label-free microscopy of living cells. We propose a new concept, spectral focusing, for
highly efficient coherent anti-Stokes Raman scattering (CARS) microscopy. It allows optimal use of the excitation
energy of femtosecond laser pulses in terms of generated CARS signal against a low background. This
is accomplished by introducing a linear chirp in the excitation pulses. The temporal delaying of the excitation
pulses can be used to record vibrational spectra of a sample. Despite the inherently broad spectral width of the
excitation pulses, the technique enables resolution of spectral features 60 times narrower than the bandwidth of
the probe light. First applications of this technique are exemplified with CARS of micron sized crystallites of
sodium nitroprusside, a commonly used hypotensive agent.
Time-efficient Monte Carlo models for fluorescence from layered tissue were developed. The computation time is reduced significantly by recognizing symmetry properties of the problem, and by reversing computation of the photon paths for the fluorescence light. Further reduction is obtained by using a white Monte Carlo approach, which enables scaling of the results to the desired optical properties after the simulation. The methods reduce computation time more than two orders of magnitude compared with conventional Monte Carlo code.