SignificanceRapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques.AimWe aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth.ApproachStandards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers.ResultsThese standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and 1240 μm, respectively. Once fabricated, the phantoms were found to be stable for many months.ConclusionsThe ability to image at depth, the phantom’s robustness and flexible layered structure, and the ready incorporation of “optical markers” make these ideal depth standards for the validation of a variety of imaging modalities.
Multiphoton microscopies are an invaluable tool in biomedical imaging given their inherent capabilities for label free imaging, optical sectioning, chemical and structural specificity. They comprise various types of Coherent Raman microscopies (CR), such as Coherent Anti-Stokes Raman Scattering (CARS), Stimulated Raman Loss (SRL) or Stimulated Raman Gain, different kinds of Harmonic Generation imaging (HG) such as Second and Third Harmonic Generation (SHG and THG respectively), and Multiphoton Autofluorescence imaging (MA) such as Two and Three Photon Excited Autofluorescence (TPEAF and ThPEAF respectively). Despite their significant advantages, multiphoton microscopies, comparably to all other types of optical microscopies, exhibit limited penetration depth in tissue due to absorption and scattering. In this work we explore the advantages of multiphoton microscopies in hard and soft deep tissue imaging when using excitation wavelengths in the range of Short-Wavelength Infrared (SWIR) windows which occur between 1000 nm and 2500 nm. These spectral windows have notable merits including longer attenuation lengths and none or very low signal absorption observed for almost all kinds of multiphoton microscopy. We show results of using excitations in the SWIR windows, generated by standard as well as novel sources, such as a thulium fibre laser, in different types of multiphoton microscopy on a variety of hard and soft tissue samples (bone, cartilage and other tissue types) and demonstrate the advantages of using excitations in this wavelength range, including longer penetration depth and high resolution for deep tissue imaging.
Multiphoton imaging methods such as Coherent Raman Scattering (CRS) microscopy which also comprises Second
Harmonic Generation (SHG) and Two Photon Excited Auto-Fluorescence (TPEAF) imaging (termed as multimodal
Coherent Raman microscopy), have greatly facilitated the advancement of biomedical research due to their unique
features. Multimodal CRS microscopy, is label free, chemically specific, inherently ‘confocal’ offering three independent
contrast mechanisms which can be associated in a composite image comprising a wide range of chemical and structural
information about the interrogated sample. The standard light source for multimodal CRS microscopy is a picosecond
pumped Optical Parametric Oscillator (OPO) which has exhibited excellent performance but due to its associated high
cost, maintenance, complexity and requirement of a dedicated optics laboratory, has hindered the wider adoption of
multimodal CRS microscopy and especially its deployment in clinical applications.
Here we present a novel, low cost Optical Parametric Amplifier (OPA) based on a MgO doped Periodically Poled Lithium
Niobate (PPLN) crystal seeded by a continuous wave (CW) laser source and pumped by a picosecond laser at 1031nm,
which removes any synchronisation requirements. We show that this OPA is a versatile light source module that can be
tailored to the tunability and affordability requirements of the specific application. We demonstrate that it can be used
either in association with an OPO or on its own as a light source for multimodal CRS microscopy and we show its
performance by imaging a variety of standards and biological samples.
We show that in the presence of fullerene complexes the optical properties of PbS QDs are significantly modified. The
absorption of the PbS QDs is observed to shift to a higher energy when fullerene complexes are introduced. Upon direct
excitation of the PbS below the fullerene absorption a corresponding blue shift in PL spectra of the PbS QDs is observed.
The strength of this blue-shift can be related to the fullerene concentration in most cases and is accompanied by a
broadening of the emission spectrum. When exciting the samples at high energy 3.4 eV (363 nm) the strength of these
effects is increased with a maximum blue-shift in the PL spectrum of 261 meV and 167 meV occurring for the C60 and
PCBM doped samples respectively. The origin of the observed behavior cannot be confirmed at this time and is the focus
of ongoing studies. However, we briefly discuss the results obtained in relation to the strong electron accepting nature of
the fullerene complexes used.