We introduce a compact attachment for microscope objectives that allows for the conversion of conventional fluorescence microscopes into Airy light-sheet microscopes. The attachment includes a one-dimensional Airy beam generator, which comprises a gradient-index collimator and a 3D nano-printed cubic phase-plate, realized through two-photon polymerization 3D nano-printing and a two-step writing process that guarantees an optical-quality surface for the phase plate. The micro-optical unit is affixed to a mechanical holder equipped with micro-stages, thereby facilitating the unit's integration into commercial microscopes. The implementation and imaging performance of this system and its fundamental imaging characteristics are discussed, with findings based on diverse samples.
Parametric imaging of attenuation using optical coherence tomography (OCT) is a powerful tool for assessing changes in tissue morphology associated with disease. Several models are available for extracting the optical properties of tissue from OCT images. However, the accuracy of these models and their dependence on the tissue optical properties has yet to be established. Here, we investigate the accuracy of several OCT models and assess their suitability for extracting optical properties. We establish that the single scattering models produce more precise results (lower variance), but the EHF model is more accurate. Furthermore, the accuracy of the single scattering model degrades as the scattering coefficient and thickness of the tissue increase. We intend that the results of this study will aid in the development of standardized protocols for extracting optical properties from OCT images.
Optical Coherence Tomography (OCT) is a powerful label-free approach for volumetric morphological imaging with numerous applications, especially within biomedicine. The penetration depth of OCT reaches well beyond conventional microscopy; however, signal reduction with depth leads to a rapid degradation of the signal below the noise level. The important pursuit of imaging at depth has been largely approached by extinguishing multiple scattering. This has been valid for many microscopies; however, here, we postulate that in OCT, multiple scattering can enhance image contrast at depth. We demonstrate this using an original geometry that completely decouples the incident and collection light fields by introducing a spatial offset between them. This approach leads to a preferential collection of multiply scattered light with depth, compensating for signal attenuation and enhancing the image contrast at depth. A wave optics model and unified theoretical framework supports our experimentally demonstrated improvement in contrast. The effective signal attenuation can be reduced by over 24 dB. Our approach reveals mesoscale features in images of ex vivo mouse bone. Considering most approaches to date have aimed to minimize multiple scattering, our results suggest that the problem of OCT imaging at depth should be distinguished from optical microscopy at depth. This facile and widely applicable geometry enables a power capacity to dynamically tune for contrast at depth.
Beam-shaping through optical fibers facilitates miniaturized light delivery in fields such as endoscopy, communication, etc. As the stability of optical beam delivery and collection depends a lot on the fiber stability, the impact of fiber deformations on mode-coupling for different types of fibers should be analyzed. To this end, we present a numerical simulation tool modeling optical field propagation through fibers with arbitrary refractive index profiles, focusing on fiber twisting, tapering, and bending. Our simulation tool is compared against the state-of-the-art simulation software evaluating computational efficiency, versatility, and user-friendliness. The simulation tool is gratis, open-source, fast, and supports optional CUDA acceleration.
Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing.
Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers.
Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio.
Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images.
Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.
Endoscopes and other optical, non-invasive diagnostic instruments require measurable parameters (biomarkers) that reliably represent early signs of cancer. These biomarkers are challenging to identify in complex tissues due to their dependence on environmental and disease specific influences. In Multiphoton Microscopy (MPM), signals are commonly separated into channels using optical filters. The choice of channels typically relies on generalized prior knowledge. In order to establish more disease specific biomarkers, a reliable cancer model is desired. We present a method to study biomarkers using spheroids as a cancer model. The spheroid development and harvesting are monitored using Optical Coherence Tomography (OCT). We further introduce a hyperspectral MPM system to investigate biomarkers in the autofluorescence of cancerous and normal cell lines. To improve the detection of the selected biomarkers, an algorithm suggests corresponding filters for diagnostic or research purposes.
Attenuation of optical fields owing to scattering and absorption limits the penetration depth into tissue. Whilst aberration correction may be used this is difficult to implement over a large field of view in heterogeneous tissue. Recently, the novel approach of attenuation-compensation of propagation-invariant light fields has shown increase in depth penetration for light-sheet microscopy. Here we show this powerful approach may be implemented in a facile manner utilizing a graded neutral density filter circumventing the need for expensive beam shaping apparatus. A ‘gold standard’ system utilizing a spatial light modulator for beam shaping is used to benchmark our low-cost implementation.
Laser lighting is an emerging technology to generate high luminance lighting. To achieve high luminance or high luminous exitance, the light emitter must have high flux and small size simultaneously. When laser light is focused to a small spot size on the phosphor material, the two main limitations are saturation of the phosphor material and the spot size of the generated light. Here, we investigate experimentally and numerically the spot size of laser lighting dependent on the spot size of the incident laser light and the material properties of the wavelength converting phosphor material. We find numerically that the spot size of the generated white light is significantly influenced by the phosphor properties. The spot size of the white light determines the étendue and thereby the possibility to collect and shape the light. This has important implications in applications of laser lighting.
Miniaturisation of endoscopes can be achieved using lensless endoscope probes, which enhances in vivo deep- tissue imaging technology. The necessity of a detailed understanding of light propagation through optical fibres is paramount, since beam focusing and scanning at tissue require beam shaping at the proximal end of the fibre. For stable light delivery and collection, the sensitivity of various fibre profiles against fibre deformations needs to be reviewed. We present a numerical simulation tool investigating optical field propagation through multimode and multicore optical fibres, emphasizing fibre-bending deformations. The simulation tool enables user to choose optimum fibre with best possible realistic parameters for any application.
We have developed a system capable of resolving Two-Photon Fluorescence Emission (TPFE) and Second Harmonic Generation (SHG) signals with high spectral resolution for the characterization of biomarkers. In Multiphoton Microscopy, those biomarkers are TPFE and SHG signals that carry valuable information on morphological and functional biological features, such as the presence of Green Fluorescent Protein (GFP) in a Zebrafish during the building of organs, or the ratio of pyridine nucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) in the classification of cancerous tissue. For this purpose, separation of different signals into channels is typically achieved by the use of optical filters. In doing so, signal spectra can be unknown or overlapping, creating a crosstalk in between the channels. Previously the choice of such filters relied on prior knowledge or heuristic testing. Our system allows for the study of biomarkers due to spectrally resolved imaging. It therefore enables the appropriate selection of channels, tailored to the application, when building fast diagnostic systems. Additionally, knowledge of the spectra can be used to avoid the crosstalk in between channels or compensate for it computationally. To demonstrate the capabilities of the system, we recorded spectrally resolved images of tissue and cell samples. Structural and functional biological features were identified and their spectra could be evaluated. Thus, appropriate filter setups for diagnostic imaging can be suggested and confirmed by means of integration over defined virtual channels.
While there exist many Monte Carlo (MC) programs for solving the radiative transfer equation (RTE) in biological tissues, we have identified a need for an open-source MC program that is sufficiently user-friendly for use in an education environment, in which detailed knowledge of compiling or UNIX command-line cannot be assumed. Therefore, we introduce MCmatlab, an open-source codebase thus far consisting of (a) a fast 3D Monte Carlo RTE solver and (b) a finite-element heat diffusion and Arrhenius-based thermal tissue damage simulator, both run in MATLAB. The kernel for both of these solvers are written in parallelized C and implemented as MATLAB MEX functions, combining the speed of C with the familiarity and versatility of MATLAB. We present example results generated by the RTE solver and the thermal model. MCmatlab is easy to install and use and can be used by students and experienced researchers alike for simulating tissue light propagation and, optionally, thermal damage.
While there exist many Monte Carlo (MC) programs for solving the radiative transfer equation (RTE) in biological tissues, we have identified a need for an open-source MC program that is sufficiently user-friendly for use in an education environment, in which detailed knowledge of compiling or UNIX command-line cannot be assumed. Therefore, we introduce MCmatlab, an open-source codebase thus far consisting of (a) a fast three-dimensional MC RTE solver and (b) a finite-element heat diffusion and Arrhenius-based thermal tissue damage simulator, both run in MATLAB. The kernel for both of these solvers is written in parallelized C and implemented as MATLAB MEX functions, combining the speed of C with the familiarity and versatility of MATLAB. We compare the RTE solver to Steven Jacques’ mcxyz, which it is inspired by, and present example results generated by the thermal model. MCmatlab is easy to install and use and can be used by students and experienced researchers alike for simulating tissue light propagation and, optionally, thermal damage.
We want to implement two-photon excitation fluorescence microscopy (TPEFM) into endoscopes, since TPEFM can provide relevant biomarkers for cancer staging and grading in hollow organs, endoscopically accessible through natural orifices. However, many obstacles must be overcome, among others the delivery of short laser pulses to the distal end of the endoscope. To this avail, we present imaging results using an all-fibre dispersion management scheme in a TPEFM setup. The scheme has been conceived by Jespersen et al. in 20101 and relies on the combination of a single mode fibre with normal and a higher order mode fibre with anomalous dispersion properties, fused in series using a long period grating. We show that using this fibre assembly, a simple and robust pulsed laser delivery system without any free-space optics, which is thus suitable for clinical use, can be realised.
Multi-photon microscopy is extensively used in research due to its superior possibilities when compared to other microscopy modalities. The technique also has the possibility to advance diagnostics in clinical applications, due to its capabilities complementing existing technology in a multimodal system. However, translation is hindered due to the high cost, high training demand and large footprint of a standard setup. We show in this article that minification of the setup, while also reducing cost and complexity, is indeed possible without compromising on image quality, by using a novel diode laser replacing the commonly used conventional solid state laser as the pump for the femtosecond system driving the imaging.
We present different methods of generating light in the blue-green spectral range by nonlinear frequency conversion of
tapered diode lasers achieving state-of-the-art power levels. In the blue spectral range, we show results using single-pass
second harmonic generation (SHG) as well as cavity enhanced sum frequency generation (SFG) with watt-level output
powers. SHG and SFG are also demonstrated in the green spectral range as a viable method to generate up to 4 W output
power with high efficiency using different configurations.
The physical mechanism underlying multiphoton luminescence in gold is still the subject of debate. To obtain a better understanding of the mechanism, experiments that study the luminescence spectra of single particles are necessary. In this study, the multiphoton luminescence spectrum was measured for surrounding media of different refractive indices. The resulting spectra of single gold nanospheres with diameters in the range of a few tens of nanometers were found to
be strongly dominated by the absorption peak of the plasmon resonance. This is in agreement with the theory proposed by Boyd et al. (1986)1. According to Lorenz-Mie Theory, an increase in the refractive index of the surrounding medium results in a redshift of the plasmon resonance spectrum; a corresponding shift in the multiphoton luminescence spectrum has now been found experimentally.
Theileria annulata is an intracellular parasite that infects and transforms bovine leukocytes, inducing continuous proliferation of its host cell both in vivo and in vitro. Theileria-infected cells can easily be propagated in the laboratory and serve as a good model for laser ablation studies. Using single pulses from an amplified ultrashort pulse laser system, we developed a technique to introduce submicrometer holes in the plasma membrane of the intracellular schizont stage of Theileria annulata. This was achieved without compromising either the viability of the organisms or that of the host cell that harbors the parasite in its cytoplasm. Multiphoton microscopy was used to generate image stacks of the parasite before and after ablation. The high axial resolution allowed precise selection of the region of the membrane that was ablated. It also allowed observation of the size of the holes generated (in fixed, stained cells) and determination of the structural changes in the parasite resulting from the laser pulses (in living cells in vitro). This technique opens a new possibility for the transfection of Theileria or delivery of molecules to the schizont that may prove useful in the study of this special host-parasite relationship.
Noble metal nanoparticles are characterized by a strong peak in the scattering and absorption spectrum, termed the
plasmon resonance. Researchers have taken advantage of this to create a new label for biological molecules. A
disadvantage of techniques based on scattering and absorption is that the detected signal is at the same wavelength
as the incident light, making it more challenging to discriminate between signal and background. Gold
nanoparticles also luminesce, suggesting an alternate method for their detection. A tightly focused ultra-short
pulse laser beam can be used to achieve multiphoton excitation of the particles; the resulting luminescence
exhibits a peak in the same region of the spectrum as the plasmon resonance. Because excitation is nonlinear,
significant luminescence is only observed when the particle is in the focus, permitting localization with both high
lateral and axial resolution. The physical mechanism underlying multiphoton luminescence in gold is still the
subject of debate. Here, we present a systematic study in single gold nanospheres with diameters between 15 nm
and 100 nm using peak laser intensities between 10 and 350 GW/cm2. A scattering confocal microscope
incorporated in the setup was used to distinguish single particles from clusters. We observed that not all gold
nanospheres have a detectable multiphoton luminescence signal; however, laser intensities above an exposure-time
dependent threshold can alter such particles so that they do. In addition, we found that gold nanoparticles
exposed to laser intensities above about 150 GW/cm2 can exhibit behavior reminiscent of the bleaching and
blinking of conventional fluorophores.
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