In fluorescence microscopy, an external source of excitation light is required for photon emission and thereby sample visualization. Even though fluorescence imaging has provided a paradigm shift for cell biology and other disciplines, the sample might suffer due to high excitation light intensities, and spurious signals originating from autofluorescence. Bioluminescence imaging, on the contrary, does not need an external source of light for photon emission and visualization, bypassing the effects of autofluorescence, phototoxicity and photobleaching. This renders bioluminescence microscopy as an ideal tool for long term imaging. A major limitation of bioluminescence, compared to fluorescence imaging, is the low quantum yield of the bioluminescent proteins, which requires long exposure times and large collecting wells. Here, we work towards universal tools to overcome the main limitations of bioluminescence imaging: low signal/noise (SNR) imaging. To enhance spatiotemporal resolution, we have designed an optimized setup that boosts the optical efficiency and combine the photon starved, low SNR output with deep learning based content aware reconstruction methods. We trained a UNet architecture neural network with augmented fluorescent experimental data to denoise low SNR bioluminescent images. In addition, we trained a subpixel convolutional network with synthetic light field data to perform 3D reconstruction from a single photographic exposure without the presence of autofluorescence. Furthermore, we compare the reconstruction time and quality improvement with classical deconvolution methods.
In recent years, deep learning has been used widely to solve a variety of digital microscopy problems. We present ZEUS as a method to correct out of focus aberrations and denoise light-sheet microscopy images. First, a convolutional neural network is used to estimate the aberrations in terms of Zernike coefficients. Then those values are used to train a UNET that outputs corrected images from noisy and aberrated ones. With this approach, we can access scanning frequencies and image qualities equivalent to the most advanced LSM systems without the need for costly equipment and complex optical setups.
LSFM is a technique that allows obtaining fast 2D images of biological samples. Its characteristic 90° geometry results in a highly efficient excitation and light collection of the generated signal, minimizing light dose onto the sample and reducing phototoxicity effects. Furthermore, by displacing the sample through the light sheet, high-resolution 3D images can also be obtained. Therefore, LSFM has been put forward as an interesting candidate for fast volumetric brain imaging. Here, I will present our results for 3D imaging of the spontaneous and dynamic calcium activity in primary neuron cultures in hydrogels. The obtained data is then processed to calculate the connectivity maps in the 3D neuron cultures in hydrogels and assess the topological properties of these maps such as the modules or highly connected subnetworks.
This abstract is part of the symposium: "Diagnosis and Prediction of Neurodegenerative Diseases using Artificial Intelligence"
We present a monolithic fiber optic configuration for generating temporally coherent supercontinuum (SC) pulsed emission with the shortest pulse duration presented to date, to our knowledge, by an all-fiber source. Few-cycle pulses as short as 14.8 fs are obtained, with central emission wavelength of 1060 nm, repetition rate of 75 MHz and average power of 250 mW. The SC generation is obtained by pumping an all-normal dispersion (ANDi) photonic crystal fiber (PCF) with a mode-locked Yb fiber laser. Spectral broadening by self-phase modulation preserves compressible pulses in the temporal domain. Compared to previously reported configurations exploiting ANDi PCFs, all stages of our source are fiber based and fiber coupled between them. Avoidance of free-space propagation between stages confers unequalled robustness, efficiency and cost-effectiveness to this novel configuration. The ANDi PCF was designed and produced to provide a convex, flat-top dispersion curve with group velocity dispersion comprised between -20 and 0 ps/nm/km in the wavelength range from 900 to 1200 nm. A d-scan system was designed and built to compress and characterize the pulses. The spectrum, wider than 150 nm, supports a Fourier limit pulse duration of 13.7 fs, and pulses have been actually compressed down to 14.8 fs, which demonstrates a high level of temporal coherence in the achieved supercontinuum; second- and third-order dispersion of the pulses are measured as low as -145 fs2 and 875 fs3, respectively. The source has been integrated in a twophoton fluorescence and second-harmonic generation microscopy setup, where 3D images of biological samples have been successfully obtained.
KEYWORDS: Microscopy, 3D image processing, Functional imaging, Luminescence, Microscopes, Neurons, Signal generators, Stereoscopy, 3D scanning, Data processing
I will present a Light-sheet fluorescence microscope (LSFM) for fast volumetric imaging during extended periods of time. In this case, the observation arm of the microscope contains an electrically tunable lens (ETL) that is used to shift the focal position of the collection lens. By moving the light sheet plane in synchrony with the ETL, it is possible to scan the full 3D sample, which remains totally static, at high speeds (25 volumes/s) [2].
This system is used to image the spontaneous Ca2+ activity, as reported by GCaMP fluorescence, in 3D of primary neuron cultures in hydrogels. The field of view is of 300µm x 300µm x 1mm. The imaging speeds allows a proper sampling of the propagation of GCaMP signal in the full observation volume [4]. The obtained data is then processed to calculate the connectivity maps in the 3D neuron cultured in hydrogels.
In this work we present a numerical analysis of the mode coupling between the pump-beam and the laser-beam in a Ti:Sapphire crystal used as a gain medium of a femtosecond laser. Using the Matrix ABCD and propagation gaussian beam models, we obtained an optimal configuration for compensate the astigmatism in the output beam laser. Also we analysed pump-beam propagation and got the settings to fix the astigmatism in the crystal. Furthermore we apply this configuration to a homemade femtosecond laser, accomplishing an overall efficiency of laser to 20% in continuum wave (CW) and 16% in mode looking (ML) operation. The femtosecond laser have 30 nm bandwidth to FWHM at 810 nm corresponding 30fs.
In this work, we analyse the use of a micro-machined deformable membrane mirror (MMDM) to shape femtosecond
pulses. We present the Spectral-Phase-Influence-Matrix constructed by an inversion method. Spectral-Phase-Influence-
Matrix represents a novel and direct method to estimates the Spectral Phase design from a given actuator voltage settings
in a single step. Numerical and experimental results are presented.
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