We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 μm × 300 μm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.
Wide field-of-view imaging of fast processes in a microscope requires high light intensities motivating the use of lasers as light sources. However, due to their long spatial coherence length lasers are inappropriate for such applications as they produce coherent noise and parasitic reflections, such as speckle, degrading image quality. Therefore, we provide a step-by-step guide for constructing a speckle-free and high contrast laser illumination setup using a rotating ground glass diffuser driven by a stepper motor. The setup is easy to build, cheap and allows a significant light throughput of 48 %, which is 40 % higher in comparison to a single lens collector commonly used in reported setups. This is achieved by using only one objective to collect the scattered light from the ground glass diffuser. We validate the stability and performance of our setup in terms of image quality, motor-induced vibrations and light throughput. To highlight the latter, we record Brownian motion of micro-particles using a 100x oil immersion objective and a high-speed camera operating at 2000 Hz with a laser output power of only 22 mW. Moreover, by reducing the objective magnification to 50x sampling rates up to 10 000 Hz are realized. To help readers with basic or advanced optics knowledge realizing this setup we provide; a full component list, 3D-printing CAD files, setup protocol, and the code for running the stepper motor.
We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke-Triplet configuration, allows for continuous beam magnifications of 2-10×, and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke-Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 μm beads shows that the Cooke-Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman-Green interferometer integrated in the Cooke-Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.
We present a versatile and fast MATLAB program (UmUTracker) that automatically detects and tracks particles by analyzing video sequences acquired by either light microscopy or digital in-line holographic microscopy. Our program detects the 2D lateral positions of particles with an algorithm based on the isosceles triangle transform, and reconstructs their 3D axial positions by a fast implementation of the Rayleigh-Sommerfeld model using a radial intensity profile. To validate the accuracy and performance of our program, we first track the 2D position of polystyrene particles using bright field and digital holographic microscopy. Second, we determine the 3D particle position by analyzing synthetic and experimentally acquired holograms. Finally, to highlight the full program features, we profile the microfluidic flow in a 100 µm high flow chamber. This result agrees with computational fluid dynamic simulations. On a regular desktop computer UmUTracker can detect, analyze, and track multiple particles at 5 frames per second for a template size of 201 × 201 in a 1024 × 1024 image. To enhance usability and to make it easy to implement new functions we used objectoriented programming. UmUTracker is suitable for studies related to: particle dynamics, cell localization, colloids and microfluidic flow measurement. Nature of problem: 3D multi-particle tracking is a common technique in physics, chemistry and biology. However, in terms of accuracy, reliable particle tracking is a challenging task since results depend on sample illumination, particle overlap, motion blur and noise from recording sensors. Additionally, the computational performance is also an issue if, for example, a computationally expensive process is executed, such as axial particle position reconstruction from digital holographic microscopy data. Versatile robust tracking programs handling these concerns and providing a powerful post-processing option are significantly limited. PROGRAM SUMMARY Friday, April 21, 2017Manuscript 2 Solution method: UmUTracker is a multi-functional tool to extract particle positions from long video sequences acquired with either light microscopy or digital holographic microscopy. The program provides an easy-to-use graphical user interface (GUI) for both tracking and post-processing that does not require any programming skills to analyze data from particle tracking experiments. UmUTracker first conduct automatic 2D particle detection even under noisy conditions using a novel circle detector based on the isosceles triangle sampling technique with a multi-scale strategy. To reduce the computational load for 3D tracking, it uses an efficient implementation of the Rayleigh-Sommerfeld light propagation model. To analyze and visualize the data, an efficient data analysis step, which can for example show 4D flow visualization using 3D trajectories, is included. Additionally, UmUTracker is easy to modify with user-customized modules due to the object-oriented programing style.
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