Figure 1.1. Preparation of grid for electron microscope-based structural determination. The procedures of making grids for (A) negative stain electron... 19

Figure 1.2. Schematics of data processing for single-particle analysis. (A) single protein particles are picked for the sequential classification for 2D and 3D, followed... 22

Figure 1.3. Microcapillary-based electron microscope sample preparation techniques. (A) A microfluidic grid preparation device assisting the negative staining protocol,... 27

Figure 1.4. Spray deposition-based sample preparation techniques. (A) A PDMS-based gas-assisted microfluidic sample spraying and grid preparation system for a... 30

Figure 1.5. Piezoelectric actuator-assisted sample preparation techniques for steady-state and transient sample strategy. (A) A single piezoelectric actuator ejects a... 33

Figure 1.6. A schematic illustration of heat transfer in cryo-EM sample preparation. A rapid heat transfer occurs from a sample-holding grid to the liquid ethane because... 36

Figure 1.7. A phase diagram for vitrification of aqueous sample. The vitrification occurs through a rapid cooling down of stable liquid at point III and I to point IV and... 38

Figure 1.8. A schematic illustration of immunoprecipitation protocol. Cell lysate including antigens (target proteins) is mixed with antibody-coated microparticles.... 41

Figure 2.1. Schematics of single-step and multi-step photolithography. A sequential single-step photolithography process for preparing master mold with (A) positive... 49

Figure 2.2. Schematic illustrations for (A) single-step and (B) multi-step soft-lithography. Complementary micropatterns of master mold are obtained by replica... 52

Figure 2.3. Electric circuit diagrams for control of solenoid valve array. A diagram showing (A) USB UART I/O communication part, (B) 12VDC external power... 54

Figure 2.4. Fabrication of electronic part of solenoid valve array control system. (A) A set of solenoid valve arrays composed of a total of 24 valves (8 solenoid valves ×... 55

Figure 2.5. A custom-built solenoid-valve control software based on LabVIEW programming. (A) The control panel of the software and (B) the block diagram of... 57

Figure 2.6. A fully-integrated plunger system controlled by microcomputer Arduino Uno board controlled by Bluetooth communication with Tablet PC. The Arduino... 60

Figure 2.7. (A) A 3D rendered image of and (B) fabricated mechanical plunger for cryo-EM sample preparation. A tweezer mountable servo holds the sprayed sample... 61

Figure 2.8. Design of environmental chamber. (A) A 3D rendering of the design of the environmental chamber with the dimension of 580 × 440 × 330 mm (width ×... 62

Figure 2.9. (A) A front panel and (B) block diagram of tablet PC application for controlling the Arduino-based integrated system in Bluetooth communication,... 64

Figure 2.10. An Arduino program code for communicating and controlling the integrated system (i - iii). The upstream (i) defines variables and designates the... 65

Figure 3.1. Design of spray deposition-based steady-state sample preparation strategy. (A) A 3D graphical illustration of and (B) the fabricated spray deposition... 71

Figure 3.2. Characterization of sample spraying with varying applied gas pressure. (A) A high-speed camera captured images of a pressure-driven variation in spraying... 74

Figure 3.3. Effect of applied gas pressure on the grid tilted angle. (A) High-speed captures of tilted grids at different gas pressures. The distance between the grid and... 75

Figure 3.4. Effect of in-line humidification on sample film formation. (A) A schematic illustration of in-line humidification of nitrogen gas for spraying.... 77

Figure 3.5. Investigation of the effect of grid types and proteins on the compatibility in cryo-EM analysis. (A) Spray sampled apoferritin sample applied on Quantifoil... 79

Figure 3.6. A comparative study on the type of Quantifoil holey carbon grids. (A) Cryo-EM images of spray sampled Quantifoil grids. (B) A histogram of quantified... 81

Figure 3.7. Investigation of particle distribution in the z-direction of the ice films. The distribution of particle location in the z-axis of (A) Vitrobot-sampled grid and... 83

Figure 3.8. Single-particle analysis of apoferritin sampled grid prepared by a spray deposition method. Image correction and particle picking process are followed by... 85

Figure 3.9. A reconstruct apoferritin 3D structures determined by spray sampling. A spherical shape of apoferritin is well resolved at 2.77 Å resolution which is a near-... 87

Figure 3.10. Design of microfluidic sample preparation for on-chip purification of biomolecules based on a loop reactor-like chamber and for direct application of the... 93

Figure 3.11. A soft-lithographic protocol for making the microfluidic device. Unlike general procedure, a separate membrane layer is subsequently bonded with a fluidic... 94

Figure 3.12. Representative operation of each process for on-chip purification of biomolecules with immunoprecipitation protocol. (A) Injection of magnetic particles... 97

Figure 3.13. Characterization of chamber mixing with the number of pump actuation. (A) fluorescent micrographs showing the mixing of red fluorescent additive inside... 99

Figure 3.14. Validation of the immunoprecipitation protocol in bulk experimental controls. ONPG assays were performed for checking the existence of β-galactosidase... 101

Figure 3.15. Validation of the immunoprecipitation protocol in the microfluidic chamber based on fluorogenic assay with resorufin β-D-galactopyranoside. The... 105

Figure 3.16. The capability of direct application of sample solution to EM grids. (A) A bright field and (B) a fluorescent micrograph showing the microfluidic chamber... 106

Figure 3.17. Single-particle analysis of negative stained 20S proteasome by cryoSPARC. (A) Extracted particle images from negative stain EM micrographs with... 107

Figure 4.1. A schematic workflow of time-resolved cryo-EM based structural determination. A microfluidic device instantly mixes two discrete solutions and... 111

Figure 4.2. Design of microfluidic sample preparation for time-resolved cryo-EM. (A) An illustration describes the rapid sample mixing and spraying by microfluidic... 114

Figure 4.3. Investigation on the effect of flow rate on the mixing efficiency. (A) spinning-disk confocal microscope-based 3D scanning of microfluidic channel... 116

Figure 4.4. Investigation of the effect of the number of 3D junctions on the mixing efficiency. (A) A representative fluorescent image obtained by 3D scanning in top... 119

Figure 4.5. A graph showing the rationale of the assumption in designing the multiscale time-resolved device that the fluid flow inside the microfluidic device is... 122

Figure 4.6. CAD drawings of the devices having different residence times (no delay, 20, 100, 200, 500, 1000 ms) based on the calculated result from Table 4 under a fixed... 124

Figure 4.7. Inspection of the fabricated device. (A) profiling the height of the channel to check the accuracy in the fabrication. The average channel height was measured... 126

Figure 4.8. Characterization of time spent in individual steps for Cryo-EM grid preparation. (A) A comprehensive indication of spent time in each step (i-v). The... 128

Figure 4.9. A simulation result of residence time distribution in the microfluidic device. (A) A color-gradient plot showing the fluid velocity simulated in COMSOL... 132

Figure 4.10. A comparative study of time-resolved sampling with Vitrobot and spray-based method. (A) A schematic showing the growth of RecA protein onto single-... 135

Figure 4.11. A proof-of-principle kinetic study for time-resolved sampling capability of the microfluidic sample preparation based RecA-ssDNA interaction. (A) Electron... 138

Figure 4.12. A group of violin plots representing the reproducibility of the time-resolved sample preparation method by independently-prepared RecA-ssDNA... 139

Figure 5.1. (A) A scheme describing a surface treatment strategy for larger coverage of EM grids. Adsorption of hydrophilic polymer (poly-L-lysine, thiolated... 148