WO2017031310A1 - Nanoparticle detection - Google Patents

Abstract

There are provided methods for detecting one or more nanoparticles within one or more liquid droplets using transmission electron microscopy.

Description

NANOPARTICLE DETECTION

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Appl. No. 62/206,690, filed August 18, 2015, the content of which is incorporated herein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under W91 lNF-15-1-0800 awarded by the U.S. Army Research Office, and under FA9550-11-1-0105 and FA9550-12-1-0414 awarded by the U.S. Air Force Office of Scientific Research. The government has certain rights in this invention.

BACKGROUND

[0003] Liquid cell transmission electron microscopy (LCTEM) is beginning to be recognized as useful in the field of nanomaterial analysis. Without wishing to be bound by any theory, it is believed that the development of LCTEM methods requires the continued development of devices, e.g., liquid cell TEM holders, and sample application methods. For example, loading and analysis of multiple samples can be problematic due to the quick evaporation of liquid samples in small volume, especially in view of the associated time and efforts required for such preparation of samples for LCTEM.

[0004] Thus, there are provided herein solutions to these and other problems in the art of nanoparticle detection.

SUMMARY

[0005] In a first aspect, there is provided a method for detecting a nanoparticle in a liquid. The method includes depositing a liquid droplet containing a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, where the volume of the liquid droplet is less than about 1000 pL. The method further includes covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover. The method further includes detecting the nanoparticle within the liquid droplet using a transmission electron microscope.

[0006] In another aspect, there is provided method for detecting a plurality of nanoparticles and an interaction thereof in a liquid. The method includes depositing a plurality of liquid droplets each containing a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, where the volume of each liquid droplet is less than about 1000 pL. The method further includes covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover. The method further includes detecting the plurality of nanoparticles and interaction thereof upon diffusion of the plurality of liquid droplets using a transmission electron microscope.

[0007] In another aspect, there is provided method of analyzing a sample using transmission electron microscope. The method includes automatically depositing a liquid droplet of the sample onto a transmission electron microscope sample holder, where the volume of said liquid droplet is less than about 1000 pL and analyzing the liquid sample using transmission electron microscope.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1F. The figures depict optical images of Hummingbird Scientific 200 nm spacer chips with a single 50 nm thick, 200 x 50 micron window (FIGS. 1 A-1C) and a Norcada 10 nm thick 100 x 100 micron, 9 window array (FIGS. 1D-1F), showing the windows pre- deposition (FIG. 1 A and FIG. ID), post-deposition of pure water to create a high humidity environment (FIG. IB and FIG. IE), and post deposition of 20 nm Au NPs (white dashed circles) and 200 nm UiO-66 (dark dashed circles) (FIG. 1C and FIG. IF).

[0009] FIGS. 2A-2G. Figure depict TEM images of 200 nm UiO-66 (FIGS. 2A-2C) and 20 nm Au NPs (FIG. 2E-2G) dispensed onto a single window on a liquid cell and allowed to dry (chip from FIGS. 1 A-1C). FIG. 2D: The tall and narrow center image is a low magnification image of the entire window area of the chip from FIG. 1C after the deposited liquid droplets have completely dried at ambient conditions. The images in the left (FIG. 2A-2C) and right (FIG. 2E- 2G) columns show clear separation between the UiO-66 and Au NPs, and solvent edges for each solution are indicated by lines. The structures of each NP are also confirmed by selected area electron diffraction. Dashed-line boxes indicate the regions on where the top row images were recorded on the window. The small circles indicate the regions where the center row images and corresponding diffraction patterns were acquired. Note the difference in scale bars for SAED patterns which is indicative of the large difference in Au and MOF lattice spacing.

[0010] FIGS. 3A-3G. The figures depict TEM images of a liquid cell sealed immediately after dispensing 200 nm UiO-66 and 20 nm Au NPs onto opposite ends of the a single window. The images show clear separation of the Ui066 (FIGS. 3A-3C) with Au (FIGS. 3E-3F) as well as an initial mixing front (FIG. 3D), "t =" refers to the time after sealing the liquid cell. The tall narrow graphic in the center (FIG. 3G) is a qualitative scheme of the liquid composition within the window region as observed over the first 20 minutes after sealing the cell. Observed are pure Au NPs, pure UiO-66 MOF and a mixed composition of Au and MOF. Labeled squares in FIG. 3G indicate the position on the window area where the respective TEM images (FIGS. 3A-3F) were acquired.

[0011] FIGS. 4A-4G. The figures depict TEM images of the same liquid cell in FIGS. 3A-3G, after full diffusion and mixing of the UiO-66 and Au NPs had occurred. The images show significant diffusion of Au into the Ui066 end of the cell (FIGS. 4A-4E) and little or no diffusion of the MOFs into the Au end of the cell (FIG. 4F). "t =" refers to the time after sealing the liquid cell. The tall narrow graphic in the center (FIG. 4G) is a qualitative scheme of the liquid composition within the window region as observed 30 minutes after sealing the cell. Observed are pure Au NPs, pure UiO-66 MOF and a mixed composition of Au and MOF (now the primary composition). Labeled squares in FIG. 4G indicate the position on the window area where the respective TEM images (FIGS. 4A-4F) were acquired. Image FIG. 4C was acquired at the same location as FIG. 4B, 90 seconds later. Circles in FIG. 4C indicate Au nanoparticles that have diffused into the field of view and stuck to the top or bottom window during the 90 second time period from image FIG. 4B.

[0012] FIGS. 5A-5C. The figures depict optical images of (FIG. 5A) Piezo Dispensing Capillary - Uncoated 80um diameter orifice Sample = Gold NP ( wt%) Volume = 316 pL, (FIG. 5B) Piezo Dispensing Capillary - Coated 80um diameter orifice Sample = MOF (wt%) Volume = 327 pL and (FIG. 5C) Sample dispense pattern of 6 drops/ spot to opposite edges of the liquid cell TEM window. The surrounding water droplets totaled 450 nL.

[0013] FIGS. 6A-6B. FIG. 6A depicts a photograph of the Sci-TEM Liquid Cell sample prep system, including enclosing chamber and Cartesian robot [element 1]. The chamber provides a local environment of near-saturated solvent vapor to control drying of volatile samples. FIG. 6B depicts a photograph of the interior of the Sci-TEM Liquid Cell sample prep system, including location of imaging systems (Global camera, Head Camera, Drop Camera) [FIG. 6B, element 2], low volume non-contact dispensing nozzles [FIG. 6B, element 3], vacuum nozzle [FIG. 6B, element 4], Microarray chamber (i.e., humidifier chamber) loaded with LC TEM stage and grids [FIG. 6B, element 5]. [0014] FIGS. 7A-7B. FIG. 7A: The figure depicts a schematic of the Sci-TEM Liquid Cell sample prep system and the processes (e.g., convection, diffusion) involved in preparation of a sample. A Controllable Heating Sleeve is depicted surrounding the enclosure holding the solvent. FIG. 7B: The figure depicts an alternative method for controlling the temperature of the enclosure, where the enclosure sits on a Controllable Heating Plate. [0015] FIGS. 8A-8B. FIG. 8A schematically depicts the robotic working area of the Sci-TEM Liquid Cell sample prep system. A photographic inset (FIG. 8B) depicts the humidifier chamber (the white cassette) and the chip positioning.

[0016] FIGS. 9A-9C. FIG. 9A: Photograph depicting relative positioning of Top Chip and Bottom Chip, also depicting 30 nm thick SiN_x "windows" and 50 μπι x 50 μπι lateral area. FIG. 9B: Photograph depicting in situ Liquid-Cel TEM (LC-TEM) grids, including the sample holder, the sample holder cover (i.e., over clamp) and the side-clamp. The area indicated in dashed rectangle is enlarged in FIG. 9C, which depicts liquid flow lines, S/TEM electron beam, silicon support chip, and further inset depicting top SiN_x, liquid sample, and bottom SiN_x regions.

DETAILED DESCRIPTION

[0017] Absent express indication otherwise, definitions used herein assume the usual and customary meaning in the art.

[0018] The term "transmission electron microscope (TEM) grid" refers, in the usual and customary sense, to a sample holding device for use in a TEM, as known in the art. The TEM grid may include equally spaced score lines (grid lines) to aid in material deposition and identification.

[0019] The term "piezo dispensing device" and the like refer, in the usual and customary sense, to a device capable of dispensing small amounts of solution using pressure waves, typically transduced via piezoelectric methodology. In embodiments, piezo dispensing devices disclosed herein and embodiments thereof can dispense sample aliquots on the picoliter scale, e.g., about 10 pL, 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL or even 100 pL or greater. [0020] The term "drop on demand" and the like refers to the methods disclosed herein which allow accurate and reproducible placement of droplets of solution for analysis in the LCTEM.

[0021] The term "multiplex liquid cell TEM," "multiple LCTEM" and the like as used herein refer to the capability of manipulating and analyzing a plurality of aliquots of solution for LCTEM, using the method and apparatus disclosed herein.

[0022] The term "nanoparticle" and the like refer, in the usual and customary sense, to particles having a largest dimension of between about 1 and 100 nm, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or even 100 nm.

[0023] Absent express indication otherwise, the term "about" in the context of a numerical value means the nominal value ± 10% thereof.

Methods

[0024] The invention described herein relates, in part, to methods for detecting nanoparticles, in particularly detecting mixing and reaction of nanoparticles using Liquid Cell Transmission Electron Microscopy (TEM) or any TEM that involves depositing a small liquid droplet sample during the sample preparation stage. Controlling the addition of multiple solutions to the liquid cell remains a hurdle (for example, due to the quick evaporation of liquid samples in small volume) in the ability to increase throughput and to study processes dependent on solution mixing, e.g., chemical reactions. Previous methods directed to related problems include mixing into wells of a TEM holder tip, or premixing solutions and flowing the solution into a TEM cell. These methods, however, fail to allow the imaging of multiple samples which mix directly within the viewing area of the TEM, or the analysis of multiple isolated samples with respect to, e.g., diffusion and mixing. These prior methods, therefore, are not able to detect the initial stage of the reactions (e.g., the initial stage within the first 15 seconds).

[0025] In embodiments, the methods described herein utilize novel approaches of depositing multiple samples in small (e.g., nanoliter or picoliter) droplets on one TEM support (e.g., TEM grid) under humidifying conditions, which significantly increases the number of samples that can be analyzed within a single measurement cycle and provides new experimental procedures, including, e.g., the ability the monitor diffusion and mixing of nanoparticle-containing solutions on the TEM support. [0026] The methods described herein can be used for analyzing a sample using TEM and/or detecting nanoscale molecules using TEM, for example, chemistry manifests on the nanoscale using TEM, such as, nanoparticles formation through block copolymer assembly, formation of a ZIF-8 metal organic-framework system, nanocrystal formation, and chemical recognition of molecules conjugated to a nanoparticle.

[0027] In a first aspect, there is provided a method for detecting a nanoparticle in a liquid. The method includes depositing a liquid droplet containing a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, where the volume of the liquid droplet is less than about 1000 pL. The method further includes covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover. The method further includes detecting the nanoparticle within the liquid droplet using a transmission electron microscope.

[0028] In another aspect, there is provided method for detecting a plurality of nanoparticles and an interaction thereof in a liquid. The method includes depositing a plurality of liquid droplets each including a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, where the volume of each liquid droplet is less than about 1000 pL. The method further includes covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover. The method further includes detecting the plurality of nanoparticles and interaction thereof upon diffusion of the plurality of liquid droplets using a transmission electron microscope. In embodiments, a plurality of liquid droplets includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or more liquid droplets, where each liquid droplet includes at least one nanoparticle.

[0029] In another aspect, there is provided method of analyzing a sample (e.g., a liquid sample) using transmission electron microscope (TEM). The method includes automatically depositing a liquid droplet of the sample onto a TEM sample holder (e.g., TEM compatible substrate), where the volume of said liquid droplet is less than about 1000 pL and analyzing the liquid sample using TEM. In embodiments, the automatically depositing is depositing within 5 microns (e.g., about 5, 4, 3, 2, or 1 micron) of a predetermined location on the TEM sample holder. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet of the sample may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In embodiments, each liquid droplet may include one type of chemicals. In embodiments, the liquid sample includes a plurality of nanoparticles or a plurality of chemicals. In embodiments, the analyzing includes detecting this plurality of nanoparticles or this plurality of chemicals. In embodiments, the method includes covering the TEM sample holder with a TEM sample holder cover prior to the analyzing. In embodiments, this covering is automated. In embodiments, the automatically depositing is performed under humidifying conditions.

[0030] In embodiments, at the time of detecting or analyzing the liquid sample with TEM according to any method described herein, the liquid sample may not be a liquid but rather a solid resulting from evaporation of said liquid.

[0031] In embodiments, the volume of each liquid droplet being deposited in any method described herein is about 1 pL to about 1000 pL. In embodiments, the volume of each liquid droplet being deposited is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,

99, 100, 101, 102, 103, 104, L05, 106, 107, L08, 109, 110, 111, 112, 113, 114, 115, 116, 117,

118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,

137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,

156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,

175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,

194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,

213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,

251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,

270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,

289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,

308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,

327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,

346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,

365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,

384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,

403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,

422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,

460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,

479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,

498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,

517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,

536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,

555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573,

574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,

593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,

612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630,

631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649,

650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,

669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687,

688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,

707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725,

726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,

745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763,

764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782,

783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801,

802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,

821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839,

840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858,

859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,

878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896,

897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915,

916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,

935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953,

954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,

973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991,

992, 993, 994, 995, 996, 997, 998, 999, 1000 pL.

[0032] In embodiments, the volume of each liquid droplet being deposited in any method described herein is about 10 pL to about 1000 pL. In embodiments, the volume of each liquid droplet being deposited is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, f

53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, '

79, 80, 8 I, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,

103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,

122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,

141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,

179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,

198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,

217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,

236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,

255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,

274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,

293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,

312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,

331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,

350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,

369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,

388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,

407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,

426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,

464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,

483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,

502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,

521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,

540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,

559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,

578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,

597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615,

616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634,

635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,

654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691,

692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,

711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729,

730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,

749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767,

768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,

787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805,

806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,

825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843,

844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862,

863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881,

882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900,

901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,

920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938,

939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957,

958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976,

977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995,

996, 997, 998, 999, 1000 pL.

[0033] The term "humidifying conditions" refers to conditions providing sufficient amounts of solvent vapor or gas (e.g., water vapor) to prevent and/or decrease evaporation of solvent during deposition and subsequent preparation of the liquid droplet (i.e., sample liquid droplet) for TEM analysis.

[0034] In embodiments, the humidifying conditions are generated by depositing a volatile solvent droplet onto the sample holder. In embodiments, the volatile solvent droplet is a plurality of volatile solvent droplets. In embodiments, the volatile solvent droplet or a plurality of volatile solvent droplets is being deposited prior to depositing the sample liquid droplet. In

embodiments, the volatile solvent droplet or a plurality of volatile solvent droplets is being deposited simultaneously with the sample liquid droplet or the first batch of sample liquid droplets. [0035] In embodiments, the volatile solvent droplet or a plurality of volatile solvent droplets are sufficiently proximal to the sample(s) to keep the sample(s) from drying out. In

embodiments, the volatile solvent droplet or a plurality of volatile solvent droplets are about 50 to about 300 μιη (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 μιη) from at least one of the samples of the interest on the sample holder.

[0036] In embodiments, the volatile solvent is compatible with the material of the sample holder and its components (e.g., the substrate material). In embodiments, the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or cyclohexanone. Exemplary alcohol includes, but is not limited to, ethanol, isopropyl alcohol, methanol, butanol, isobutanol, and glycerol. In embodiments, the volatile solvent is water.

[0037] In embodiments, the humidifying conditions are generated by a humidifier chamber. In embodiments, the humidifier chamber surrounds the sample holder as illustrated in FIG. 8B. The humidifier chamber can be made from any suitable materials, for example, acetal (Delrin®), aluminum, stainless steel, glass. In embodiments, the humidifier chamber is made from acetal (Delrin®). In embodiments, the humidifier chamber is made from aluminum:: In embodiments, the humidifier chamber includes a volatile solvent reservoir that provides sufficient vapor to prevent the samples from drying out. In embodiments, the volatile solvent reservoir is underneath the transmission electron microscope sample holder. In embodiments, the volatile solvent is compatible with the material of the humidifier chamber. In embodiments, the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or cyclohexanone. Exemplary alcohol includes, but is not limited to, ethanol, isopropyl alcohol, methanol, butanol, isobutanol, and glycerol. In embodiments, the volatile solvent is water.

[0038] In embodiments, the volatile solvents compatible for glass include, but are not limited to, the following in Table 1 below. A-Excellent is defined as no effect, no detectable corrosion or discoloration.

Table 1. Volatile solvents compatible for glass

Chemical Compatibility Chemical Compatibility

Acetic Acid A-Excellent Oils:Aniline A-Excellent

Acetic Acid 20% A-Excellent Oils: Anise A-Excellent

Acetic Acid 80% A-Excellent Oils:Bay A-Excellent

Acetic Acid, Glacial A-Excellent Oils:Bone A-Excellent

Antifreeze A-Excellent Oils:Castor A-Excellent

Asphalt A-Excellent Oils: Cinnamon A-Excellent

Beer A-Excellent Oils:Citric A-Excellent

Beet Sugar Liquids A-Excellent Oils:Clove A-Excellent

(Paper Mill)

[0039] In embodiments, the volatile solvents compatible for stainless steel (e.g., 316 Stainless Steel) include, but are not limited to, the following in Table 2 below. A-Excellent is defined as no effect, no detectable corrosion or discoloration.

Table 2. Volatile solvents compatible for 316 stainless steel

Chemical Compatibility Chemical Compatibility

Acetaldehyde A-Excellent Mash A-Excellent

Acetamide A-Excellent Mayonnaise A-Excellent

Acetate Solvent A-Excellent Mercurous Nitrate A-Excellent

Acetic Acid 20% A-Excellent Mercury A-Excellent

Acetic Acid, Glacial A-Excellent Methane A-Excellent

Acetic Anhydride A-Excellent Methanol (Methyl A-Excellent

Alcohol)

Acetone A-Excellent Methyl Acetone A-Excellent

Acetyl Chloride (dry) A-Excellent Methyl Alcohol 10% A-Excellent

Acetylene A-Excellent Methyl Bromide A-Excellent

Acrylonitrile A-Excellent Methyl Butyl Ketone A-Excellent

Adipic Acid A-Excellent Methyl Chloride A-Excellent

Alcohols: Amyl A-Excellent Methyl Ethyl Ketone A-Excellent

Alcohols: Butyl A-Excellent Methyl Isopropyl A-Excellent

Ketone

Alcohols: Di acetone A-Excellent Methylamine A-Excellent

Borax (Sodium Borate) A-Excellent Oils: Lemon A-Excellent

Cyanic Acid A-Excellent Plating Solutions: A-Excellent

Bronze: Cu-Cd Bronze

Bath R.T.

Cyclohexane A-Excellent Plating Solutions: A-Excellent

Bronze: Cu-Sn Bronze

Bath 160°F

Cyclohexanone A-Excellent Plating Solutions: A-Excellent

Bronze: Cu-Zn Bronze

Bath 100°F

Detergents A-Excellent Plating Solutions: A-Excellent

Cadmium: Cyanide Bath

90°F

Diesel Fuel A-Excellent Plating Solutions: A-Excellent

Cadmium: Fluoborate

Bath 100°F

Diethylamine A-Excellent Plating Solutions: A-Excellent

Copper (Cyanide):

Copper Strike Bath

120°F

Diethylene Glycol A-Excellent Plating Solutions: A-Excellent

Copper (Cyanide):

High-Speed Bath 180°F

Diphenyl Oxide A-Excellent Plating Solutions: A-Excellent

Copper (Cyanide):

Rochelle Salt Bath

150°F

Di sodium Phosphate A-Excellent Plating Solutions: A-Excellent

Copper (Misc): Copper

Pyrophosphate

Dyes A-Excellent Plating Solutions: Gold: A-Excellent

Cyanide 150°F

Ethane A-Excellent Plating Solutions: Silver A-Excellent

Plating 80-120°F

Ethanol A-Excellent Plating Solutions: Zinc: A-Excellent

Alkaline Cyanide Bath

R.T.

Ethanolamine A-Excellent Potassium Chloride A-Excellent

Ether A-Excellent Potassium Hydroxide A-Excellent

(Caustic Potash)

Ethyl Chloride A-Excellent Potassium Iodide A-Excellent

Ethylene Bromide A-Excellent Potassium Sulfate A-Excellent

Fatty Acids A-Excellent Propane (liquefied) A-Excellent

Ferric Sulfate A-Excellent Propylene A-Excellent

Fluorine A-Excellent Pyridine A-Excellent

Formaldehyde 100% A-Excellent Rosins A-Excellent

Formaldehyde 40% A-Excellent Rum A-Excellent

Formic Acid A-Excellent Rust Inhibitors A-Excellent

Freon® 11 A-Excellent Salad Dressings A-Excellent

[0040] In embodiments, the volatile solvents compatible for acetal (Delrin®) include, but are not limited to the ones in the Table 3 below. A-Excellent is defined as no effect, no detectable corrosion o rdiscoloration.

Alcohols: Octyl A-Excellent Milk A-Excellent

Alcohols: Propyl A-Excellent Mineral Spirits A-Excellent

Aluminum Hydroxide A-Excellent Molasses A-Excellent

Ammonium Nitrate A-Excellent Naphtha A-Excellent

Amyl Alcohol A-Excellent Naphthalene A-Excellent

Amyl Chloride A-Excellent Nickel Chloride A-Excellent

Aniline A-Excellent Nickel Sulfate A-Excellent

Aromatic Hydrocarbons A-Excellent Nitromethane A-Excellent

Barium Carbonate A-Excellent Oils: Castor A-Excellent

Barium Chloride A-Excellent Oils: Citric A-Excellent

Barium Sulfide A-Excellent Oils: Coconut A-Excellent

Beer A-Excellent Oils: Corn A-Excellent

Benzaldehyde A-Excellent Oils: Cottonseed A-Excellent

Benzene A-Excellent Oils: Crude Oil A-Excellent

Benzol A-Excellent Oils: Ginger A-Excellent

Benzyl Chloride A-Excellent Oils: Linseed A-Excellent

Boric Acid A-Excellent Oils: Mineral A-Excellent

Butadiene A-Excellent Oils: Olive A-Excellent

Butane A-Excellent Oils: Palm A-Excellent

Butanol (Butyl Alcohol) A-Excellent Oils: Peanut A-Excellent

Butter A-Excellent Oils: Pine A-Excellent

Buttermilk A-Excellent Oils: Rapeseed A-Excellent

Butyl acetate A-Excellent Oils: Silicone A-Excellent

Butyl ene A-Excellent Oils: Soybean A-Excellent

Butyric Acid A-Excellent Oils: Transformer A-Excellent

Calcium Carbonate A-Excellent Oils: Turbine A-Excellent

Calcium Chlorate A-Excellent Oleic Acid A-Excellent

Calcium Hydroxide 10% A-Excellent Palmitic Acid A-Excellent

Calcium Oxide A-Excellent Paraffin A-Excellent

Calgon A-Excellent Picric Acid A-Excellent

Cane Juice A-Excellent Plating Solutions: A-Excellent

Antimony Plating 130°F

Carbon Bisulfide A-Excellent Plating Solutions: A-Excellent

Arsenic Plating 110°F

Carbon Dioxide (dry) A-Excellent Plating Solutions: Brass: A-Excellent

High-Speed Brass Bath

110°F

Carbon Dioxide (wet) A-Excellent Plating Solutions: Brass: A-Excellent

Regular Brass Bath 100°F

Carbon Disulfide A-Excellent Plating Solutions: A-Excellent

Bronze: Cu-Cd Bronze

Bath R.T.

Carbon Monoxide A-Excellent Plating Solutions: A-Excellent

Bronze: Cu-Zn Bronze

Bath 100°F

Carbon Tetrachloride A-Excellent Plating Solutions: A-Excellent

(wet) Cadmium: Cyanide Bath

90°F

Carbonated Water A-Excellent Plating Solutions: Copper A-Excellent

(Acid): Copper Sulfate

Bath R.T.

Chlorine, Anhydrous A-Excellent Plating Solutions: Copper A-Excellent

Liquid (Cyanide): Copper Strike

Bath 120°F

Chloroform A-Excellent Plating Solutions: Copper A-Excellent

(Misc): Copper

Pyrophosphate

Chocolate Syrup A-Excellent Potassium Bromide A-Excellent

Cider A-Excellent Potassium Chloride A-Excellent

Coffee A-Excellent Potassium Dichromate A-Excellent

Copper Chloride A-Excellent Potassium Hydroxide A-Excellent

(Caustic Potash)

Copper Cyanide A-Excellent Potassium Nitrate A-Excellent

Copper Nitrate A-Excellent Potassium Permanganate A-Excellent

Cream A-Excellent Propane (liquefied) A-Excellent

Cyclohexane A-Excellent Rum A-Excellent

Cyclohexanone A-Excellent Rust Inhibitors A-Excellent

Detergents A-Excellent Salad Dressings A-Excellent

Dichloroethane A-Excellent Sea Water A-Excellent

Diesel Fuel A-Excellent Shellac (Bleached) A-Excellent

Diethylene Glycol A-Excellent Shellac (Orange) A-Excellent

Ethane A-Excellent Silicone A-Excellent

Ethanol A-Excellent Silver Nitrate A-Excellent

Ether A-Excellent Soap Solutions A-Excellent

Ethyl Acetate A-Excellent Soda Ash (see Sodium A-Excellent

Carbonate)

Ethyl Chloride A-Excellent Sodium Bicarbonate A-Excellent

Ethyl Ether A-Excellent Sodium Bromide A-Excellent

Ethylene Chloride A-Excellent Sodium Carbonate A-Excellent

Fatty Acids A-Excellent Sodium Chlorate A-Excellent

Fluoboric Acid A-Excellent Sodium Chloride A-Excellent

Fluosilicic Acid A-Excellent Sodium Cyanide A-Excellent

Formaldehyde 100% A-Excellent Sodium Ferrocyanide A-Excellent

[0041] In embodiments, the volatile solvents compatible for aluminum include, but are not limited to the ones in the Table 4 below. A-Excellent is defined as no effect, no detectable corrosion or discoloration.

Chromium: Chromic- Sulfuric Bath 130°F

Formic Acid A-Excellent Plating Solutions: A-Excellent

Chromium: Fluoride Bath

130°F

Fruit Juice A-Excellent Plating Solutions: A-Excellent

Chromium: Fluosilicate

Bath 95°F

Furan Resin A-Excellent Plating Solutions: Copper A-Excellent

(Acid): Copper Fluoborate

Bath 120°F

Furfural A-Excellent Plating Solutions: Copper A-Excellent

(Acid): Copper Sulfate

Bath R.T.

Gasoline, leaded, ref. A-Excellent Plating Solutions: Copper A-Excellent

(Cyanide): High-Speed

Bath 180°F

Gasoline, unleaded A-Excellent Plating Solutions: Copper A-Excellent

(Cyanide): Rochelle Salt

Bath 150°F

Gelatin A-Excellent Plating Solutions: Copper A-Excellent

(Misc): Copper

(Electroless)

Glucose A-Excellent Plating Solutions: Copper A-Excellent

(Misc): Copper

Pyrophosphate

Glue, P.V.A. A-Excellent Propane (liquefied) A-Excellent

Glycerin A-Excellent Propylene A-Excellent

Heptane A-Excellent Shellac (Bleached) A-Excellent

Hexane A-Excellent Shellac (Orange) A-Excellent

Honey A-Excellent Silicone A-Excellent

Hydraulic Oil (Petro) A-Excellent Sodium Benzoate A-Excellent

Hydraulic Oil A-Excellent Sodium Ferrocyanide A-Excellent (Synthetic)

Hydrocyanic Acid A-Excellent Sodium Hydrosulfite A-Excellent

Hydrogen Gas A-Excellent Sodium Silicate A-Excellent

Hydrogen Peroxide A-Excellent Sodium Sulfate A-Excellent 10%

Hydrogen Peroxide A-Excellent Sodium Thiosulfate (hypo) A-Excellent 100%

Hydrogen Peroxide A-Excellent Soy Sauce A-Excellent 30%

Hydrogen Peroxide A-Excellent Starch A-Excellent 50%

Iodine A-Excellent Stoddard Solvent A-Excellent

Isooctane A-Excellent Styrene A-Excellent

Isopropyl Ether A-Excellent Sugar (Liquids) A-Excellent

[0042] In embodiments, the humidifier chamber includes a lid. In embodiments, the humidifier chamber includes a controllable heating device (e.g., a controllable heating sleeve, see FIG. 7A; controllable heating plate, see FIG. 7B). In embodiments, the heating element may be a base plate connected to a recirculating water bath.

[0043] Further to any method set forth above and embodiments thereof, in embodiments sample holder includes a first chip, where the liquid droplet is being deposited onto. In embodiments, the method further includes placing a second chip onto the first chip after the liquid droplet and optionally the volatile solvent droplet(s) are being deposited onto the first chip, and thereby forming a chip-sample-chip sandwich.

[0044] In embodiments, the chip is a silicon supporting chip or includes silicon nitride as substrate material. In embodiments, the chip includes silicon, silica, graphene, graphene oxide, cellulose, Gold-coated silicon nitride, Titanium-coated silicon nitride, nucleic acid-coated silicon nitride, peptide-coated silicon nitride, or protein-coated silicon nitride as a substrate material. [0045] In embodiments, the sample holder/cover and the chips (e.g., the first chip and the second chip) are together referred as TEM grids. In embodiments, the TEM grids are liquid flow grids (e.g., for Liquid Cell TEM (LCTEM)). In embodiments, the TEM grids are

electrochemical grids. In embodiments, the TEM grids are heating grids. In embodiments, the TEM grids are gas flow grids. Various commercial TEM grids can be purchased, for example, from Hummingbirds Scientific. [0046] In embodiments, the methods described herein include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) steps of the following steps: (1) placing a first chip onto the TEM sample holder; (2) conducting Fiducial recognition of the first chip; (3) taking a volatile solvent by a depositing device; (4) depositing one or more volatile solvent droplets onto the sample holder/first chip; (5) taking a liquid sample or a plurality of liquid samples by a depositing device; (6) conducting a drop calibration for the depositing device; (7) depositing a liquid droplet or a plurality of liquid droplets onto the TEM sample holder/first chip; (8) taking images of deposited grids (i.e., sample holder/first chip); (9) placing a second chip onto the sample holder/first chip, thereby forming a sandwich assembly (i.e., first chip-sample-second chip); (10) aligning the windows of the first chip and the second chip; (11) covering the TEM sample holder with a TEM sample holder cover (i.e., over clamp); and (12) inserting side-clamp into holder to seal the assembly, thereby providing a sealed TEM holder assembly. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In embodiments, each liquid droplet may include one type of chemicals.

[0047] In embodiments, the methods described herein include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) steps of the following steps: (1) placing a first chip onto the TEM sample holder; (2) conducting Fiducial recognition of the first chip; (3) taking a liquid sample or a plurality of liquid samples by a depositing device; (4) conducting a drop calibration for the depositing device; (5) depositing a liquid droplet or a plurality of liquid droplets onto the TEM sample holder/first chip; (6) taking images of deposited grids (i.e., sample holder/first chip); (7) placing a second chip onto the sample holder/first chip, thereby forming a sandwich assembly (i.e., first chip-sample-second chip); (8) aligning the windows of the first chip and the second chip; (9) covering the TEM sample holder with a TEM sample holder cover (i.e., over clamp); and (10) inserting side-clamp into holder to seal the assembly, thereby providing a sealed TEM holder assembly. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In embodiments, each liquid droplet may include one type of chemicals.

[0048] In embodiments, one or more of the steps of the methods derided herein are automated.

For example, in embodiments, one or more or all (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) steps of the following steps are automated: (1) placing a first chip onto the TEM sample holder; (2) conducting Fiducial recognition of the first chip; (3) taking a volatile solvent by a depositing device; (4) depositing one or more volatile solvent droplets onto the sample holder/first chip (as above); (5) taking a liquid sample or a plurality of liquid samples by a depositing device; (6) conducting a drop calibration for the depositing device; (7) depositing a liquid droplet or a plurality of liquid droplets onto the TEM sample holder/first chip; (8) taking images of deposited grids (i.e., sample holder/first chip); (9) placing a second chip onto the sample holder/first chip, thereby forming a sandwich assembly (i.e., first chip-sample-second chip); (10) aligning the windows of the first chip and the second chip; (11) covering the TEM sample holder with a TEM sample holder cover (i.e., over clamp); and (12) inserting side-clamp into holder to seal the assembly, thereby providing a sealed TEM holder assembly. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In embodiments, each liquid droplet may include one type of chemicals. [0049] In embodiments, one or more of the steps of the methods derided herein are automated. For example, in embodiments, one or more or all (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) steps of the following steps are automated: (1) conducting Fiducial recognition of the first chip; (2) taking a volatile solvent by a depositing device; (3) depositing one or more volatile solvent droplets onto the sample holder/first chip; (4) taking a liquid sample or a plurality of liquid samples by a depositing device; (5) conducting a drop calibration for the depositing device; (6) depositing a liquid droplet or a plurality of liquid droplets onto the TEM sample holder/first chip; (7) taking images of deposited grids (i.e., sample holder/first chip); (8) placing a second chip onto the sample holder/first chip, thereby forming a sandwich assembly (i.e., first chip-sample-second chip); (9) aligning the windows of the first chip and the second chip; (10) covering the TEM sample holder with a TEM sample holder cover (i.e., over clamp); and (11) inserting side-clamp into holder to seal the assembly, thereby providing a sealed TEM holder assembly. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In

embodiments, each liquid droplet may include one type of chemicals.

[0050] In embodiments, one or more of the steps of the methods derided herein are automated.

For example, in embodiments, one or more or all (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) steps of the following steps are automated: (1) conducting Fiducial recognition of the first chip; (2) taking a liquid sample or a plurality of liquid samples by a depositing device; (3) conducting a drop calibration for the depositing device; (4) depositing a liquid droplet or a plurality of liquid droplets onto the TEM sample holder/first chip; (5) taking images of deposited grids (i.e., sample holder/first chip); (6) placing a second chip onto the sample holder/first chip, thereby forming a sandwich assembly (i.e., first chip-sample-second chip); (7) aligning the windows of the first chip and the second chip; (8) covering the TEM sample holder with a TEM sample holder cover (i.e., over clamp); and (9) inserting side-clamp into holder to seal the assembly, thereby providing a sealed TEM holder assembly. In embodiments, each liquid sample may contain one type of nanoparticles. In embodiments, each liquid droplet may include one type of nanoparticle. In embodiments, each liquid sample may contain one type of chemicals (e.g., reactants that have size of smaller than lnm). In embodiments, each liquid droplet may include one type of chemicals.

[0051] Further to any method set forth above and embodiments thereof, in embodiments the depositing device is a piezo dispensing device. [0052] Further to any method set forth above and embodiments thereof, in embodiments automated step or steps are performed by a machine using a computer controller. In

embodiments automated step or steps are carried out by a robot (e.g., a Catesian robot) that is controlled by a computer.

[0053] Automated steps (placements and loadings) may be used for high throughput TEM analysis. In embodiments, a plurality of liquid droplets each including a nanoparticle (i.e., a plurality of samples) is being deposited onto the sample holder at the same time. In

embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or more liquid droplets samples are being deposited onto the sample holder at the same time.

[0054] In embodiments, every 1000 samples can be deposited and assembled in 15 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes or less. [0055] In embodiments, the position of each liquid droplet sample can be predetermined. The term "predetermined" in this context means that the location of the deposition of the liquid droplet on the TEM sample holder (or grid) is at a desired location (e.g., the location is determined prior to deposition and then deposited at the location). In embodiments, the deposited position is within 5 μιη (e.g., about 5 μιτι, 4 μιτι, 3 μιτι, 2 μιτι, 1 μιη) of the desired location. In embodiments, a desired location can be adjacent to, or distal from, the

predetermined position of another droplet. In embodiments, different liquid droplets are deposited at predetermined positions adjacent one another. In embodiments, liquid droplets diffuse from the initial predetermined position. In embodiments, different liquid droplets are deposited at predetermined positions which are close enough to afford diffusion of the solutions and mixing of the liquid droplets. In embodiments, placement of liquid droplets at a

predetermined position can be reproducibly achieved with an accuracy and precision of ± 5 μιτι, e.g., about 5 μιτι, 6 μιτι, 7 μιτι, 8 μιτι, 9 μιτι, 10 μιτι, 15 μιτι, 20 μιτι, 25 μιτι, 30 μιτι, 35 μιτι, 40 μιτι, 45 μιη, 50 μιη, 60 μιτι, 70 μιτι, 80 μιτι, 90 μιτι, 100 μιτι, or even greater. In embodiments, placement of two liquid droplets can be as close as 5 μιτι, 6 μιτι, 7 μιτι, 8 μιτι, 9 μιτι, 10 μιτι, 15 μιη, 20 μιη, 25 μιτι, 30 μιτι, 35 μιτι, 40 μιτι, 45 μιτι, 50 μιτι, 60 μιτι, 70 μιτι, 80 μιτι, 90 μιτι, 100 μιη, or even greater. In embodiments, the position of the deposition of the liquid droplet on the TEM sample holder is predetermined by headcam Fiducial recognition of chips (e.g., z- calibration, target recognition of chip features).

[0056] Further to the method and any embodiment thereof, in embodiments the transmission electron microscope is a liquid cell transmission electron microscope (LCTEM).

EXAMPLES

[0057] Example 1 - Picoliter Drop-on-Demand Dispensing for Multiplex Liquid Cell TEM

[0058] Abstract: Liquid Cell Transmission Electron Microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for the mixing of multiple solutions directly within the viewing area. This technique permits the deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, prior to assembly of the cell in a fully controlled manner. This study demonstrates the great success of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients. [0059] Introduction.

[0060] Liquid Cell Transmission Electron Microscopy (LCTEM) is bringing about a paradigm shift in the analysis of nanomaterials in solution. For the first time, we can use electron microscopy to not only characterize critical features including particle size and morphology, but to observe liquid phase dynamics, in real-time with nanometer resolution (de & Ross, 2011;

Williamson, et al., 2003). LCTEM has shown great potential for advancing our understanding of nanoparticle growth (Evans, et al., 2011; Liao & Zheng, 2013; Liao, et al., 2014; Patterson, et al., 2015a; Smeets, et al., 2015; Woehl, et al., 2012; Woehl, et al., 2014; Zheng, et al., 2009b) and particle-particle interactions. (Chen, et al., 2015a). There has been a particular focus on inorganic nanocrystalline materials such as Au, Pt, and Pd because these materials provide high contrast and the ability to undergo electron beam induced growth via radiolysis and metal complex reduction of precursor solutions during the imaging experiments (Evans, et al., 2011; Liao & Zheng, 2013; Liao, et al., 2014; Woehl, et al., 2014). However, increasingly other materials, including ones containing organic components (Proetto, et al., 2014a) and having low contrast, are being studied.

[0061] In a typical LCTEM experiment, a liquid sample is deposited onto the surface of a flat silicon nitride chip (ca. 2.5 x 2.5 mm square) and subsequently sealed off to prevent exposure to the internal vacuum of the electron microscope. The seal is made by placing a second chip on top of the solution and enclosing the chips within a liquid cell TEM holder (de & Ross, 2011). Therefore, the liquid thickness of the cell is set by the size of nanoparticles within the sample, which physically hold the chips apart. If a thicker cell is required, so-called 'spacer-chips', which have raised columns on the silicon nitride surface, can be used to physically separate the two silicon nitride surfaces. These spacer-chips allow for the cells to be assembled "in air" (no liquid initially between the chips), and then flow external liquid into the window region of the cell after insertion in the microscope. While many reports discuss the great potential of LCTEM for the study of nanoparticle systems formed through the mixing of solutions containing various required components or conditions (Chen, et al., 2015b; de & Ross, 2011), there has been no demonstration whereby two solutions have been mixed within the viewing windows of the liquid cell, which is believed to be largely due to technical limitations in dispensing and circulating separate liquids within the confined nanoliter volumes typical of the cells. Furthermore, while several holder designs allow the flow of multiple liquids into the tip region of the LCTEM holder using inlet/outlet lines, mixing of the liquids by this method occurs in the collection wells prior to the mixture reaching the window region of the cell (Nielsen, et al., 2014). This inability to control the location and extent of liquid mixing significantly limits the ability to study how chemistry manifests itself on the nanoscale using LCTEM. For example, nanoparticles formed through block copolymer assembly are typically synthesized through a solvent mixing process(Barnhill, et al., 2015; Choucair & Eisenberg, 2003; Mai & Eisenberg, 2012; Proetto, et al., 2014b), whereby particle assembly occurs within seconds of reaching a certain mixing ratio, which is followed by a period of particle relaxation(Barnhill, et al., 2015). Unless solution mixing is initiated directly in the window region and in the TEM field of view, initial particle assembly cannot be observed. Furthermore, we recently studied the formation of a ZIF-8 metal organic-framework system, which again occurs through solution mixing (Patterson, et al., 2015a). Due to the inability to mix the components directly in the viewing region of the cell, the initial stages of particle formation which can occur within the first 15 seconds (Cravillon, et al., 2011) cannot be observed.

[0062] We demonstrate herein that picoliter (pL) drop-on-demand dispensing can be used to controllably load multiple samples onto a single liquid cell window or a cell with an array of windows under humidifying conditions, which can provide the ability to pattern samples for both high throughput liquid cell experiments, as well as to perform mixing of multiple different solutions inside the cell. We demonstrate herein methods of dispensing and imaging solutions of two types of crystalline particles amenable to selected area diffraction for verification of structure during LCTEM. The two particles can also be chosen to be morphologically distinguishable by TEM imaging. Therefore, we employed gold nanoparticles (Au Ps) and nanocrystals of the metal-organic-framework (MOF) UiO-66 (Patterson, et al., 2015a). These two particle types were dispensed onto a single liquid cell window where initial mixing occurs upon assembly of the cell and continues through diffusion of the components during in situ LCTEM imaging. Without wishing to be bound by theory, we believe that the LCTEM technique disclosed herein can facilitate in situ nanomaterial synthesis studies that require the mixing of precursor solutions directly in the viewing area, and for studies of responsive nanomaterials that undergo dynamic transformation upon changes in solution conditions and/or constituents.

[0063] Material and Methods.

[0064] Transmission electron microscopy was performed on a FEI Sphera microscope operated at 200 keV. Micrographs were recorded on a 2K X 2K Gatan CCD. Movies were recorded at dose rates between 15.0 e.nm².s-l (6.7 xlO⁵ Gy.s^"1) and 0.2 e.nm².s-l (1.2 xlO⁵ Gy.s^"1). Ui066 was synthesized and characterized as previously reported (Patterson, et al., 2015a), and dispersed in water at 5 mg/mL. 20 nm gold nanoparticles were purchased from BB International,

EM.GC20, and used as received. LCTEM experiments were carried out with a Hummingbird Scientific Liquid Flow TEM Holder.. Drop-On-Demand dispensing was performed on a Sci- TEM (SCIENION AG, Berlin, Germany). The Sci-TEM is a liquid handling platform designed for dispensing volumes as low as 35 pL with a lateral precision of ±2 μπι. These capabilities enable the user to spot up to 10 x 10 individual spots on a single TEM grid (dimension 1 mm²). The spot positions can be easily positioned using a multi-point touch screen and optical camera system which is specifically designed to recognize TEM grids. All steps are captured and recorded by three independent cameras: the DropCam controls drop formation, stability, and volume; the GlobalCam enables intuitive selection of the target grids; and the high resolution

HeadCam allows precise fiducial-aided drop positioning. Unlike other picoliter printing systems, the Sci-TEM' s unique nozzle design, which is made out of medical-grade borosilicate glass, exhibits a round orifice with no edges. This eliminates the onset of crystallization, minimizing the chance for drop deviation. The nozzles have an orifice ranging between 50 - 80 μπι, depending on the nozzle type. This allows the Sci-TEM to dispense both nanomaterials and microstructures up to 20 μπι in diameter without clogging (i.e. carbon nanotubes, catalysts, and quantum dots). In contrast to other dispense technologies, SCIENION' s dispensers do not impose a strong shock wave on the sample and thus create minimal shear forces: e.g. living Eukaryotic cells can be dispensed without altering their viability. All liquid handling parts are composed of inert materials: PEEK®, TEFLON® and glass. The liquid phase can be freely changed from aqueous to organic solvents to allow printing of aqueous and organic samples respectively. Four coatings have been developed to modify the nozzle's surface energy to enable stable drop formation of various sample types without the need to change the sample properties. The Sci-TEM can dispense solutions within a viscosity range of 0.4 - 6 mPa. [0065] Results and Discussion.

[0066] Dispensing of low volume liquids can be used for many scientific applications including high-throughput mass spectrometry analysis (Aerni, et al., 2006), low volume synthesis

(Wixforth, et al., 2004), the preparation of materials through inkjet printing of polymer/inorganic materials (Calvert, 2001; de Gans, et al., 2004; Tekin, et al., 2008), and biosensing applications (Li, et al., 2015). The development of these approaches is of interest to the field of LCTEM for their ability to fully automate the dispensing of nano- or picoliter volumes of liquid in a controlled manner. The Sci-TEM (SCIENION AG, Berlin, Germany) used herein is an automated picoliter drop-on-demand dispensing instrument capable of recognizing and aligning TEM grids to be decorated with multiple and varied samples by dispensing liquid droplets down to 35 pL and allowing them to dry directly on the grid. Depending on the surface hydrophobicity of the substrate, droplets will either spread over large areas or remain as high contact angle ellipsoids. Typically, an array of such low volume droplets on a hydrophobic surface (e.g., silicon nitride) will evaporate within a few seconds (and even faster for hydrophilic surfaces), much too quickly to allow sealing of the liquid cell after deposition without complete or significant dehydration of the samples. However, if an array of water droplets is first dispensed around the sample area, the evaporation of these water droplets creates a local high humidity microenvironment, greatly reducing evaporation rates of the sample droplets (FIGS. 1A-1F). This method of controlled water array dispensing to create stable droplets of sample-solution, which persist for approximately 1 minute, provides sufficient time to seal the liquid cell and ensure that the samples remain hydrated. Various array designs can be imagined, utilizing different TEM grids, in order to dispense multiple picoliter sample volumes onto a single channel for controlled mixing (FIGS. 1 A-1C) or, to dispense multiple separate samples onto a single grid for high- throughput analysis (FIGS. 1D-1F). In a proof-of-concept demonstration, we used this method to observe progressive diffusion mixing of a 20 nm Au P solution and a MOF UiO-66 solution (both in H₂0). For the initial test, after dispensing the array (FIG. 1C), the solution droplets were left to dry, and the window was imaged under vacuum to show full separation of the Au NPs and MOF nanoparticles (FIGS. 2A-2G) that match very well the optical images of the dispensed droplets (FIG. 1C). Particle type was confirmed both by imaging the morphology and size of the particles and via selected area diffraction (FIGS. 2A-2G). This demonstrates that even with conventional, commercially available liquid-cell chips, two different liquid samples can both be dispensed over the imaging window (200 μπι x 50 μπι) while still being isolated from each other prior to sealing the holder, at which point mixing would be initiated due to the lateral spreading of each droplet.

[0067] To create a hydrated liquid cell for in situ TEM observation, we dispensed picoliter droplets of the two samples onto a single chip using the same two sample array and 200 μπι x 50 μπι imaging window (FIG. 1C), intending to quickly seal the cell prior to sample evaporation. The sample and water droplets were all dispensed within a 2 minute time period, and the cell was sealed in the liquid stage tip approximately 20 seconds after the two sample droplets were dispensed. At the moment of sealing the sample droplets under the top chip, both droplets were visually still hydrated on the bottom chip surface. For this experiment, the droplets were dispensed onto a 200 nm height spacer-chip, and the cell was sealed with a flat chip (windows aligned parallel) in order to set the liquid thickness to roughly the same as the MOF nanoparticles (200 nm). The spacer chip was not subjected to plasma treatment to avoid the droplets spreading immediately after dispensing. However, the flat chip was plasma treated to help the spreading of the liquid upon sealing the cell and to create a mixing front within the window. Due to the surrounding water droplets, which create the high humidity environment, sealing the cell is believed possibly to create some dilution in the NP solutions. However, for this experiment, the demonstration of mixing was the aim, rather than loading particles of a specific concentration. The liquid cell was then loaded into the microscope to start imaging within 7 minutes of sealing the cell. The imaging was performed intermittently at electron doses between 15.0 e.nmV¹ (6.7 xlO⁵ Gy.s^"1) and 0.2 e.nmV¹ (1.2 xlO⁵ Gy.s^"1) in order to limit beam damage. We have previously shown these dose rates are low enough to prevent beam damage to the MOFs

(Patterson, et al., 2015a), which are the more beam sensitive materials in the experiment. Without wishing to be bound by theory, it is believed that the beam has no effect on particle size or morphology, although some charging of the particles can be envisaged to occur. [0068] Initial imaging of the cell (FIGS. 3A-3G) showed discrete areas of MOFs nanocrystals at the 'top' of the cell (FIGS. 3A-3C), AuNPs at the 'bottom' of the cell (FIGS. 3E-3F), and an area with initial mixing of the two components at the center of the cell (FIG. 3D). After approximately 30 minutes following cell assembly, we observed AuNPs (based on their distinctive size, morphology and contrast) at the top of the cell mixed with the MOF

nanoparticles (FIGS. 4A-4G) indicating AuNP migration across the cell. The number of detected AuNPs at the top of the cell continued to increase over time during our observation. This observed movement / mixing is much slower than would be expected based on bulk diffusion of these nanomaterials, but is consistent with other recent work studying AuNP dynamics in a similar LCTEM assembly (Verch, et al., 2015; Woehl & Prozorov, 2015). It is well know that near surface effects in the liquid cell can dramatically reduce diffusion rates (Proetto, et al., 2014b; Zheng, et al., 2009a). Motion of individual Au NPs was observed, as well as their adherence to the surface of the MOFs, which can be clear indicators of a fully hydrated cell. No migration of MOF particles was observed to the bottom end (i.e. the location initially patterned with AuNPs) of the liquid cell. We recently reported that the assembly of liquid cell immediately after plasma treatment (as in these experiments) resulted in the observation of MOF particles stuck to the liquid cell windows; however, assembly of the cell 1-2 hours post plasma treatment resulted insignificant motion if particles inside the cell (Patterson, et al., 2015a). Therefore the lack of motion observed here can be likely due to either their adherence to the plasma treated SiN_x membrane and/or to their large size (roughly equal to the spacer height). Controlling particle motion inside the cell is of paramount importance for the observation of dynamics.

Particles moving too quickly will not be able to be observed, either due to their motion exceeding the maximum framerate of the CCD camera, or due to decreased signal-to-noise when using high framerates. However, if particle motion is too slow, then the time scales over which mixing or dynamic process (e.g., particle collisions or interactions) will occur can exceed the threshold timescales related to beam damage. Further work in controlling particle and small molecule motion in the cell is clearly needed (Patterson, et al., 2015b). This can involve not only differences in plasma coating times, intensities and surface aging, but also more permanent surface coatings and functionalization. Furthermore, detailed experiments involved controlled liquid thickness and beam doses can be conducted. However, it is believed that controlled motion combined with the mixing procedures outlined here can enable much more detailed experiments involving solution mixing and chemistry inside the cell. The ability to spot two different liquids at μιη-scale separation distances on LCTEM chips greatly improves the probability for solution mixing over the viewing window for directly observing chemical reactions by LCTEM, and can help overcome the limitations associated with the sub-diffusion motion of nanomaterials inside liquid cells, shorting the imaging times (radiolysis doses) required to observe particle migration and mixing.

[0069] Conclusion. These experiments demonstrate that automated picoliter drop-on-demand dispensing, combined with automated grid recognition and alignment in the Sci-TEM instrument can be used to mix multiple solutions within the liquid cell, allowing assembly of liquid cells to observe nanoparticle migration and interaction, and potentially chemical reactions, while imaging in the microscope. Furthermore, using this dispensing technology and imaging strategy, one can conceive of many array designs where the mixing time of multiple different solutions could occur within one experiment. From a fundamental standpoint, technology for controlling the dispensing of low liquid volumes is essential for the advancement of LCTEM and this study shows the great success to perform direct solution mixing and/or high-throughput in situ experiments. This study also demonstrated a new approach that enables researchers to study processes involving mixing of materials and/or reagents. [0070] References (Example 1).

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[0101] Overview. As depicted in FIGS. 6A-6B, the Sci-TEM Liquid Cell sample prep system includes a Cartesian robot [FIGS. 6A-6B, element 1] equipped with imaging systems (Global camera, Head Camera, Drop Camera) [FIG. 6B, element 2], low volume non-contact dispensing nozzles [FIG. 6B, element 3], vacuum nozzle [FIG. 6B, element 4], Microarray chamber loaded with LC TEM stage and grids [FIG. 6B, element 5].

[0102] Instrument. The depicted chamber (FIG. 6A) provides a local environment of near- saturated solvent vapor to control drying of volatile samples. As depicting in FIGS. 7A-7B, liquid sample arrays can be loaded on a variety of supports including liquid cell stages inserted through a stage port located in the front of the chamber. Liquids are introduced through a small opening in the top of the chamber with liquid dispensing nozzles. This vertical opening is also used as line of sight for targeting the grid, analyzing deposited droplets, and placement of the top chip. Access to the liquid cell stages can be through a hole large enough for imaging and printing (e.g., deposition) to the whole grid with a single nozzle. Physiochemical processes depicted in FIGS. 7A-7B include e.g., diffusion and convection of solvent. A controllable heating sleeve can be employed as indicated in FIG. 7A. A controllable heating plate can be employed as indicated in FIG. 7B.

[0103] Instrument Setup. Setup times for 2.5 nL drops of H₂0 were determined for the SciTEM Liquid Cell sample prep system as depicted in FIGS. 6A-6B and 7. The drying after droplet deposition is tabulated in Table 5 following.

Table 5. Setup drying times for SciTEM Liquid Cell sample prep system

[0104] Loading the SciTEM Liquid Cell sample prep system

[0105] The processing can begin when all necessary components are loaded into the robotic working area. This includes liquid samples in open container on Plate Holder; Microarray chamber reservoir filled with solvent: LC stage loaded with bottom chip in the Microarray chamber; top chip located face down in the chip cradle. FIG. 8A schematically depicts the robotic working area. A photographic inset (FIG. 8B) depicts the humidifying chamber and the chip positioning.

[0106] The process of loading takes the following steps. Steps 2-6 can be done a variety of orders based on the needs.

[0107] 1. The Headcam Fiducial recognition of chips - z-calibration, target recognition of chip features.

[0108] 2. Aspiration of samples into nozzles.

[0109] 3. Drop calibration using recognition and actuation software.

[0110] 4. Deposition of droplets onto grid with each nozzle.

[0111] 5. Optionally, acquisition of images from Headcam of printed guid.

[0112] 6. Moving pick-up tool to top-chip position and activation of vacuum pump to pick up top-chip.

[0113] 7. Moving top chip to location above bottom chip and encapsulating droplets in patterned thin liquid films.

[0114] 8. Removal of LC-Stage from microarray chamber, finishing assembly of the LC stage, insertion into a TEM device.

[0115] Using these steps, it has been found that the total robot time can be about 2-min, and the total time from robot to TEM imaging can be about 1-5 min.

[0116] Example 3 - In situ Liquid-Cell TEM (LC-TEM)

[0117] FIG. 9A provides a photographic depiction of relative positioning of Top Chip and Bottom Chip, showing 30 nm thick SiN_x "windows" and 50 μπι x 50 μπι lateral area. FIG. 9B provides a photographic depiction of probe for in situ Liquid-Cel TEM (LC-TEM) grids

(including the sample holder, the over clamp and the side clamp). The area indicated in dashed rectangle is enlarged in FIG. 9C, which depicts liquid flow lines, S/TEM electron beam, silicon support chip. The further inset to FIG. 9C depicts relative positioning of top SiN_x, liquid sample, and bottom SiN_x regions. [0118] Embodiments

[0119] Embodiments disclosed herein include embodiments PI to P7 following.

[0120] Embodiment PI . A method for depositing a solution directly on a transmission electron microscope (TEM) grid, the method including: 1) loading a solution into a piezo dispensing device; and 2) depositing an aliquot of the solution from the piezo dispensing device directly onto the TEM grid.

[0121] Embodiment P2. The method of embodiment PI, wherein step 1) is repeated for each of a plurality of solutions. [0122] Embodiment P3. The method of embodiment P2 ,wherein step 2) is repeated for each of the plurality of solutions.

[0123] Embodiment P4. The method of embodiment P3, wherein each depositing for each of the plurality of solutions is at a predetermined position on the TEM grid.

[0124] Embodiment P5. The method of embodiment P4, wherein the predetermined position is suitable to afford mixing of a plurality of solutions on the TEM grid.

[0125] Embodiment P6. The method of embodiment PI, wherein the solution includes a nanoparticle.

[0126] Embodiment P7. A method for liquid cell transmission electron microscopy (LCTEM), the method including: 1) preparing a TEM grid as set forth in any one of embodiments PI to P6; and 2) conducting LCTEM on the TEM grid in a LCTEM instrument.

[0127] Further embodiments includes embodiments 1 to 40 following.

[0128] Embodiment 1. A method for detecting a nanoparticle in a liquid, the method including: a. depositing a liquid droplet including a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, wherein the volume of the liquid droplet is less than about 1000 pL; b. covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover; and c. detecting the nanoparticle within the liquid droplet using a transmission electron microscope.

[0129] Embodiment 2. The method of embodiment 1, wherein the depositing and the covering are automated. [0130] Embodiment 3. The method of embodiment 1, wherein the humidifying conditions are generated by depositing a volatile solvent droplet onto the sample holder.

[0131] Embodiment 4. The method of embodiment 3, wherein the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitnle or cyclohexanone.

[0132] Embodiment 5. The method of embodiment 3, wherein the volatile solvent is water.

[0133] Embodiment 6. The method of embodiment 3, wherein the volatile solvent droplet is a plurality of volatile solvent droplets.

[0134] Embodiment 7. The method of embodiment 1, wherein the humidifying conditions are generated by a humidifier chamber.

[0135] Embodiment 8. The method of embodiment 7, wherein the humidifier chamber is surrounding the sample holder.

[0136] Embodiment 9. The method of embodiment 7, wherein the humidifier chamber includes a volatile solvent reservoir. [0137] Embodiment 10. The method of embodiment 9, wherein the volatile solvent reservoir is underneath the transmission electron microscope sample holder.

[0138] Embodiment 11. The method of embodiment 10, wherein the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitnle or cyclohexanone. [0139] Embodiment 12. The method of embodiment 10, wherein the volatile solvent is water.

[0140] Embodiment 13 . The method of embodiment 1, wherein the sample holder includes a first silicon support chip.

[0141] Embodiment 14. The method of embodiment 13, further including placing a second silicon support chip onto the first silicon support chip. [0142] Embodiment 15. The method of embodiment 14, wherein the placing a second silicon support chip is automated.

[0143] Embodiment 16. The method of embodiment 1, wherein the transmission electron microscope is a liquid cell transmission electron microscope (LCTEM). [0144] Embodiment 17. A method for detecting a plurality of nanoparticles and an interaction thereof in a liquid, the method including: a. depositing a plurality of liquid droplets each including a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, wherein the volume of each liquid droplet is less than about 1000 pL; b. covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover; and c. detecting the plurality of nanoparticles and interaction thereof upon diffusion of the plurality of liquid droplets using a transmission electron

microscope.

[0145] Embodiment 18. The method of embodiment 17, wherein the depositing and the covering are automated.

[0146] Embodiment 19. The method of embodiment 17, wherein the humidifying conditions are generated by depositing a volatile solvent droplet onto the sample holder.

[0147] Embodiment 20. The method of embodiment 19, wherein the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or cyclohexanone.

[0148] Embodiment 21. The method of embodiment 19, wherein the volatile solvent is water.

[0149] Embodiment 22. The method of embodiment 17, wherein the volatile solvent droplet is a plurality of volatile solvent droplets.

[0150] Embodiment 23. The method of embodiment 17, wherein the humidifying conditions are generated by a humidifier chamber.

[0151] Embodiment 24. The method of embodiment 23, wherein the humidifier chamber is surrounding the sample holder.

[0152] Embodiment 25. The method of embodiment 23, wherein the humidifier chamber includes a volatile solvent reservoir. [0153] Embodiment 26. The method of embodiment 25, wherein the volatile solvent reservoir is underneath the transmission electron microscope sample holder.

[0154] Embodiment 27. The method of embodiment 26, wherein the volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or cyclohexanone [0155] Embodiment 28. The method of embodiment 26, wherein the volatile solvent is water.

[0156] Embodiment 29 . The method of embodiment 17, wherein the sample holder includes a first silicon support chip.

[0157] Embodiment 30. The method of embodiment 29, further including placing a second silicon support chip onto the first silicon support chip.

[0158] Embodiment 31. The method of embodiment 30, wherein the placing a second silicon support chip is automated.

[0159] Embodiment 32. The method of embodiment 17, wherein the transmission electron microscope is a liquid cell transmission electron microscope (LCTEM). [0160] Embodiment 33. A method of analyzing a sample using transmission electron microscope, the method including: a. automatically depositing a liquid droplet of the sample onto a transmission electron microscope sample holder, wherein the volume of the liquid droplet is less than about 1000 pL; b. analyzing the liquid sample using transmission electron microscope. [0161] Embodiment 34. The method of embodiment 33, wherein the automatically depositing is depositing within 10 microns of a predetermined location on the transmission electron microscope sample holder.

[0162] Embodiment 35. The method of embodiment 33, wherein the liquid sample includes a plurality of nanoparticles. [0163] Embodiment 36. The method of embodiment 35, wherein the analyzing including detecting the plurality of nanoparticles.

[0164] Embodiment 37. The method of embodiment 33, further including covering the transmission electron microscope sample holder with a transmission electron microscope sample holder cover prior to the analyzing. [0165] Embodiment 38. The method of embodiment 37, wherein the covering is automated.

[0166] Embodiment 39. The method of embodiment 33, wherein the automatically depositing is performed under humidifying conditions.

[0167] Embodiment 40. The method of embodiment 39, wherein the humidifying conditions are generated by a humidifier chamber.

Claims

WHAT IS CLAIMED IS: 1. A method for detecting a nanoparticle in a liquid, the method comprising: a. depositing a liquid droplet comprising a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, wherein the volume of said liquid droplet is less than about 1000 pL;

b. covering said transmission electron microscope sample holder with a transmission electron microscope sample holder cover; and

c. detecting said nanoparticle within said liquid droplet using a transmission electron microscope.

2. The method of claim 1, wherein said depositing and said covering are automated.

3. The method of claim 1, wherein said humidifying conditions are generated by depositing a volatile solvent droplet onto said sample holder.

4. The method of claim 3, wherein said volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or

cyclohexanone.

5. The method of claim 3, wherein said volatile solvent is water.

6. The method of claim 3, wherein said volatile solvent droplet is a plurality of volatile solvent droplets.

7. The method of claim 1, wherein said humidifying conditions are generated by a humidifier chamber.

8. The method of claim 7, wherein said humidifier chamber is surrounding said sample holder.

9. The method of claim 7, wherein said humidifier chamber comprises a volatile solvent reservoir.

10. The method of claim 9, wherein said volatile solvent reservoir is underneath said transmission electron microscope sample holder.

11. The method of claim 10, wherein said volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or

cyclohexanone.

12. The method of claim 10, wherein said volatile solvent is water.

13 . The method of claim 1, wherein said sample holder comprises a first silicon support chip.

14. The method of claim 13, further comprising placing a second silicon support chip onto said first silicon support chip.

15. The method of claim 14, wherein said placing a second silicon support chip is automated.

16. The method of claim 1, wherein said transmission electron microscope is a liquid cell transmission electron microscope (LCTEM).

17. A method for detecting a plurality of nanoparticles and an interaction thereof in a liquid, the method comprising:

a. depositing a plurality of liquid droplets each comprising a nanoparticle onto a transmission electron microscope sample holder under humidifying conditions, wherein the volume of each liquid droplet is less than about 1000 pL;

b. covering said transmission electron microscope sample holder with a transmission electron microscope sample holder cover; and

c. detecting said plurality of nanoparticles and interaction thereof upon diffusion of said plurality of liquid droplets using a transmission electron microscope.

18. The method of claim 17, wherein said depositing and said covering are automated.

19. The method of claim 17, wherein said humidifying conditions are generated by depositing a volatile solvent droplet onto said sample holder.

20. The method of claim 19, wherein said volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or

cyclohexanone.

21. The method of claim 19, wherein said volatile solvent is water.

22. The method of claim 17, wherein said volatile solvent droplet is a plurality of volatile solvent droplets.

23. The method of claim 17, wherein said humidifying conditions are generated by a humidifier chamber.

24. The method of claim 23, wherein said humidifier chamber is surrounding said sample holder.

25. The method of claim 23, wherein said humidifier chamber comprises a volatile solvent reservoir.

26. The method of claim 25, wherein said volatile solvent reservoir is underneath said transmission electron microscope sample holder.

27. The method of claim 26, wherein said volatile solvent is water, alcohol, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), acetone, acetonitrile or

cyclohexanone

28. The method of claim 26, wherein said volatile solvent is water.

29 . The method of claim 17, wherein said sample holder comprises a first silicon support chip.

30. The method of claim 29, further comprising placing a second silicon support chip onto said first silicon support chip.

31. The method of claim 30, wherein said placing a second silicon support chip is automated.

32. The method of claim 17, wherein said transmission electron microscope is a liquid cell transmission electron microscope (LCTEM).

33. A method of analyzing a sample using transmission electron microscope, the method comprising:

a. automatically depositing a liquid droplet of said sample onto a transmission electron microscope sample holder, wherein the volume of said liquid droplet is less than about 1000 pL;

b. analyzing the liquid sample using transmission electron microscope.

34. The method of claim 33, wherein said automatically depositing is depositing within 10 microns of a predetermined location on said transmission electron microscope sample holder.

35. The method of claim 33, wherein said liquid sample comprises a plurality of nanoparticles.

36. The method of claim 35, wherein said analyzing comprises detecting said plurality of nanoparticles.

37. The method of claim 33, further comprising covering said transmission electron microscope sample holder with a transmission electron microscope sample holder cover prior to said analyzing.

38. The method of claim 37, wherein said covering is automated.

39. The method of claim 33, wherein said automatically depositing is performed under humidifying conditions.

40. The method of claim 39, wherein said humidifying conditions are generated by a humidifier chamber.