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1 Supporting information Vacuum ultraviolet laser desorption/ionization mass spectrometry imaging of single cells with submicron craters Jia Wang, 1, + Zhaoying Wang, 2, + Feng Liu, 1 Lesi Cai, 2 Jian-bin Pan, 3 Zhanping Li, 2 Sichun Zhang, 2 Hong-Yuan Chen, 3 Xinrong Zhang, 2* Yuxiang Mo 1* 1 Department of Physics and State Key Laboratory of Low-Dimensional Quantum Physics, Tsinghua University, Beijing , China 2 Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, , P.R. China. 3 The State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Jiangsu , China. *To whom correspondence should be addressed: Prof. Yuxiang Mo Department of Physics and State Key Laboratory of Low-Dimensional Quantum Physics Tsinghua University Beijing , China ymo@mail.tsinghua.edu.cn Prof. Xinrong Zhang Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation Tsinghua University Beijing, , China. xrzhang@mail.tsinghua.edu.cn Table of Contents Design and implementation for focusing VUV laser.. S-2 VUV laser ablation with submicron craters S-2 Simple model for the depth estimation of VUVDI.....S-3 Figure S-1....S-4 Table S S-5 Table S S-6 Figure S S-7 Figure S S-7 Figure S S-7 Figure S S-8 Figure S-6...S-8 References...S-8 S-1

2 Design and implementation for focusing VUV laser. The MgF 2 windows and lenses were used in the experiments because conventional glass and quartz are opaque at nm. We chose the co-axis optics system to focus the VUV laser beam. The gratings or single off axis lens are simple and widely used, when considering the separation of VUV laser beam from other fundamental laser beams in four wave mixing experiments. 1, 2 However, it is inconvenient to focus VUV laser to a tiny spot and ultimately to diffraction limit due to two reasons: (1) the intensities of fundamental laser are high enough to damage the gratings; (2) the light path could not be collimated and the windows and lens need to be cleaned frequently, which results in lots of time to re-align the optics. Instead, we chose the co-axis optics system with a pair of MgF 2 lenses (L 1 (f 1 = 49 mm), L 2 (f 2 = 65 mm)) and an aperture (Ф 20 µm) at the focus point as a space filter to block parts of VUV laser, which was necessary to focus the VUV laser beam to diffraction limit. The coaxial design makes VUV light path alignment more efficient. The VUV was then focused on the interest region by an aspheric MgF 2 lens L 3 (f 3 = 47 mm). The L 3 and sample were mounted on five piezo motor driven linear stages and three nano-positioner linear piezo stages, respectively. The distances of D 1, D 2, D 3 and D 4 were designed as 145, 191, 450, 49 mm respectively. The relationships are (1) 1.5 (f 1 + f 2 ) > D 1 > f 1 + f 2 ; (2) D 1 > 2f 1 ; (3) f 2 > f 1 ; (4) D 2 = (D 1 - f 1 ) f 2 / (D 1 - f 1 - f 2 ). The whole optics system was simulated by Zemax software, the spot size of VUV laser at aperture was ~ Ф 40 µm, which was in good agreement with experiment result. The aspherical lens L 3 eliminated the spherical aberration of lenses L 1 and L 2. The whole system was designed to reach spot size of 500 nm in diameter. For the tunable wavelength of VUV laser, the refractive index of MgF 2 lens are slightly different. However, the submicron spot can be achieved within the range of refractive index (more than 80% energy is concentrated in a 1 µm spot). Regardless of the birefringence effect and machining errors of MgF 2 lenses, the assembly errors also should take into account. Based on the analysis of assembly errors, the L 3 is more sensitive to pitch angles, while L 1 and L 2 are insensitive. In addition, L 1 and L 2 are more sensitive to eccentricity, and the diffraction limit can be achieved when the eccentric distance is less than 10 µm. The YAG: Ce crystal as a fluorescent material was chosen to measure the spot size of VUV laser because of its high luminescence efficiency, relatively long wavelength (520 nm 630 nm) emission and good stability of lifetime. 3 The YAG: Ce crystal sample is only 50 µm thick to make the fluorescence measurements more accurate. The yellow fluorescence from YAG: Ce crystal was collected by a homebuilt microscopic observation system including a CCD camera, an objective lens (20, working distance 31 mm), a filter ( nm) and a zoom lens (up to 7 ). The schematic diagram of the experimental setup is shown in Figure 1c. VUV laser ablation with submicron craters. We have produced a series submicron craters on YAG: Ce crystal by focusing the coherent VUV laser at nm (Figure S-1). The atomic force microscope (AFM) image of the ablated craters was measured (Figure S-1a). The submicron craters ablated by VUV laser formed a clear pattern of an Arabic numeral 0 (horizontal view). The pulse-to-pulse reproducibility of VUV laser ablation was found to be good, as illustrated by AFM image. All ablated craters were created with 10 pulses. The distance between each crater is 1 µm. The profile of 11 submicron craters (red dash line in Figure S-1a) is presented in Figure S-1b. The minimum and maximum diameter of craters are 440 nm and 880 nm respectively. The smallest crater has a volume of ~ 0.5 al. The diameter of the minimum crater created by VUV laser ablation was roughly 16 % of the spot size, which is similar to the result of quartz. 4 However, the smallest diameter produced on metal film with wavelength of 800 nm was roughly 60% of the spot size. 5 For these craters, the average diameter, etch depth and volume rate are 740 ± 120 nm, 3.6 ± 1.1 nm/pulse and ~ 1.0 al/pulse respectively. The production of these small features is possible because (1) VUV laser ablation is a photochemical interaction process and the effects of thermal S-2

3 diffusion are minimized; (2) VUV laser ablation patterns have Gaussian distribution, only parts of spot with high fluence could ablate the samples. As for nanosecond laser ablation process with longer wavelength radiation, some of laser energy is absorbed for thermal process result in heating, melting and damaging samples. 6 However, AFM images show that the profile of VUV laser ablation is very smooth with no signs of thermal damage due to the strong absorption. Eight ablation craters on YAG: Ce crystal by different VUV laser pulses were produced to measure the etch depth. Figure S-2c shows the optical image of craters at the focal plane of L 3. These ablation craters shown in Figure S-2c were produced with 20, 40, 60, 80, 100, 200, 400, 600 laser pulses respectively. The VUV laser fluence to ablate each crater is W/cm 2. The increase of VUV laser pulses caused the amplification of the depth and diameter of craters (Figure S-2d). The ablated volume was varied from 20 al (10 pulses) to 800 al (600 pulses). The inset shows the evolution of the etch depth as a function of pulse numbers. The etch rate of the craters ablated for 600 pulses is ~ 1 nm/pulse, which is twice lower than that of 20 pulses. We noticed the crater diameter was much larger when attaining 600 pulses, which would decrease the lateral resolution for scanning experiments. The scanning step should be within tens of pulses rather than hundreds of pulses for VUVDI-MSI experiments. Simple model for the depth estimation of VUVDI. Many kinds of molecules have quite high absorption efficiencies for VUV laser, especially in our case (125.3 nm, 9.9 ev/photon). We assumed the absorption efficiencies for every molecule (m/z < 400) are the same. Therefore, the number of ablated molecules N are the same for each VUV laser pulse. We have N=V*n=0.67*S*D*n, where, n is molecular population density per unit volume, V is ablated volume of each pulse (V 0.67* S*D is obtained, S is the ablated area of VUV laser, D is the depth of each pulse). This can be expressed as D*n=1.5*N/S=constant, which shows that the depth of each pulse D is inversely proportional to the molecular population density per unit volume n. For a molecule with density ρ and molecular mass M, molecular population density per unit volume n = ρn A /M is obtained, where N A is the Avogadro s number. For two molecules (molecule1 and molecule 2), we can get the depth of each pulse for molecule 2 n1 ρ1 M 2 D2 = D1 = D1. n2 ρ2 M1 We have found that each VUV laser pulse ablated ~ 1 nm layer of quartz material (SiO 2 ). [3] For quartz, the density ρ 1 is ~ 2.65 g/cm 3 and the molecular mass M 1 is ~ 60. As for cholesterol, the density ρ 2 is ~ 1.05 g/cm 3 and the molecular mass M 2 is ~ 386. Then the depth of each pulse D 2 is ~ 16 nm according to the equation above. Similarly, for the alanine molecule, the density ρ 2 is ~ 1.44 g/cm 3 and the molecular mass M 2 is ~ 89. Then the depth of each pulse D 2 is ~ 3.7 nm. S-3

4 Figure S-1. Characterization of focusing VUV laser at submicron level. (a) Atomic force microscope (AFM) image of the ablated craters of an Arabic numeral 0 (horizontal view). Created by 10 pulses with fluence of 5.0ⅹ10 8 W/cm 2. (b) Profile of ablation craters along with red dash line in (a). (c) Optical image of craters produced by VUV laser ablation on YAG: Ce crystal with 20, 40, 60, 80, 100, 200, 400, 600 pulses. (d) Depth profiling according to the craters shown in (c). The inset in (d) shows the evolution of the etch depth as a function of pulse numbers. See Supplementary section "VUV laser ablation with submicron craters" for details. S-4

5 Table S-1. Calculations of the limit of detection for VUVDI and ToF-. Samples Methods Number of molecular ions DHB (at m/z 155) CHCA (at m/z 190) vitamin E (at m/z 431) Rhodamine B (at m/z 444) Janus Green B(at m/z 476) Ir(dtbpa) 3 (at m/z 1223) Ir(Ftbpa) 3 (at m/z 1613) Area probed (µm 2 ) Depth probed (µm) Volume probed (al) Ion yield (al -1 ) Ratio of Ion yield (VUVDI/ ) VUVDI TOF VUVDI TOF VUVDI TOF VUVDI TOF VUVDI TOF VUVDI TOF VUVDI TOF Note that the limit of detection of azithromycin was not shown here due to the low signals. S-5

6 Table S-2. Calculation of fragmentations for VUVDI and ToF- of seven organic molecules. Sample m/z (counts) VUV (mv) DHB (C 7 H 6 O 4 ) MW: Ratio ([m/z 137]/ [m/z 155]) CHCA (C 10 H 7 NO 3 ) MW: Ratio ([m/z 146]/ [m/z 190]) Vitamin E (C 29 H 50 O 2 ) MW: Ratio ([m/z 165]/ [m/z 431]) Rhodamine B (C 28 H 31 ClN 2 O 3 ) MW: Ratio ([m/z 400]/ [m/z 444]) Janus Green B (C 30 H 31 ClN 6 ) MW: Ratio ([m/z 327]/ [m/z 476]) Ir(dtbpa) 3 (C 60 H 33 IrN 6 S 6 ) MW: Ratio ([m/z 878]/ [m/z 1223]) Ir(Ftbpa) 3 (C 72 H 33 F 18 IrN 6 S 3 ) MW: Ratio ([m/z 1132]/ [m/z 1613]) S-6

7 Figure S-2. The intensity evolution as a function of VUV laser fluence for organic molecules. The LATs of glycerol (1), CHCA (2), palmitic acid (3), cholesterol (4), vitamin E (5) and Rhodamine B (6) are 2.3 ⅹ10 7, 7.5ⅹ10 7, 3.8ⅹ10 7, 3.8ⅹ10 7, 2.3ⅹ10 7, 5.0ⅹ10 7 W/cm 2 respectively. Figure S-3. The fragment and [M+H] + signals of DHB as a function of laser fluence. The signal of fragment (m/z 41) is less than molecular ions (m/z 155) at low fluence, while the opposing trend appears in the high fluence range. Figure S-4. A sequence of 2D ion images of HeLa cells. MSIs of HeLa cells were obtained by (two cells) and VUVDI (sample 1 with single cell; sample 2 with two cells) according to different m/z. VUVDI-MSI was obtained with fluence of 1.7ⅹ10 7 W/cm 2. The units of signals for VUVDI and ToF- are mv and counts respectively. S-7

8 Figure S-5. MSIs of two HeLa cells obtained with (upper) and VUVDI (down) according to different m/z. VUVDI-MSI was obtained with fluence of 1.2ⅹ10 8 W/cm 2. Figure S-6. VUVDI mass spectra of MSI for HeLa, POPC and DOPC. For the convenience of comparison, the total MSI signal was divided by 10 4 (black line). References [1] Albert, D. R.; Proctor, D. L.; Davis, H. F., Rev. Sci. Instrum. 2013, 84, ; [2] Riedel, D.; Castex, M. C. Appl. Phys. A 1999, 69, [3] Bachmann, V.; Ronda, C.; Meijerink, A. Chem. Mater. 2009, 21, [4] Wang, J.; Liu, F.; Mo, Y.; Wang, Z.; Zhang, S.; Zhang, X. Rev. Sci. Instrum. 2017, 88, [5] Pronko, P. P.; Dutta, S. K.; Squier, J.; Rudd, J. V.; Du, D.; Mourou, G. Opt. Commun. 1995, 114, [6] Garrison, B. J.; Srinivasan, R. J. Appl. Phys. 1985, 57, S-8

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