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Multi-frequency optomechanical stiffness measurement of single adherent cells on a solid substrate with high throughput Ali Mehrnezhad, and Kidong Park Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02356 • Publication Date (Web): 12 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017
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Multi-frequency optomechanical stiffness measurement of single adherent cells on a solid substrate with high throughput Ali Mehrnezhad and Kidong Park* Division of Electrical and Computer Engineering, Electrical Engineering Building, Louisiana State University, Baton Rouge, LA, 70809, U.S.A. E-mail:
[email protected] Keywords: cell mechanics, cancer, laser interferometry, single cell analysis, HT-29
Abstract
Mechanical properties of a cell reflect its biological and pathological conditions and there have been active research efforts to develop high-throughput platforms to mechanically characterize single cells. Yet, many of these research efforts are focused on suspended cells and use a flowthrough configuration. In this paper, the stiffness of single adherent cells are optomechanically characterized using the vibration-induced phase shift (VIPS) without detaching them from the substrate. With the VIPS measurements, the frequency and amplitude dependency of the cell stiffness is investigated and statistically significant difference in the cell stiffness is confirmed after exposure to various drugs affecting cytoskeleton network. Furthermore, a 3-dimensional finite element model of a cell on a vibrating substrate is developed to extract the mechanical property from the measured VIPS. The developed technique can characterize the mechanical properties of single adherent cells at multiple frequencies with high throughput and will provide valuable clues in understanding cell mechanics.
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Mechanical properties of living cells are closely related to the physiological and pathological status of the cells.1 Cancerous cells in many types of tumors such as, breast cancer,2,3 prostate cancer,4 thyroid cancer,5 and kidney cancer,6 were reported to be softer than their benign counterparts. Even among cancerous cells, highly metastatic cancer cells tend to be softer and more deformable than non-invasive ones. Cross et al.3 reported that metastatic cancer cells taken from the patients with lung, breast, and pancreatic cancer were significantly softer than corresponding normal cells. Corbin et al.2 showed that non-malignant human breast cancer cells, MCF-10 were stiffer than corresponding non-invasive breast cancer cells, MCF-7 and that highly-invasive breast cancer cells, MDA-MB-231 were softer than MCF-7. Human red blood cells infected by malaria7 and diabetic cardiomyocytes8 showed increased stiffness in comparison to healthy counterparts. Inflammation of neutrophil cells after treatment with inflammatory mediators reduced the deformability of the cells.9 Stem cell differentiation is also associated with changes of the mechanical properties.10–12 These studies established mechanical properties of cells as a biomarker of cells’ biological conditions,1,13 which can be used to investigate various cellular disorders and essential cellular processes.
Various methods to characterize mechanical properties of single cells were developed. These methods can be largely divided into two categories, one that requires suspended cells and one that measures cells attached on substrate. The former includes microfluidic flow cytometry,14–17 suspended microchannel resonator,18 optical stretcher,19,20and solid-state micropores21,22. These methods are based on a flow-through configuration and exhibit very high throughput. Thus, they are suitable to obtain a snapshot of a large cell population and identify a rare subgroup in a heterogeneous population. When assessing the effects of external stimuli such as cancer drugs or inhibitors of key pathways, these methods provide a statistical inference by comparing two cell 2 ACS Paragon Plus Environment
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groups with and without the stimuli. However, with the flow-through configuration, it is difficult to directly compare the mechanical properties of a same cell before and after the exposure to external stimuli with high throughput.
On the other hand, techniques in the second category measure adherent cells without detaching them from the substrate and usually provide more detailed characterization on a small number of cells. These techniques include AFM indentation,23,24 micropipette aspiration,25,26 magnetic bead twisting,27,28 and microrheology.29 Many of these techniques can provide the mechanical characterization on a sub-cellular level. As these techniques can repeat the measurement on a same cell, the temporal dynamics of cell mechanics can be characterized. However, these techniques induce significant deformation on the cells, which can damage the cells or changes their biological or physical properties. Also, the observations can be often obscured by cell-tocell variations, due to their limited throughput.
In this study, we measured vibration induced phase shift (VIPS) of human colon adenocarcinoma cells (HT-29), to demonstrate mechanical characterization of single adherent cells. VIPS does not require direct contact and produces small deformation less than 1 nm. As such, it is non-invasive and can characterize same single cells repeatedly. In our earlier work,30 a MEMS resonator array oscillated cells for VIPS measurement. However, the use of the MEMS resonator array limited the assay throughput, as only several cells attached properly on the MEMS resonators could be characterized. Moreover, the mechanical properties of the target cells could be measured only at the first resonant frequency of the MEMS resonators, significantly limiting the measurement flexibility. To address these issues, a new actuation unit based on a bare silicon chip and a commercial piezoelectric plate was developed in this study. The new actuation unit was based on off-the-shelf components and enabled the mechanical characterization at various frequencies. 3 ACS Paragon Plus Environment
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With the new actuation unit, any cells on the silicon substrate can be measured, substantially increasing the measurement throughput and flexibility. Measurements are done in a sequential point-by-point manner with throughput of ~25 points/minute. It can provide high throughput measurement if the number of scanning points per cell is reduced. Using the new measurement setup, many VIPS profiles of live, fixed, and chemically treated cells are obtained to statistically show how cytoskeleton structure affects the mechanical properties of the cell. We also investigate how the actuation frequency and amplitude affect the VIPS measurements. Lastly, we setup a 3dimensional numerical model to extract the mechanical properties from the measured VIPS.
Experimental Section Measurement principles: The vertical vibration of a silicon substrate deforms the cells on it. This deformation produces the oscillation in the cell height, which is inversely proportional to the cell stiffness. A laser Doppler vibrometer (LDV) and a lock-in amplifier are used to measure the amplitude and phase of the cell height oscillation to characterize the mechanical properties of the cell, similarly to our earlier report.30
As shown in Figure 1, adherent single cells are growing on a bare silicon chip. In this figure, ncell, nGM, h, As, Ac, ω and ϕ represent the refractive index of the cell, the refractive index of the growth media, the cell height, the amplitude of the silicon chip’s vibration, the amplitude of the cell height oscillation, the angular frequency of vibration, and the phase of height oscillation, respectively. The silicon chip is vibrating vertically with amplitude of As (0.5 - 4 nm) and angular frequency of ω (90 - 120 kHz), as shown in Equation 1,
PositionSilicon (t ) = D + As sin ωt
(1)
where D is a static distance from the silicon chip to the LDV. Unless explicitly stated, As was set 4 ACS Paragon Plus Environment
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to 1 nm in the VIPS measurement.
The vibration of the silicon chip causes the cell to vibrate and the cell height is oscillating with the same angular frequency, but with different amplitude Ac and phase ϕ, as shown in Equation 2.
HeightCell (t ) = h + Ac sin(ωt + φ )
(2)
The vibration amplitude and phase of the cell, which are indicators of cell’s mechanical properties, can be extracted with a LDV and a lock-in amplifier, as ncell (=1.38) is larger than nGM (=1.35).31,32 The LDV measures the instantaneous velocity of a reflective surface from the changes of the optical path length of a laser beam.33 When the laser beam is located outside the cell, the total optical path length, OPL(t) is defined as shown in Equation 3. In this case, the vibration amplitude of the silicon chip can be obtained from the velocity signal of the LDV, which is time-derivative of OPL(t).
OPL(t ) = ∑ ni d i (t ) = nGM × PositionSilicon (t ) = nGM As sin ωt + const
(3)
When the laser passes through the cell, OPL(t) is affected by height oscillation of the cell as shown in Equation 4.
OPL(t ) = ∑ ni d i (t ) = nGM × ( Position Silicon (t ) − Height Cell (t )) + nCell × Height Cell (t ) ≈ nGM As × (1 + ∆A) × sin(ωt + ∆θ ) + const
∆A =
(nCell − nGM ) × Ac nGM × As
(4)
(5)
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n −n A ∆θ = arctan Cell GM × c × sinφ As nGM
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(6)
The lock-in amplifier extracts the apparent amplitude increase ∆A and the apparent phase shift ∆θ from the LDV’s velocity output. This apparent phase shift, ∆θ is termed as VIPS. VIPS is determined by the amplitude and the phase of the cell height oscillation. In this paper, Young's moduli at multiple frequencies are extracted from the VIPS at multiple frequencies with numerical analaysis.
Experimental setup: The experimental setup is shown in Figure 2. An aluminum box is used to provide a standard cell culture condition (37°C and 5% CO2). A rectangular window is cut on the box lid and it is closed with a glass coverslip for optical access. To vertically vibrate the silicon chip, to which the cells are attached, the acoustic wave is generated by a piezoelectric plate (20 x 20 mm, T180-A4E-602, Piezo Systems, Inc., USA) and propagates to the silicon chip via silicone oil (50 cSt). The piezoelectric plate with electrical wiring is attached to the bottom of a petri dish (90 x 90 mm) and immersed in the silicone oil. A PDMS well or a 4 mm thick PDMS slab with a 6 mm hole is attached to the silicon chip and the silicon chip with the PDMS well or a culture well is attached on a small petri dish (diameter = 36 mm), as shown in Figure S1a. The petri dish is attached to the lid of the box with four magnets as shown in Figure S1b, so that the bottom of the petri dish is immersed in the silicone oil with a 4 mm gap to the piezoelectric plate. This configuration minimizes the transmission of the vibration through solid objects and most of the vibration energy is acoustically transferred to the silicon chip through the silicone oil. As the acoustic wave propagates through the silicone oil, its shear wave is quickly attenuated,34 and only the longitudinal wave can reach the silicon chip, so that the silicon chip is actuated with a purely vertical vibration. Further details of other experimental 6 ACS Paragon Plus Environment
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setup are provided in Figure S2 and Supporting information.
The average value of VIPS at the four corners of the measurement area is defined as the reference VIPS and the measured VIPS is subtracted by the reference VIPS for better comparison. Also, the difference between the average of the top 6% peaks of VIPS and the reference VIPS is defined as the VIPS peak value. Averaging multiple points suppresses measurement noise in VIPS. The data analysis can be carried out in real-time, but in this study, it is done after the experiments.
Cell culture and assay protocols: HT-29 is cultured in DMEM (Dulbecco’s modified eagle medium, Thermo Fisher Scientific, USA) with 10% FBS (Fetal bovine serum, Thermo Fisher Scientific, USA) and 1% penicillin streptomycin (Thermo Fisher Scientific, USA). A 10 x 10 x 2 mm silicon chip cut out from a 2-mm thick 4-inch single crystalline (100) silicon wafer, a 10 x 10 x 4 mm PDMS slab with a 6 mm hole, and a glass coverslip are sterilized with 70% ethanol for 30 minutes and carefully dried in a sterile environment. The width and height of the silicon chip are the minimum dimensions required for cell culture area and PDMS well attachment. The PDMS slab is attached on the silicon chip to form a culture well. Then, a 50 μg/mL solution of collagen type I (Thermo Fisher Scientific, USA) is introduced into the culture well to functionalize the silicon chip for 1 hour at room temperature. After the functionalization, the culture well is washed with PBS and HT-29 cell suspension is added with a final concentration of 10,000 cells per each well. The growth media is added to fill the culture well, which is then closed with a glass coverslip. The prepared samples are kept in the CO2 incubator for at least 2 hours before the measurement. The cells in the culture well are fixed with 4% paraformaldehyde aqueous solution (Electron microscopy sciences, USA) for 30 minutes. Then, the cells are gently washed 5 times with DPBS (Dulbecco's Phosphate Buffered Saline, Thermo Fisher Scientific, USA) and the growth media is 7 ACS Paragon Plus Environment
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added to the culture well for the measurement.
Noc (Nocodazole, Sigma-Aldrich, USA), Cyto-D (Cytochalasin-D, Sigma-Aldrich, USA), calyculin A (Abcam, USA), blebbistatin (MedChem Express, USA), Y-27632 (MedChem Express, USA) are purchased and stock solutions are made with concentration of 5 mg/mL for Noc, 30 µg/mL for Cyto-D and 1 µM for calyculin A in DMSO. Then final solutions for the experiment are made in the growth media with concentration of 10 µg/mL for Noc35, 0.1 µg/mL for Cyto-D35, 0.5 nM for calyculin A, 10 µM for blebbistatin36 and 10 µM for Y-2763237. The drug concentrations are from the literature cited above, except calyculin A36,38. For calyculin A, we experimentaly found a maximum concentration, which does not cause cell damages or detachment. The measurement is started 30 minutes after exposing cells to each drug.
Result Vertical vibration of a silicon substrate with spatially uniform phase: For precise VIPS measurement, purely vertical vibration of the substrate is essential. Otherwise, the lateral vibration translates the cells horizontally and additional phase shift will be generated by the slope of the cell contour. Also, the phase of the substrate vibration should be spatially uniform, so that the reference phase can be properly subtracted from the measured phase to calculate VIPS at each point. We characterized the lateral vibration of the silicon chip with a 2-dimensional VIPS profile of a water microdroplet immersed in mineral oil (See Supporting Information.). As the microdroplet is circular and homogeneously filled with water, it should show a non-directional and circular 2D VIPS profile if the substrate is vibrating only vertically. When a silicon chip is directly attached to a piezoelectric plate with an adhesive tape, the 2D VIPS profile of a water microdroplet has a line-symmetrically sloped profile with positive value on one side and negative value on the other 8 ACS Paragon Plus Environment
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side, as shown in Figure S3a. Furthermore, the VIPS profiles of nearby microdroplets exhibited same direction of symmetricity. This lateral vibration of the silicon chip is believed to originate from the lateral vibration of the piezoelectric plate, as its d31 coefficient (-190 pm/V ), the ratio of the vertical electric field to the lateral strain, is comparable to the d33 coefficient (390 pm/V), the ratio of the vertical electric field to the vertical displacement.
In order to prevent the piezoelectric plate actuating the silicon chip laterally, the direct mechanical contact between the silicon chip and the piezoelectric plate is removed by attaching the chip to the lid of the culture box, so that the vibration of the piezoelectric plate is forced to propagate acoustically through silicone oil, as shown in Figure 2. With these modifications, the 2D VIPS profiles of the water microdroplet become circularly symmetric, as shown in Figure S3c, confirming the vertical vibration.
The phase of the velocity measured on the silicon chip with LDV should be spatially uniform for the precise measurement of VIPS. As shown in Figure S1c, the phase of the velocity measured on the 10 x 10 x 0.3 mm silicon chip has about 50° variation in a 4 x 4 mm area at 90 kHz, as shown in Figure S1c. We believe that this is caused by the vibration of the silicon chip above its first resonant frequency, where the first resonant frequency of the silicon chip is only 24 kHz. To address this issue, a silicon chip with the thickness of 2 mm is used to increase its first resonant frequency (160 kHz). With the thick silicon chip, the variation of the phase is mostly eliminated as shown in Figure S1d.
Typical VIPS measurements of cells and water microdroplet: Typical 2D VIPS profiles of a live HT-29 in growth media and a water microdroplet in mineral oil are presented in Figure 3. The VIPS profiles are captured while the substrate is vibrated with amplitude of 1 nm at 120 kHz. The 9 ACS Paragon Plus Environment
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VIPS values in the area between the measurement points are obtained by linear interpolation for better visualization. As shown in Figure 3a, the VIPS profile of the live cell contains mostly positive VIPS with high values near the edge and low but still positive values in the middle. We believe that the cell oscillates in both vertical and radial direction during the deformation and that this complicated oscillation pattern causes more changes in OPL(t) and higher VIPS value near the cell boundary than in the middle. On the other hand, the VIPS profile of the water microdroplet shown in Figure 3b contains the positive VIPS near the boundary and the negative VIPS in the middle. The negative VIPS value in the middle indicates that the oscillation of the middle part has 180° phase difference with respect to the oscillation at other parts of the microdroplet.
Repeatability of the VIPS measurement: VIPS measurements are repeated on fixed cells and live cells three times with 20-minute intervals, as shown in Figure 4. The three 2D VIPS profiles of a fixed cell in Figure 4a are consistent to each other and show very similar VIPS peak values (0.98° ± 0.06°). However, multiple VIPS measurements on a live cell with same time intervals, exhibit similar VIPS peak values (2.25° ± 0.25°) but dissimilar VIPS patterns, as shown in Figure 4b. We believe that the dynamic nature of cytoskeletons and focal adhesions as well as the movement of cytoplasm and intercellular compartments cause the variation in the VIPS profile. VIPS measurement of chemically treated cells: Typical VIPS profiles of live, fixed, and Noc & Cyto-D treated cells measured at 90 kHz and 120 kHz are presented in Figure 5a and 5b. The cells treated with Noc & Cyto-D, which are known to be softer than live cells,35,39,40 exhibit the highest VIPS peak in their VIPS profiles while, fixed cells, which are known to be stiffer than live cells,41,42 show the lowest VIPS peak. To characterize how cytoskeleton networks affect the cell mechanics, the VIPS profiles are 10 ACS Paragon Plus Environment
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measured on eight groups of cells treated with different drugs that affect cytoskeleton networks. In addition to fixed (n90kHz=45, n120kHz=30) and live cells (n90kHz=50, n120kHz=50), six groups of cells treated with Noc (n90kHz=35, n120kHz=38), Cyto-D (n90kHz=40, n120kHz=24), Noc & Cyto-D (n90kHz=50, n120kHz=50), calyculin A (n90kHz=38, n120kHz=29), blebbistatin (n90kHz=39, n120kHz=30), and Y-27632 (n90kHz=30, n120kHz=23) are used. The VIPS peak values of these eight cell groups at 90 kHz and 120 kHz are shown in Figure 5c and 5d, respectively. At 90 kHz, the VIPS peak value of the live cells is significantly larger (p
