Technical Reference #1651

1651.

Electron Microscopy of Whole Cells in Liquid with Nanometer Resolution N. de Jonge; D.B> Peckys; G.J. Kremers. And D.W. Piston, Vanderbilt University, PNAS, 106(1651), (2009)
Link To Paper

Abstract:
Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells wereimaged in liquid with a scanning transmission electron microscope(STEM). The cells were placed in buffer solution in a microfluidi

Keywords:
cellular imaging; molecular labels

Materials & Methods:
Construction and Preparation of the Silicon Chips. Custom designed silicon chips with silicon nitride windows (Protochips Inc NC) were used to form a liquid enclosure. They were fabricated using low stress silicon nitride of 50-nm thickness deposited with a low-pressure chemical vapor deposition process onto 300- m-thick silicon wafers. Openings in the silicon wafer were obtained by anisotropic etching in a heated bath of KOH leaving open silicon nitride membrane windows with an area of 0.2 0.05mm2. One set of chips was used for cell growth. A chip was cleaned with acetone and ethanol. It was then plasma cleaned and coated with a thin layer of polyL-lysine applied to make the window hydrophilic and to enhance the attachment of the cells. Another set of chips was used as covers of the liquid enclosure. For spacing between a chip with cells and a cover chip a solution of polystyrene microspheres of 10- m thickness was placed in droplets at the 4 corners of the chip and the solution was dried. Placement of a Liquid Sample in the Electron Microscope. A STEM/TEM specimen holder (Hummingbird Scientific) was designed for placing a liquid sample in the vacuum of the electron microscope. The specimen holder fitted 2 chips in a slot with their silicon nitride surfaces facing such that the silicon nitride windows in the middle of both chips overlapped. The holder contained 2 fluid lines (one input and one output) connected to the chips. The fluid lines were fed to plastic tubing for microfluidics (Upchurch Scientific). One tube was connected to a syringe pump (Harvard Scientific MA). Liquid flow occurred between the 2 chips and through a bypass channel. For loading of the sample in the fluid holder a cover chip was first placed in the slot of the holder with its silicon nitride surface facing up. A droplet of 50/50 glycerol/10% PBS buffer was placed on the cover chip. The chip with cells was transferred from its storage in liquid to the fluid holder and placed on top of the cover chip with the cells facing down. The silicon nitride surfaces of both chips were kept under liquid continuously. The use of 50% glycerol prevented rapid evaporation of the liquid during loading. The tip of the fluid holder was closed by a coverlid and a vacuum seal was obtained using O-rings above and below the silicon chips. A flow of 10% PBS buffer of 2 L/min was initiated at the input tube of the fluid holder until liquid appeared at the exit tube typically a few minutes later. The holder was then inspected for leaks visually in a vacuum test chamber. After this inspection the flow rate was maintained at 2 L/min and the holder was transferred to the electron microscope. Imaging and Analysis. Electron microscope images were recorded with a CM200 TEM/STEM (Philips) at 200 kV using the ADF detector. The microscope was set for an electron probe semiangle of 11 mrad a probe current of 0.59 nA (measured on the phosphor screen) a detector semiangle of 70 mrad and a pixel dwell time of 20 s. The probe diameter obtained with these settings was 1 nm. The images on liquid biological specimen were recorded at various magnifications and with pixel sizes between 2.9 and 5.7 nm. A conservative estimate of the electron dose is thus 7.4 102 electrons/ Å2. Liquid STEM imaging of the COS7 cells took place in 10% PBS buffer in water at magnifications varying between 8000 and 48000 while the flow rate in the fluid system was 0.1 L/min. Regions of interest were selected from the recorded images of a size of 1024 1024 pixels. The contrast and brightness were adjusted for maximal visibility of the labels and the cells. The liquid STEM images presented throughout this article represent the original data and no procedures were applied to reduce the noise. STEM images of dried samples in Figs. S1–S4 were enhanced by applying a despeckle procedure to reduce the noise. Confocal imaging was performed on a Zeiss LSM510 microscope equipped with a 40 1.3 NA oil-immersion objective. A 488-nm argon laser was used for excitation of the quantum dots and fluorescence was detected through a 650- to 710-nm band-pass emission filter. The image size was 1024 1024 pixels. Images at a single focal plane were selected. The thickness of the fixed COS7 cells was determined by recording z stacks and determining the distance between the lowest and the highest focal plane still containing fluorescent signals. A total of 44 cells were imaged and the average thickness was 7.0 0.7 m. Labeling of COS7 Cells with EGF-Gold Nanoparticles. We optimized existing procedures (34 35) to gold-label EGF receptors on living COS7 cells. Goldlabeled streptavidin (KPL) was diluted in PBS containing 0.5% BSA (PBS-BSA). The gold particles were washed twice by centrifugation and resuspension of the gold pellet. A 16 nMgold nanoparticle solution in PBS-BSA was incubated with 0.4 MBiotin-EGF (Invitrogen) for 1 h at 35 °C. Unbound biotin-EGF was removed using a size exclusion column. The filtrate containing EGF-gold nanoparticles (EGF-Au) was diluted with Tyrode’s buffer supplemented with 50 mM glucose and 0.5% BSA (Tyrode’s-BSA) washed once and resuspended to yield 5 nM EGF-Au in Tyrode’s-BSA. COS7 cells (African Green Monkey fibroblast) were cultured in DMEM (ATCC) supplemented with 10% FBS in a 5% CO2 atmosphere at 37 °C. Confluent COS7 cells were harvested by rinsing in Dulbecco’s PBS and dissociating the adherent layer with CellStripper (Mediatech) followed by a quench in supplemented media. Poly-L-lysine coated silicon chips were placed at the bottom of the wells of a 96-well plate filled with 200 L of supplemented media 20 L of harvested cells in suspension were added per chip. The chips with the cells were incubated for at least 4 h or overnight in a 5% CO2 atmosphere at 37 °C. For EGF-Au labeling the medium in the wells was exchanged by serum free DMEM. After 4 h of incubation in serum free medium cells were washed once with Tyrode’s-BSA. Eleven-microliter droplets of EGF-gold nanoparticle solution were placed in plastic wells and 1 silicon chip per droplet was placed inclined upside down on the droplet. The setup with droplets and chips was then stored in a closed box with a 100% humidity environment. The chips remained in this environment for either 5 or 10 min at room temperature with slight agitation. The 5-min samples were washed 3 times with PBS and fixed for 15 min in 4% glutaraldehyde diluted in PBS. The 10-min samples were placed in a new well filled with Tyrode’s-BSA (without EGF-gold nanoparticles) and incubated for an additional 15 min before washing and fixation. The chips were then washed 3 times with PBS once with 10% PBS in water incubated for 5 min in 100 mM Glycine to quench un-reacted aldehyde groups after fixation washed twice with 10% PBS and left in this solution at 4 °C until imaging. STEM images were recorded of 3 dried samples with (i) 5-min incubation with EGF-Au (ii) 10-min incubation with EGF-Au and an additional 15-min incubation in buffer and (iii) a control with 10-min incubation with streptavidin- Au in buffer and 15-min incubation in buffer. Figs. S1–S4 demonstrate that specific labeling of the EGF receptor was obtained. The existence of circular vesicles after 10  15 min incubation indicates that internalization of the receptor took place (10). The size of the gold labels was determined from Fig. S3 and amounted to 10 nm (full width at half maximum of a line scan over the image of a gold nanoparticle). Labeling of COS7 Cells with EGF-QD. EGF-Qdot655 (6:1) labels were prepared by mixing 10 L of 6 M EGF-biotin (Invitrogen) and 10 L of 1 M Qdot655- streptavidin (Invitrogen) in 80 L of 50 mM sodiumborate pH 8.3. After 2 h shaking at room temperature unbound EGF-biotin was removed using a Microcon YM-100 column (Millipore). The EGF-Qdot655 pellet was resuspended in 100 L of sodium borate buffer to make a 100 mM stock solution. COS7 cells were grown in 35-mm dishes containing No. 1 glass coverslips (MatTek) in standard DMEM (Invitrogen) supplemented with 10% FBS and were incubated in serum-free medium for 4 h before the labeling. Cells were labeled with 5 nM EGF-Qdot655 in BMHH buffer [125 mM NaCl 5.7 mM KCl 2.5 mM CaCl2 1.2 mM MgCl2 0.1% BSA 10 mM glucose 10 mM Hepes (pH 7.4)] for 5 min at 37 °C. The cells were then washed 3 times with BMHH buffer and either fixed immediately with 2% formaldehyde or incubated another 25 min at 37 °C before fixation.

Microscopic Technique
Electron Microscopy

Cell Type(s)
COS-7