Nano Letters few minutes, the microchips were transferred into new medium and incubated overnight at 37 C in a 5% CO2 environment. Figure 1. Principle of liquid scanning transmission microscopy (STEM) of live eukaryotic cells. A cell (orange) is enclosed in a microﬂuidic chamber between two 50 nm thin silicon nitride membranes supported by silicon microchips, protecting the cell from the vacuum (gray) inside the STEM. Gold nanoparticles (Au-NPs) accumulate in clusters of Au-NP- ﬁlled vesicles. Continuous ﬂow of buﬀer (blue) keeps the cell alive until scanning with the electron beam (black) is started. LETTER To ensure that the cells could be kept alive inside the microﬂuidic chamber, we ﬁrst examined the cell survival in the chamber, independently of the recording of STEM images. Cells that had settled on microchips overnight were incubated for 1 h with the live/dead indicator calcein AM (Invitrogen), 1 μM in Tyrode’s buﬀer (TB) (Sigma Aldrich). Calcein AM is an initially nonﬂuorescent, cell permeable compound that is converted into green ﬂuorescent calcein when hydrolyzed by intracellular esterases in live cells. To enclose the cells in a microﬂuidic chamber, two microchips, one with cells and one with spacer, were assembled in the liquid ﬂow EM specimen holder (Protochips Inc.). First, the dry microchip with spacer was placed in the slot for the microchips in the tip of the holder, with the spacer side up. The wet microchip with adhering cells upside down was placed (rapidly to prevent drying) on the spacer microchip. Due to the design of the slot, this “sandwiching” procedure aligned the microchips with a precision of 20 μm such that their SiN windows overlapped to a degree of at least 80%, providing visibility of the cells enclosed between the membranes. The microﬂuidic compartment was closed with a lid, and the ﬂow of the imaging buﬀer (1 μM Calcein AM in TB) was initiated. The total assembly time amounted to 1 2 min. The cells were imaged with an inverted ﬂuorescence micro- scope (Nikon Diaphot 300), equipped with 20 (Nikon Plan 20/0.40 phase 2 160/1.2) and 40 (Nikon Plan 40/0.55 phase 3 160/0 2.5) air lenses and an EXFO X-Cite 120PC XL illumina- tion system that included a 120 W mercury arc lamp. The ﬂuorescence image of Figure 2a shows the cells on a microchip before enclosure. Figure 2b is the same sample in a closed chamber, ∼2 min after assembly. Figure 2c was taken 40 min after the microﬂuidic chamber had been reopened; the cells were enclosed for 4 mins total. None of the cells displayed signs of damage. Several other samples were tested as well. Cells in the Figure 2. Assessment of cell viability. (a) Fluorescence microscopy of live COS-7 cells on a microchip. The cells were stained with the live/dead indicator calcein AM (green ﬂuorescence indicates undamaged membrane integrity). (b) The same cells 2 min after enclosure in a microﬂuidic chamber. (c) The cells after opening of the microﬂuidic chamber 40 min later. (d) Phase contrast image of a diﬀerent sample of live COS-7 cells 39 h after opening of the microﬂuidic chamber. The dotted oval in the upper left image area indicates the cell that underwent mitosis. (e, f) Cells division occurring in a time period of 2 h. 1734 dx.doi.org/10.1021/nl200285r |Nano Lett. 2011, 11, 1733–1738