Journal: Lab on a Chip

Article Title: Single and few cell analysis for correlative light microscopy, metabolomics, and targeted proteomics †

doi: 10.1039/d4lc00269e

Figure Lengend Snippet: Workflow overview, single-cell lysis principles, and light microscopy using the cryoWriter setup. a) Overall workflow for the correlative analysis. Live cell imaging by (fluorescence) light microscopy (LM) is used for the structural and functional characterization of individual cells (1). The microscope is used for target selection, the monitoring of the single-cell lysis, and the cell contents' uptake. The cell lysate is dispensed onto a carrier slide for subsequent analysis by reverse-phase analysis (RPPA) and liquid-chromatography mass spectrometry (LC-MS, see for details). The slides can either be analyzed by RPPA (2) or LC-MS (3) alone or in a combined mode (4), where first LC-MS is performed with subsequent analysis by RPPA. b) Light- and fluorescence microscopy imaging stage for live-cells, integration of the single-cell lysis setup, and handover system. In a live-cell incubator, cells are grown in a PDMS well on an ITO-coated slide (S), lysed and aspirated with a microcapillary electrode (nozzle, N), and spotted on the adjacent microarray slide (C). c) Adherent eukaryotic cells are grown on functionalized and electrically conducting ITO-coated glass slides (S). The cells are imaged using a (fluorescence) LM. An individual cell is located in the LM and lysed by electropulses ( E⃑ ) between the ITO-coated slide and the electrically conductive microcapillary for single-cell lysis. Simultaneously, the lysate is aspirated in a volume of ≈3 nL into the microcapillary nozzle (N). Figure not in scale. d) Cell imaging and lysis monitoring by differential interference contrast (DIC) and fluorescence light microscopy. For a movie see also the ESI Fig. S2. As an application example, undifferentiated cells (SH-SY5Y) were incubated with fibrillated α-synuclein, which was fragmented by freeze–thaw cycles and fluorescently labeled with an NHS-Alexa dye. This fluorescence signal guides and triggers the cell selection. The nozzle (N) targeted the cell for lysis by the combined forces of electroporation and friction by suction (see panel c). The arrow marks the target cell before and after lysis. Scale bars: 100 μm.

Article Snippet: Furthermore, correlative measurements were performed using NHS-functionalized glass slides (NEXTERION® Slide H, Schott, Germany).

Techniques: Lysis, Light Microscopy, Live Cell Imaging, Fluorescence, Functional Assay, Microscopy, Selection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Imaging, Microarray, Incubation, Labeling, Electroporation

Journal: Lab on a Chip

Article Title: Single and few cell analysis for correlative light microscopy, metabolomics, and targeted proteomics †

doi: 10.1039/d4lc00269e

Figure Lengend Snippet: Single-cell RPPA. a) One to six HEK cells were lysed, aspirated and dispensed as spots on an NC-pad on the carrier slide. The slide was scanned after incubation with an anti-actin antibody and a fluorescently labeled secondary antibody. The total fluorescence intensity was plotted against the number of lysed cells dispensed in each spot. The average intensities and the standard deviation are indicated in black to show the variation. The individual cells are shown in gray. A linear regression is shown as a dashed line. b) Single-cell RPPA dot-blot using an NHS-functionalized glass slide for covalent immobilization of the proteins. The left panel shows the detection of actin (in red), and the right panel of GAPDH (in green). Single cells were individually lysed and dispensed. This cycle was repeated for every spot for 1, 5, and 10 cells. Cumulated volumes of 3 nL, 15 nL or 30 nL of lysate were dispensed, depending on the number of lysed cells. The number of individually lysed cells per spot is indicated in the left column. The last row depicts the negative control (NC), where 10 nL, 20 nL or 30 nL of the buffer surrounding the cells were dispensed.

Article Snippet: Furthermore, correlative measurements were performed using NHS-functionalized glass slides (NEXTERION® Slide H, Schott, Germany).

Techniques: Incubation, Labeling, Fluorescence, Standard Deviation, Dot Blot, Negative Control