Figure 1.
Overview of correlative PALM and SEM procedure and the PALM imaging system.
(A) Flow diagram showing the steps involved in correlating PALM and SEM images. (B) PALM imaging set-up showing the 100 nm thick cryosection on an ITO coated coverslip with 80 nm Au beads that are used for image registration and alignment.
Figure 2.
Correlated images of TFAM-mEos2 PALM data with electron micrographs.
(A) PALM image of TFAM-mEos2. (B) SEM image of mitochondria (M) in the same area imaged in A. (C) Correlated image of TFAM-mEos2 PALM data with SEM data. TFAM-mEos2 resides in the mitochondrial matrix surrounded by boundary and cristae membranes.
Figure 3.
Correlated images of lamin B1-mEos2 PALM data with electron micrographs.
(A) Correlated image of Lamin B1-mEos2 signal with electron micrograph. White arrows point to Au beads used for alignment. (B) Higher magnification view of an area from A showing a possible nuclear pore (red arrowhead) with corresponding gap in Lamin B1-mEos2 signal. (C) SEM only image of panel B to show cellular ultrastructure. M, mitochondrion. G, Golgi apparatus.
Figure 4.
Correlated images of peroxisome-localized mEos2-SKL PALM data with electron micrographs.
(A, C) SEM image of cells showing mitochondria (M) and other membranous organelles. Red arrowheads indicate peroxisomes as seen by overlaying PALM data of mEos2-SKL on electron micrographs (B, D).
Figure 5.
Two-color correlative PALM and SEM data of nuclear lamina proteins.
(A) PALM image of lamin A-PS-CFP2. (B) PALM image of lamin B1-mEos2. (C) Combined lamin A-PS-CFP2 and lamin B1-mEos2 PALM data. (D) Combined two-color lamin protein PALM data correlated with electron micrograph. (E-H) Higher magnification views from boxed area in D of PALM image of lamin A-PS-CFP2 (E), PALM image of lamin B1-mEos2 (F), combined lamin A-PS-CFP2 and lamin B1-mEos2 PALM data (G), and combined two-color lamin protein PALM data correlated with electron micrograph (H). Note the cytoplasmic organelles such as mitochondria (M). White arrows in panels A-D indicate Au beads that are used to align the datasets.
Figure 6.
Caged dye-phalloidin labeling of actin and correlated PALM/SEM.
(A) Maximum intensity projection image of confocal z-stack of a cell with actin labeled using NVOC2-carborhodamine110–PEG8–phalloidin (magenta) and the nuclear stain 4',6-diamidino-2-phenylindole (blue). (B) PALM image of caged dye labeled actin. (C) SEM of area imaged by PALM in panel B. (D) Correlated PALM and SEM of caged dye labeled actin.
Table 1.
PALM imaging acquisition parameters.