Figure 1.
Schematic representation of the correlative light and electron microscopy software CLEMSite. (a) Overview of the different elements of CLEMSite, CLEMSite-LM, and CLEMSite-EM. CLEMSite-EM is divided into three modules: the Navigator, which allows to store and move to different positions in the SEM, then Multisite, which drives the FIB-SEM acquisitions, and the Run Checker, which controls and reports during the FIB-SEM runs. (b) Workflow for the automated acquisition of multiple correlated datasets. Light microscopy is performed to find specific phenotypes (“LM phenotyping”). From them, individual cells are selected (“LM targets”) and their corresponding landmarks and positions are recorded using CLEMSite-LM. (i) This scheme illustrates that for “LM targets, the low magnification overview shows the selected cellular targets (green circles), the landmarks (pink circles) used for correlating across imaging modalities, and the alphanumeric coordinate system that is patterned on the cell culture dish. On the right, a higher magnification image shows more clearly the Golgi as the cellular target (green circle), and the landmark used (pink circle) provided by the patterned culture dish, whose position is referred to the closest alphanumeric coordinates of the culture dish. (ii) Inside the FIB-SEM, “EM targets” refers to the process of obtaining the positions of the cells in the EM (stage coordinates). For that, a transformation matrix T is calculated based on the respective landmark positions of LM and EM (LM landmarks list in pink and EM landmarks list in black). This matrix transforms an LM Target list (cell positions in LM stage coordinates in green) into an EM target list (cell positions in EM stage coordinates in orange). On the right, the blue trapezoid and rectangle represent the milled and targeted region on the surface of the sample, inside the FIB-SEM. The black circle indicates the target coordinates in EM for the landmark, which should have its equivalent pink circle on LM stage coordinates. All of this correlation work is performed using the Navigator. (iii) Finally, in “FIB-SEM acquisitions,” cell image volumes are acquired at the “EM target” positions using Multisite and Run Checker. At each location of interest, the focused ion beam (red arrowhead) and the electron beam (blue arrowhead) are iteratively used to acquire datasets. The acquired data is finally analyzed to characterize different phenotypes (“EM phenotyping”).

Schematic representation of the correlative light and electron microscopy software CLEMSite. (a) Overview of the different elements of CLEMSite, CLEMSite-LM, and CLEMSite-EM. CLEMSite-EM is divided into three modules: the Navigator, which allows to store and move to different positions in the SEM, then Multisite, which drives the FIB-SEM acquisitions, and the Run Checker, which controls and reports during the FIB-SEM runs. (b) Workflow for the automated acquisition of multiple correlated datasets. Light microscopy is performed to find specific phenotypes (“LM phenotyping”). From them, individual cells are selected (“LM targets”) and their corresponding landmarks and positions are recorded using CLEMSite-LM. (i) This scheme illustrates that for “LM targets, the low magnification overview shows the selected cellular targets (green circles), the landmarks (pink circles) used for correlating across imaging modalities, and the alphanumeric coordinate system that is patterned on the cell culture dish. On the right, a higher magnification image shows more clearly the Golgi as the cellular target (green circle), and the landmark used (pink circle) provided by the patterned culture dish, whose position is referred to the closest alphanumeric coordinates of the culture dish. (ii) Inside the FIB-SEM, “EM targets” refers to the process of obtaining the positions of the cells in the EM (stage coordinates). For that, a transformation matrix T is calculated based on the respective landmark positions of LM and EM (LM landmarks list in pink and EM landmarks list in black). This matrix transforms an LM Target list (cell positions in LM stage coordinates in green) into an EM target list (cell positions in EM stage coordinates in orange). On the right, the blue trapezoid and rectangle represent the milled and targeted region on the surface of the sample, inside the FIB-SEM. The black circle indicates the target coordinates in EM for the landmark, which should have its equivalent pink circle on LM stage coordinates. All of this correlation work is performed using the Navigator. (iii) Finally, in “FIB-SEM acquisitions,” cell image volumes are acquired at the “EM target” positions using Multisite and Run Checker. At each location of interest, the focused ion beam (red arrowhead) and the electron beam (blue arrowhead) are iteratively used to acquire datasets. The acquired data is finally analyzed to characterize different phenotypes (“EM phenotyping”).

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