FIB-SEM - TFS Hydra Bio: Simple CLEM workflow

Ideally, you collect information about your grids before your milling session, to use the instrument efficiently.

One example, is a fluorescent atlas overview of our sample.

Load your sample in the Hydra.

Follow steps 1-5 in the basic lamella milling protocol.

Briefly: i) open Maps, ii) create a new project, iii) take SEM snapshot of your grid, iv) add and acquire atlas tileset

Import previously acquired fluorescent atlas of your grid.

Adjust opacity of this image to 50%.

If Alignment Wizard doesn't appear automatically, right-click on the imported image -> Alignement -> Fine Alignment

Use 2-point alignment, if necessary apply mirroring/flipping to your fluorescent atlas.

To select matching features between SEM atlas and imported image, right-click -> Place Point. Select the same feature on both images.

Using the fluorescent atlas you can already select areas of interest and place lamella site over them.

Either based on the fluorescent atlas image (previous step) or based on SEM atlas, select cells for milling.

Right-click on the cell -> Add lamella site here

You may be able to fit 15-20 lamellae in one session.

Select live Maps project and import lamella sites.

Apply your preferred template for the selected lamella sites and start lamella preparation.

It is recommended to switch on milling of artificial features, that will not only enchance lamella targeting, but can serve as CLEM fiducial in the following step.

Start lamella site preparation. (see basic workflow here)

Revisit lamella sites in Maps software and take high magnification snapshot with all fiducials in the field of view.

Open iFLM software.

Switch microscope to LM mode.

Make sure to switch to LM mode ONLY, when the stage is in mapping position (sample is perpendicular to SEM).

Bring sample in focus (ca. 2500um) while using reflection mode, 20x magnification (bin 4x), 625nm wavelength, 0.1% LED power and 8 ms exposure time.

Set focus distance and link Z-height to Y axis. Setup destination path for your Z-stacks and switch on auto-saving. If you need MIP or TIFF images as well, you will need to save them after each acquisition.

Navigate to first lamella site, setup Z-stack imaging parameters and acquire Z-stack. Repeat this step for each lamella site.

For a 20x, 10um Z-stack with GFP-labeled sample, these parameters work well. Fluorescence: 470nm, 1% LED power, 50ms exposure || Reflection: 625nm, 0.1% LED power, 8ms exposure || Start: 0um, End: 10um, Step: 0.5um

Make sure the previously milled artifical fiducials appear in focus somewhere in your Z-stack. This will be crucial for the next correlation steps.

Import the previous acquired iFLM Z-stacks from your project folder, by clicking on the import button and selecting the the .tfs files.

Right click on the Z-stack and select fine alignment. Change Z-slice to set artificial or natural fiducials in focus and use them to correlate to SEM atlas.

Select your Z-stack and switch to the Z-slice where your artificial or natural fiducial is in focus.

Right-click on fiducial -> Select as fiducial -> Select corresponding lamella site from the drop down list.

Select your Z-stack and switch to the Z-slice where your region of interest is in focus.

Right-click on ROI -> Select as ROI -> Select corresponding lamella site from the drop down list.

In AutoTEM Cryo the correlation between fiducial and ROI will be calculated from X,Y coordinates in SEM atlas and the Z-coordinate based on the plane number in the Z-stack.

Enable correlation in Preparation step.

Place red cross-hair over you r selected fiducial. The calculated position of ROI will be marked by a green rectangle.

Select Correct lamella position

Adjust rough milling pattern height.

Continue lamella milling with milling and polishing steps.

If needed, set delay time on the first lamella on top of the list.

Safe stage position and Beams off options cause an error, make sure to disable them if you use delay.