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  1. eLife assessment

    This paper is of particular interest to researchers who plan to use focused-ion beam scanning electron microscopes (FIB-SEMs) and require fluorescent data to guide the milling process. The authors describe a valuable after-market upgrade that allows fluorescent data acquisition during FIB-milling without stage repositioning. Technical details of the fluorescent module upgrade together with the sample stage redesign are compellingly documented and will enhance the implementation of this important technology.

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  2. Reviewer #1 (Public Review):

    This paper described in considerable detail the extension of the FIB milling technique to incorporate an in-chamber fluorescent light microscope that is coincident with the FIB and SEM beams. Existing instruments either rely on an external FLM system (requiring a specimen transfer step that may result in additional contamination) or an integral FLM that required cumbersome and inaccurate movement of the stage inside the SEM chamber. Coincident beams would thus be very welcome to all practitioners of the challenging art of making cryo lamella. While not novel in concept the authors had to develop several innovations inside the chamber to make all this work.

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  3. Reviewer #2 (Public Review):

    The authors report here on the development of an integrated, on-axis fluorescent module as an upgrade to existing FIB-SEMs. The optical axis of the new fluorescent module is designed to be coincident with both SEM and FIB beams, thus allowing imaging of the same spot of the specimen with three beams (i-beam, e-beam, and light beam), all within the chamber of a FIB-SEM and without any stage movement. The authors show a detailed design of the FLM module, together with the complete redesign of the specimen-holding stage. A new specimen stage is needed to accommodate the objective lens of the FLM that must be positioned within a few millimeters from the sample and would not fit into the already crowded upper part of most FIB-SEM chambers. In such a setup, the sample is observed from the top with i-beam and e-beam and with a light beam from underneath.

    The design of the piezo positioning stage is well presented together with the results of the stage performance. It has very low repositioning error and resistance to mechanical vibrations. With five degrees of freedom, the sample at this stage can be accurately positioned for specific milling geometry. It is unclear what are the stage limits and if, for example, 90 degrees (orthogonal) FIB-milling is achievable with this stage.

    The second part of the paper showcases two results utilizing the coincident beam setup for fluorescence-guided lamellae preparation. The authors describe the successful preparation of several lamellae while guided by the fluorescent signal from the area of thinning. Subsequent TEM data acquisition showed that a) the target of interest was present in the lamella after the final thinning; b) lamellae were sufficiently thin for tomographic data acquisition; c) ice remained vitreous and with minor contamination.

    Advantages:
    In the described setup, all three beams inside the FIB-SEM chamber are coincident and can be centered on the same area of the specimen given the correct Z-height. This greatly simplifies and accelerates the acquisition of the fluorescent signal that is currently done in either a) an external fluorescent microscope, which involves additional time-consuming sample transfer steps prone to contamination; or b) integrated off-axis FLM, which involves large stage movements with limited precision. Additionally, since there is no need for stage movement, fluorescent data can be acquired without interrupting the milling process, enabling real-time monitoring of the presence of the fluorescent label. Reported Z-resolution for the light microscope module, coupled with the precision of the piezo stage enables accurate positioning of the sample for the targeted milling with 100 nm accuracy in Z using the specimen's fluorescent signal without the need for additional fiducials.
    The proposed setup comes as a complete solution: FLM module + custom cryo-cooled piezo stage + modified Quorum sample shuttle transfer + Odemis imaging software to control the microscope as well as all custom components. This setup has the potential to modernize older FIB-SEMs that don't have a cryo stage at all, lack integrated FLM, have stage issues, or run outdated software. However, it is unclear how compatible this system is FIB-SEM manufacturers other than TFS.

    Limitations:
    The described stage is designed from the ground up to work with the standard TEM AutoGrids, thus limiting the type of the compatible sample to the prepared on-the-grid (i.e. plunge-frozen grids or grids prepared following the waffle method). It looks like the standard SEM stub cannot be used in this system, however, a 3 mm standard HPF type-B can potentially be accommodated (perhaps additional modification is needed). Even if 3 mm HPF hats can be used, positioning of the FLM objective below the specimen makes fluorescent imaging impossible, thus lift-out will rely on external fluorescence imaging.
    Another concern is the possibility of automated FIB-milling using Odemis software. Modern proprietary software, such as TFS AutoTEM, Zeiss' SmartFIB, or open-source Autoscript-based solutions such as SerialFIB, offer the GUI-based user-friendly automated milling setup suitable for unsupervised overnight lamellae preparation. It is unclear whether Odemis software would allow a similar level of automation.

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