1. Imaging guidelines

Image requirements:
Max total pixel size per image is 30,000,000 which is equivalent to 6.8 X 6.8 inch area at 800 dpi resolution. The input file format can be JPG,PNG, and TIF. JPG has the lowest file size given the same pixel size, minimizing data upload time. The samples should be visible to human eyes within the image, and the lower limit for size is 200~300 μm. The image should include 3~4 visible fiducial points. The background should be uniformly dark or uniformly bright compared to the samples. Note that for old PCs, lower image resolution will result in improved frame rate for the image navigator.
Imaging by scanner
800 dpi is normally fine for samples larger than 500 μm. Scan the ITO slide in a dark room, while the scanner cover is fully lifted. This results in black background which aids target identification. For a stainless target plate, darkening the room will not help. Greater contrast between the background and sample can be achieved by setting brightness thresholds in the scanner software.
It is better to reduce tilting of the glass within the image. Push the target towards the plastic frame of the scanner glass to align the sample plate straight.
Because of the requirement of having to have the sample side of the glass face toward the scanner, the samples will make a physical contact the scanner glass. This can be avoided by three ways.
  1. 1.
    If the target plate is an ITO glass for TLC adapter, mount the glass slide onto the adapter, and it can be imaged facing down without making a contact.
  2. 2.
    Set a small thin wedge on one side of the ITO glass (i.e. 2~3 layers of duct tape attached to the scanner glass), creating a gap between the target slide and scanner glass.
  3. 3.
    If the target plate is an ITO glass for PAC adapter, the user can choose to scan with the sample side facing away from the scanner. In this case, from the point of view of an imaging device, the glass can be flipped horizontally, not vertically.
This flipping event should be reported to macroMS by clicking the ‘Mirror across vertical axis’ button in the macroMS user interface, before generating the output file.
Imaging by cellphone camera
Construct a black box where the inner surface of the top plane is covered with black paper. Flocked light trap sheet is recommended (FPR-01, Protostar, New Albany, Ohio). The height of the box should be 2.5~5″. Put the sample in the box, and the image is taken through a hole on the top plane with flashlight. For the capacity to focus at a close distance of 2.5″ by cellphone, an app can be used (Manual Camera DSLR Lite, Lenses Inc). To remove the reflection of the flashlight, the camera can be tiled at an angle, or a piece of black paper can be set on the ITO glass to remove the reflection. The image can be taken without the black box, 1)if the samples are to be found manually by clicking in macroMS, or 2) if an array is to be added by the array handling functionality (See section 4 of this manual)
Maintaining orientation
On the backside of the target plate, draw an upward arrow that indicates the orientation of the glass. Use this arrow mark to maintain the up-down orientation of the target plate. This means the up-down orientation of the sample slide within the image should be equal to the orientation of the slide within the MALDI plate holder, like below.
Fiducial points
Fiducial points are critical locations that allow converting pixel coordinates of the found targets into physical coordinates within the target stage of the MALDI-MS instrument. These points should be visible in the image and also by the video screen for the instrument stage. For images acquired by a scanner, three fiducials are needed, and they can be the corner edges of the target slide, center point of an 'X' mark that is etched by a knife, salt particles, etc. These should be close to the corner edge of the target slide. For images acquired by a camera, four are needed, and these should be in precise rectangular arrangement such as the corner edges of the target slide or the marks created by a CNC machine with precise X-Y control.
When higher spatial precision is needed for small sample targets (~300 μm), smaller fiducial marks are needed. Salt particles less than 50 μm can be applied by a molecular sieve. The particles will appear both in the image and the stage video within the ultrafleXtreme, and they can be used as fiducial points. When applying salts, sample area should be covered by paper, and tap the ITO glass to remove particles with weak attachment. Alternatively, ~50 μm etch points created by UltraFlex laser ('Medium' mode) on sputter coated glass slide can be used as well, example of which are visible in Figure S8 of the macroMS paper. Even 50 μm dust particles present in the ITO glass have been used as fiducials successfully.