Specimen Devices for LMD
Glass membrane slides
For laser microdissection special membranes mounted to a glass slide are used to achieve highest quality results. Glass slides covered with a membrane with high UV absorption, making them particularly suitable for cold photo ablation (vaporization), are called glass membrane slides. This type of slide is shown on the left in Figure 1a.
Applied to the upper surface of the glass slide, the membrane is cemented to the glass at its edges. Thus, a rectangular area to mount the specimen is created where air is between the membrane and the glass backbone. The membrane is specially coated to ensure optimal adhesion of the specimens.
In general, glass membrane slides have versatile application potential in membrane-assisted laser microdissection (LMD) and come with various coatings for different applications.
Careful treatment of the foils is essential to avoid damage to the membrane. The accumulation of moisture between the glass and the membrane should be avoided, as this can cause the dissectates to stick to the glass backbone. There are two different membrane types for glass slides:
- PPS (polyphenylene sulfide)
- PEN (polyethylene naphthalate)
Figures 1a - 1c each show a standard specimen holder for a motor stage, scanning stage, or ITK LMT350 ultra stage with different slides in place. Fig. 1 shows a glass slide (left), a frame slide (center) and an Ibidi® slide (right).
PEN and PPS membrane slides
(Slide size: 26 x 76 mm, usable area for LMD 19 x 41 mm)
The standard slides have PEN membranes. These can be used for most LMD applications with the exception of the extremely high laser energies used in plant research, for example. The PEN membranes are available in two different thicknesses: 2.0 µm or 4 µm. The 4 µm is a special invention to be combined with Leica LMD systems: the membrane is stronger compared to the 2 µm standard which allows easier section mounting and a lower risk of membrane injury during the mounting procedure and the membrane itself is heavier and thus easier to collect via gravity.
PEN membrane slides are also available in a certified RNase- and DNase-free version in order to guarantee contamination-free work in DNA and RNA research. Like PEN-membrane slides, PPS-coated membrane slides can also be used in almost all laser-assisted microdissection techniques. PPS membrane slides have less autofluorescence and a smoother surface structure than slides with PEN membranes, but are less resistant than PEN slides to chemical treatment with xylol, for instance. When using glass slides, it is important to put them in the specimen holder with the tissue facing downwards. Otherwise the desired area will be dissected but dropped on the glassbackbone between the membrane and capture device instead of captured in the collection vessel itself.
Large PEN and PPS slides
(Slide size: 52 x 76 mm, usable area for LMD 39 x 45 mm)
These are PEN or PPS membrane glass slides for especially large specimens as used, for example, in the form of tissue sections in neurosciences (cf. Allan Jones’ video). Special specimen holders are available for microdissection. Large glass slides can be seen in Figures 2a and 2b.
(Slide size: 26 x 76 mm, usable area for LMD 16 x 45 mm)
Frame Slides consist of a metal frame which can be covered with various membranes (Figure 4b). The following membranes are available for this type of slide:
- PEN (polyethylene naphthalate)
- PPS (polyphenylene sulfide)
- PET (polyethylene terephthalate)
- POL (polyester)
- FLUO (fluocarbon)
Combined with the special 150x dry objective (Figure 3) that offers sub-cellular resolution without requiring immersion oil, frame slides are excellent for single-cell and chromosome applications. As the dissectates adhere directly to the foil when using frame slides, they are particularly suitable for the dissection of extremely small samples, e.g. for chromosome dissection. A frame support with a raised surface structure that fits exactly into the cavity of the front of frame slides facilitates the mounting of tissue sections to the membrane. This creates a flat surface and supports the membrane. The frame support forms the backbone of the membrane, as it were, by providing adequate resistance and tension for applying the sample to the membrane during sample preparation (Figure 4).
However, the frame slides can be used to mount tissue sections even without the frame support.
Like appropriately coated glass membrane slides, frame slides with PPS membranes can be used for practically any application.
Whereas frame slides with POL membranes are mainly used for chromosome dissection (Post et al., MolEcoNotes, 2006) in combination with the 150x dry objective, frame slides with PET membranes are excellent for follow-on mass spec analysis techniques (Drummond et al. Acta Neuropathol 2017). PET is in contrary to the other available membranes almost free of softeners which can be purified next to proteins using HPLC. These softeners might block a portion of the MS spectra so that some bands cannot be observed in the blocked spectra area. Similar phenomena can be observed in terms of the PCR tubes which are used for collection.
Special FLUO membranes are particularly suitable for fluorescence, PH and true DIC.
Frame slides can also be used as sample carriers for live cell culture. Therefore, the rectangular cavity of the slide is used as culture dish. It is recommended for such cultivations to put the slides into bigger Petri dishes and cover completely with media after plating the cells onto the membrane in the cavity. The morphology of the cultured cells will not change if the PEN membrane is used, other membrane types could lead to different morphologies of the cultured cells due to different surface properties of the distinct membranes.
A sandwich of two frame slides can be created for samples which are hard to attach to the membrane: simple clamp the sample between the membranes of two frames, the laser is strong enough to cut such sandwich stacks. Droplet or smear preparations are as well easy applied to laser microdissection with frame slides.
For plant applications as well as for other hard tissues like bone, teeth etc. frame slides are recommended as those are much better compatible to high laser powers than glass slides. High laser power can edge glass, making the material, which is mounted on it, harder to cut.
For plant samples like leaves no membrane slide as carrier is needed at all, as such material can directly be applied to laser microdissection (Hölscher, Rec Adv Polyphenol Res (2019)).
Collection Devices for LMD
After dissection by LMD, the sample drops by the force of gravity into a collection vessel, for example standard thin walled microcentrifuge tubes and their caps. The combination of different slides with caps is one of the most frequent applications in laser microdissection. Mostly, standard 0.2 ml and 0.5 ml tubes are used, either with an empty (dry) cap or with a cap filled with a suitable buffer or similarly suitable medium. For example, a solution with RNase inhibitors in the cap may prevent the decomposition of nucleic acids immediately after the laser microdissection process. Equally, proteins can be kept in a stable state with buffer solutions. Empty, dry caps offer a good way of identifying the dissectates and can be easily compared by taking before/after pictures. Basically, the microdissectate can be transferred to any standard cap. Figures 5a and 5b show matching collectors for caps and microcentrifuge tubes. Although it is possible to use special OptiCaps (#11505169) even these are not necessary for Leica laser microdissection. OptiCaps have an integrated optical element to ensure optimal illumination homogeneity and may also be "adhesive". However, the usage of expensive nonstandard tube caps is not needed for Leica LMD systems as the collection process is gravity driven and does not need to overcome gravity.
In addition, there are 1.5 mL tubes with a silicone filled cap. With the help of the lever in the Multi-Collector of the LMT350 ultra stage, this filled cap can be brought into contact with the tissue on the membrane. This set-up helps to collect challenging, sticky specimen.
There is also the possibility to collect into special sample devices, such as PCT microtubes for tissue homogenization, protein extraction, and digestion by Pressure Cycling Technology (PCT, PressureBioSciences Inc.). Directly collecting into these devices saves time since no re-pipetting is necessary.
Universal Holder for 8-well strips, 8-well &12-well strip caps and multi-well slides like 18-well Ibidi slides or LOC (Lab on a Chip) slides
The universal holder for different collection devices fitting in the same carrier is unique for Leica and offers the option for multiple different collection devices suitable for distinct applications. In addition, the Universal Holder is height-adjustable to adjust the chosen collector close to the specimen carrier for a minimal falling distance.
8-well strips can be used for high volume applications (e.g. for live cell collection into culture media), 8-well strip caps are ideal for high throughput applications. In addition the universal holder is suitable to carry chamber slides, multi-well slides as well as LOC (Lab on a Chip) devices such as the Ibidi® slide. All devices can be used as collection device for laser microdissection.
8-well strips LCC
Collectors such as these were developed to obtain a higher sample throughput of subsequent examinations of dissected live cells and can be joined together to form 96-well trays. Figures 6a and 6b each show a suitable holder with 8-well strips LCC that can be joined to form well trays. Well trays are the basis for carrying out high-throughput screening for cell cultures.
Using such screening techniques, enormous quantities of single-cell cultures can be analyzed and, if necessary, recultivated within an extremely short time.
8-well & 12-well strip caps
(for molecular biology/PCR)
Caps of 0.2 ml 8-well strips can be used as collectors for laser microdissection. Following laser microdissection, the 8-well strip caps can be placed on 0.2 ml 8-well strips or 96-well PCR-plates for direct insertion into PCR machines (available on request). The use of 8-cap strips has proved ideal for subsequent PCR, quantitative PCR or other heating or cooling processes, particularly for large quantities of samples.
The height-adjustable 48-well collector (Figure 8) allows to use six 8-well strips on a time so that with a minimum amount of effort during one experiment a 96-well plate can be filled easily.
The universal holder is also capable for other LOC devices, e.g. 18-well Ibidi® slides and chamber slides for live cell culture applications (Figure 9). Any self-made collection device with the x-y-dimensions of a slide could also be used in combination with this holder.
Consumables for use in LCC mode
There is a variety of specimen carriers compatible with laser dissection, manipulation and selection techniques in LCC (live cell culture). Leica offers LMD specific consumables for live cell treatment:
- Petri dishes (with or without PEN membranes)
- Ibidi® slides (with or without PEN membrane)
These consumables with PEN membrane can serve as specimen carrier for dissection, or, and even without PEN membrane, as specimen carriers for live cell laser manipulation or also as collector vessels for re-cultivation, cloning or downstream analysis.
The rectangular cavity of frame slides can also be used for cell cultivation (see above → Frame slides).
Further, the Petri dishes, as well as the Ibidi® slides with PEN membrane can be combined with commonly used collectors (0.5 ml tubes, 0.2 ml tubes, 8-well strips, 8-strip tube caps, Ibidi slides, conventional Petri dishes, multiple chamber culture slides, and 96-well plates).
Petri dishes are generally suitable for live cell cultivation (LCC) and are available with or without PEN membranes. Figure 10b shows the specimen holder already illustrated in Figures 2a and 2b for large PEN/PPS slides and Petri dishes; a Petri dish with PEN membrane has been inserted instead of a large slide. Figure 10c shows the use of a Petri dish as a collector.
Petri dishes without PEN membrane
(Ø 50 mm)
Conventional Petri dishes without a PEN membrane are used to collect and recultivate the dissectate. Petri dishes without a PEN membrane cannot be used in the specimen holder for laser microdissection, but for live cell manipulation experiments. However, selected areas can be manipulated with the LMD laser and observed over long periods of time using the time-lapse movie function.
Petri dishes with PEN membrane
(Ø 50 mm, usable area for LMD Ø 30 mm)
Cells cultivated in Petri dishes with a PEN membrane can be dissected in LCC mode and transferred to other Petri dishes (with or without a PEN membrane) or other suitable collection vessels. The membrane can be coated with poly-L-lysine or collagen for better adhesion and cell growth.
(Slide size: 26 x 76 mm, 30 µl liquid applicable per well)
These slides are equipped with wells that are specifically designed for the laser microdissection of living cells. Each slide has 18 recesses with a PEN membrane base.
Ibidi® slides can be combined with the entire range of collectors suitable for the LCC mode.
Another contamination-free selection variant are so-called slide stacks (Figures 11b, 11c), consisting of 2 Ibidi slides arranged on top of each other in sandwich technique. Cells dissected from such a stack fall by the force of gravity straight into the next lower Ibidi slide, which is fixed in a special stack holder (Figures 11b, 11c). Subsequent recultivation is possible as well as the downstream analysis of the extracted cells.
A matching cap offers a further contamination protection option.
Ibidi® slides can also be used as collection devices in combination with the universal holder as LOC device (Fig 11c).
There is also a solution for experiments which demand a higher throughput. 96-well standard PCR plates (semi-skirted, non-skirted, or fully skirted) enable users to collect material for up to 96 different experiments in parallel. These plates can be loaded directly into standard PCR machines. Additionally, there are holders for 96-well plates for live-cells, e.g. for cloning purposes.
There is also a collector option to directly dissect into special 96-well plates made by the company Covaris® for ultra-sonication.
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