Glass membrane slides
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 Fig. 1a.
Applied to the upper surface of the glass slide, the membrane is cemented to the glass at its edges so that a minimal space is left between the glass and the membrane. 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. There are two different membrane types for glass slides:
- PPS (polyphenylene sulfide)
- PEN (polyethylene naphthalate)
- Figs. 1a, 1b: Each photo shows a standard specimen holder for the scanning stage accommodating 3 slides. Fig. 1a shows a glass slide (left), a frame slide (center) and an Ibidi slide (right).
PEN & PPS membrane slides
(Slide size: 26x76 mm, usable area for LMD 19x41 mm)
The standard slides have PEN membranes. These can be used for most LMD applications with the exception of DIC mode and the extremely high laser energies used in plant research, for example.
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. In particular, PPS is more suitable than PEN for use in combination with the HY-LITER-Kit. It is also possible to use oil immersion objectives together with glass slides for laser microdissection. When using glass slides with oil immersion objectives, the importance to put them in the specimen holder with the tissue facing downwards is underlined, otherwise the dissectate cannot be collected via gravity and would get in contact with immersion oil.
Large PEN slides
(Slide size: 52x76 mm, usable area for LMD 39x45 mm)
These are PEN membrane glass slides for especially large specimens as used, for example, in the form of tissue sections in neurosciences (cf. Allan Jones’ video). A special specimen holder is available for microdissection. Large glass slides can be seen in Figs. 2a and 2b.
- Figs. 2a and 2b: This specimen holder can be used for large PEN slides as well as for Petri dishes (cf. Fig. 7a).
Frame slides
(Slide size: 26x76 mm, usable area for LMD 16x45 mm)
Frame Slides consist of a metal frame which can be covered with various membranes (Fig. 3b). The following membranes are available for this type of slide:
- PPS (polyphenylene sulfide)
- PET (polyethylene terephthalate)
- POL (polyester)
- FLUO (fluocarbon)
Combined with a special 150x dry objective that offers extremely high 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 application 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 (Fig. 3a).
Like appropriately coated glass membrane slides, frame slides with PPS membranes can be used for practically any application except in DIC mode.
Whereas frame slides with POL membranes are mainly used for chromosome dissection, frame slides with PET membranes are excellent for follow-on MALDI-TOF analysis techniques.
Special FLUO membranes are particularly suitable for fluorescence, PH and DIC.
- Fig. 3a: Metal frame with membrane and frame supporter as a “backbone“
- Fig. 3b: Complete frame slide with frame supporter inserted
DIRECTOR slides
DIRECTOR slides from expression pathology are a special solution for membrane free collection using the “draw & scan” mode. This method is well described:
http://www.expressionpathology.com/director_slides.shtml
The “draw & scan” mode is also applyable to usual standard glass slides, but therefore much more energy is needed for sample ablation compared to DIRECTOR slides.
Caps/microcentrifuge tubes
After dissection by LMD, the sample drops by the force of gravity into a collection vessel, for example 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. Figs. 4a and 4b show matching collectors for caps and microcentrifuge tubes. Although it is possible to use special OptiCaps, 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“.
- Figs. 4a and 4b: Special collection devices for caps and microcentrifuge tubes can be used in the normal sizes 0.2 ml (Fig. 4a, top) and 0.5 ml (Fig. 4a, bottom). The collectors are situated immediately under the sample, dissectates fall straight into the tube caps due to the force of gravity.
8-well strips
(Use as collectors in LCC mode)
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. Figs. 5a and 5b each show a suitable holder with 8-well strips 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.
- Figs. 5a and 5b: 8-well strips as dissectate collectors enable high throughput screening of cell cultures.
8-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.
LOC (Lab on a Chip) – AmpliGrid
(Slide size: 26x76 mm)
The AmpliGrid is a special form of the LOC (Lab-on-a-chip), and is another alternative for collecting LMD dissectates. The AmpliGrid is a chemically structured slide with hydrophobic and hydrophilic areas that serves as an amplification platform for ultra-low-volume applications. It consists of a glass slide of a standard size, its special feature being its 48 reaction compartments which are also suitable for collecting dissected single cells (Figs. 6a, 6b). As dissectates are transferred to the AmpliGrid by the force of gravity when using the Leica LMD6500/7000 microscope, liquid does not necessarily have to be applied to the reaction sites in advance, as is the case with instruments that employ the catapult technique. The dissected cells can be collected “dry”. Although it is possible to use a buffer solution, it is not mandatory.
After cell dissection, PCR or RT(reverse transcriptase)-PCR is immediately possible on the 48 reaction sites of the AmpliGrid using an ultra-low-volume amplification technique. Created by hydrophobic and hydrophilic interactions, the micellar surface structure keeps a drop (usually 1 µL) of a reaction mix (which is directly applied to the AmpliGrid for amplification) in shape.
- Abb. 6a, 6b: Inserted in special collection holders, “LOC catchers“ (AmpliGrid) guarantee high sample throughput for PCR analysis or RT-PCR approaches.
Consumables for use in LCC mode
The following slides are compatible with laser dissection, manipulation and selection techniques in LCC (living cell culture) mode:
- Petri dishes (with PEN membranes)
- Stackable membrane rings
- Ibidi slides
These also serve as collector vessels for recultivation.
Further, the above-mentioned slides can be combined with commonly used collectors (0.5 ml tubes, 0.2 ml tubes, 8-well strips, LOC (Lab on a Chip), Ibidi slides, conventional Petri dishes and multiple chamber culture slides).
Petri dishes
Petri dishes are generally suitable for cell cultivation (LCC) and are available with or without PEN membranes. Abb. 7a shows the specimen holder already illustrated in Figs. 2a and 2b for large PEN slides and Petri dishes; a Petri dish with PEN membrane has been inserted instead of a large slide. Fig. 7b shows the use of a Petri dish as a collector.
- Fig. 7a: Here, a Petri dish is used in the specimen holder for large slides and Petri dishes shown in Fig. 2. Stackable membrane rings can also be combined with this holder.
- Fig. 7b: The dissectate from the laser microdissection is collected by a Petri dish here.
Petri dishes without PEN membrane
(Ø 50mm)
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. 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 50mm, 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.
Stackable membrane rings
(Ø 50mm, usable area for LMD Ø 50 mm)
These are rings covered with a membrane that are inserted into standard cell culture dishes of 50 mm diameter. Stackable membrane rings enable laser microdissection to be performed in the living cell sector, and are particularly useful for the selection of single cells. Stackable membrane rings can also be connected to form a duplex ring system in order to enable contamination-free transfer of the dissectate. 2 stackable membrane rings are screwed together for this purpose. Immediate further cultivation of the dissectate is also possible with the sandwich technique. Stackable membrane rings can be used both as specimen holders and as collection holders for the dissectates. Screwed together as a sandwich, they do not require a special collection holder – dissectates are directly captured in the bottom membrane ring. Combination with a suitable cap or optional climate chamber for the dissection system enables a contamination-free selection process.
Ibidi slides
(Slide size: 26x76 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 (Fig. 8a, 8b), 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 (Fig. 8a, 8b). Subsequent recultivation is possible.
A matching cap offers a further contamination protection option.
- Fig. 8a: Slide stack composed of 2 Ibidi slides on top of each other provides another contamination-free selection option with laser microdissection.
- Fig. 8b: An Ibidi slide used as collector in the LOC holder.
- Table 1: Membrane Slides and Petri Dishes for Laser Microdissection – Comparison of cutting performance, depending on membrane type, membrane support, and objective used. Laser Microdissection Performance: – not suitable; + satisfactory; ++ good; +++ very good. ªThese objectives possess improved optics for UV-light transmission in conjunction with the best image quality. D stands for the position of the correction ring.
Order no. | Product description/quantity | Usable area for LMD (approx.) | Membrane thickness | Objectivesª | Fluorescence applicability | ||||||
5x | 6.3xª | 10x | L20x CORRª
| L40x CORRª | L63x CORRª | 150x | |||||
11505273 | Glass, PPS-membrane | 19 mm x 41 mm | 1.2 μm | ++ | ++ | + | +++ | +++ | +++ | – | + |
11505268 | Steel frames, PPS-membrane | 16 mm x 45 mm | 1.2 μm | ++ | ++ | + | +++ | +++ | +++ | +++ | ++ |
11505158 | Glass, PEN-membrane | 19 mm x 41 mm | 2 μm | ++ | ++ | + | +++ | +++ | +++ | – | + |
*11505189 | |||||||||||
11505151 | Steel frames, PET-membrane | 16 mm x 45 mm | 1.4 μm | ++ | ++ | + | +++ | +++ | +++ | ++ | + |
*11505190 | |||||||||||
11505188 | Steel frames, POL-membrane | 16 mm x 45 mm | 0.9 μm | ++ | ++ | + | +++ | +++ | +++ | +++ | ++ |
*11505191 | |||||||||||
11505215 (sterile) | LARGE Glass PEN-membrane | 39 mm x 45 mm | 2 μm | ++ | ++ | + | +++ | +++ | +++ | – | + |
11505172 | 50 mm Petri dish with PEN-membrane | 30 mm diameter | 2 μm | + | + | ++ | +++ | +++ | – | – | + |
11505240 (sterile) | μ-Slide, 18-well with PEN-membrane | 30 μl volume per well | 2 μm | + | + | ++ | +++ | +++ | – | – | + |
11600250 | Steel frames, FLUO-Membrane | 16 x 45 mm |
| – | + | + | +++ | +++ | +++ | + | + |
11532638 (sterile) | Stackable membrane rings 50 mm Petri dish, PEN-membrane | 50 mm diameter | 2 μm | + | + | ++ | +++ | +++ | – | – | + |
