Application-specific Consumables for Laser Microdissection

The right solution for every application (updated version)

May 04, 2015

There are many different types of consumables for laser microdissection instruments. They cover a wide range of applications from basic to highly specialized, enabling scientists to choose their own individual configuration whatever their field of research.

The most common consumables are various glass membrane slides in combination with standard caps or cap strips to collect the dissectates. Frame slides, Petri dishes with and without PEN membrane, Ibidi slides and multiple chamber culture slides are also used. Further the flexible software setup of the Leica LMD system allows to use self-made or customized specimen carriers or collection devices for laser microdissection.

In general Leica Microsystems offers demo slide kits which contain different slide types to test the different available membranes for different applications to find the best suitable membrane slide for any application. Especially for fluorescence the test of different slides can make your life easier. PPS for example offers less autofluorescence compared to PEN, but also POL or PET might be suitable for your fluorescent dye.

Seeing is believing, so try, see and believe!

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 and 1b each show a standard specimen holder for a scanning stage with different slides in place.

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 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 DIC mode and 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.

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. It is also possible to use oil immersion objectives together with glass slides for laser microdissection. When using glass slides (with or without oil immersion), 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). A special specimen holder is available for microdissection. Large glass slides can be seen in Figures 2a and 2b.

Figs. 2a and 2b: This specimen holder can be used for large PEN or PPS slides as well as for Petri dishes (cf. Fig. 10a).

PEN coverslip slides

(Slide size: 26 x 76 mm, usable area for LMD 19 x 41 mm)

These are glass coverslip slides with PEN membranes and can be used for most LMD applications like the PEN glass slides. In addition to standard PEN glass slides the coverslip slides can be used for high energy applications, in combination with oil immersion objectives and in combination with the 150x dry objective.

The PEN coverslips are only available upon request. The special 150x dry objective (Figure 3) makes the use of oil immersion and the fragile PEN coverslip slides obsolete.

Frame slides

Fig. 3: The 150x dry objective for combination with POL membrane slides dedicated for dissection of every tiny samples.
Fig. 3a: Metal frame with membrane and frame supporter as a “backbone“
 

(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 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 during sample preparation (Figure 4a).

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 except in DIC mode.

Whereas frame slides with POL membranes are mainly used for chromosome dissection in combination with the 150x dry objective, frame slides with PET membranes are excellent for follow-on MALDI-TOF analysis techniques. PET is in contrary to the other available membranes almost free of softeners which are purified next to proteins using HPLC. Those softeners will block a portion of the MS spectra so that some bands cannot be observed in the blocked spectra area.

Special FLUO membranes are particularly suitable for fluorescence, PH and DIC.

Fig. 4a: Metal frame with membrane and frame supporter as a “backbone“

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.

For plant samples like leafs no membrane slide as carrier is needed as all as such material can directly be applied to laser microdissection.

Caps/microcentrifuge tubes

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, 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 non standard tube caps is not needed for Leica LMD systems as the collection process is gravity driven and does not need to overcome gravity.

Figs. 5a and 5b: Special collection devices for caps and microcentrifuge tubes can be used in the normal sizes 0.2 ml (Fig. 5a, top) and 0.5 ml (Fig. 5a, bottom). The collectors are situated immediately under the sample, dissectates fall straight into the tube caps due to the force of gravity.

Universal Holder for 8-well strips, 8-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 device 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

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 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. 6a and 6b: 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.

Figs. 7a and 7b: 8-well strip caps

The new height-adjustable 48-well collecter (Figure 8) allow 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.

Fig. 8: 48-well collector
Fig. 9: Universal holder with a chamber slide as collection device.

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 recultivation, cloning or downstream analysis.

The rectangular cavety 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 and multiple chamber culture slides).

Petri dishes

Petri dishes are generally suitable for live cell cultivation (LCC)  and are available with or without PEN membranes. Figure 10a 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 10b shows the use of a Petri dish as a collector.

Fig. 10a: Here, a Petri dish is used in the specimen holder for large slides and Petri dishes shown in Fig. 2.
Fig. 10b: The dissectate from the laser microdissection is collected by a Petri dish here.

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.

Ibidi® slides

(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 11a, 11b), 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 11a, 11b). Subsequent recultivation is possible as well as the downstream analysis of the extracted cells.

A matching cap offers a further contamination protection option.

Fig. 11a: Slide stack composed of 2 Ibidi slides on top of each other provides another contamination-free selection option with laser microdissection.
Fig. 11b: An Ibidi slide used as collector in the LOC holder.

Ibidi® slides can also be used as collection devices in combination with the universal holder as LOC device (see above).

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. * certified RNAse-free

 Order no.
 (*RNase-free slides)
Product description/quantity Usable area for LMD (approx.) Membrane thickness Objectivesª
Recommended objective CORR settings for high-quality images
Fluorescence applicabilityRemarks
 5x 6.3xª 10x L20x
CORRª 
L40x CORRª L63x CORRª 150x 
11600288
50 slides/box
Glass, PEN-membrane (25 x 76 mm) 19 mm x 41 mm4 µm ++ ++ +

+++

D = 1.0

+++

D = 1.0

+++

D = 1.0
+The most popular slides; improved with a thicker membrane.

11600289
50 slides/box

Steel frames, PEN-membrane (25 x 76 mm) 16 mm x 45 mm 4 µm ++ ++ +

+++

D = 1.0

+++

D = 1.0

+++

D = 1.0
+The most popular slides; improved with a thicker membrane and on a steel frame.
11505273
50 slides/box
Glass, PPS-membrane
(25 x 76 mm)
19 mm x 41 mm 1.2 μm ++ ++ + +++
D = 1.0
+++
D = 1.0
+++
D = 1.0
+Suitable for almost all applications (except DIC); PPS is semi-permeable for water, do not dip into water/water-containing solution
11505268
50 slides/box
Steel frames, PPS-membrane
(25 x 76 mm)
16 mm x 45 mm 1.2 μm ++ ++ + +++
D = 1.0
+++
D = 0
+++
D = 0
+++ ++ Suitable for almost all applications (except DIC)
11505158 Glass, PEN-membrane
(25 x 76 mm)
19 mm x 41 mm 2 μm ++ ++ + +++
D = 1.0
+++
D = 1.0
+++
D = 1.0
+ The most popular slides: standardly used in 95 % of all applications
*11505189
50 slides/box
11505151 Steel frames, PET-membrane
(25 x 76 mm)
16 mm x 45 mm 1.4 μm ++ ++ + +++
D = 0
+++
D = 0
+++
D = 0
++ + Recommended for MALDI-TOF
*11505190
50 slides/box
11505188 Steel frames, POL-membrane
(25 x 76 mm)
16 mm x 45 mm 0.9 μm ++ ++ + +++
D = 0
+++
D = 0
+++
D = 0
+++ ++ Recommended for chromosome spreads
*11505191
50 slides/box
1505215 (sterile)
100 slides/box
LARGE glass PEN membrane
(50 x 76 mm)
39 mm x 45 mm 2 μm ++ ++ + +++
D = 0
+++
D = 0
+++
D = 0
+ For large tissue sections (e.g. in neuro-biology for brain slides), requires optional slide holder
1532860 (sterile)
100 slides/box
LARGE glass PPS membrane
(50 x 76 mm)
39 mm x 45 mm 1.2 μm ++ ++ + +++
D = 0
+++
D = 0
+++
D = 0
+
1600250
5 slides/box
Steel frames, FLUO-membrane
(25 x 76 mm)
16 mm x 45 mm + + +++
D = 0
+++
D = 0
+++
D = 0
+ +++ For fluorescence and DIC-contrast
1505172
20 dishes/box
50 mm Petri dish with PEN-membrane 30 mm diameter 2 μm + + ++ +++
D = 0
+++
D = 0

D = 0
+ For live cell cutting; direct LMD of adherent cells for recultivation or analysis
1505240 (sterile)
15 slides/box
μ-slide, 18-well with PEN-membrane 30 μl vol. per well 2 μm + + ++ +++
D = 0
+++
D = 0

D = 0
+

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