CLEM (correlative light and electron microscopy) and LMD – fast and precise tracking of marked structures
The increasing complexity of protein and organelle investigations in cell biology sometimes demands a combination of different imaging approaches. The CLEM method in particular couples technologies such as light and electron microscopy. The key aim is to display images obtained with a light microscope at electron microscopy level, i.e. to correlate dynamic events in living cells with ultrastructural and 3D information. Using LMD, a system of reference coordinates can be produced on the surface of a culture substrate to enable quick and precise location of labeled details during sample preparation. This process is impressively illustrated in Figure 1.
Fig. 1: Reference grid imprinted onto the cell culture substrate. (A) Scheme of the reference grid for the laser microdissection microscope. The patterned aclar substrate was imaged under an epifluorescence microscope using excitation filters BP 545/30 (B), BP 480/40 (C) and BP 360/40 (D). The grid fluorescence is much fainter than the GFP signal of tagged proteins. (E) The reference grid is visible in brightfield and by scanning electron microscopy (F). (G–I) Due to the melting of the aclar, positive and negative patterns are imprinted as shown by scanning EM (G). As a result, after polymerization, and removal of the culture substrate (H), the pattern appears as negative and positive marks leaving visible holes (I) on the first EM sections. (J) Pictures of the pre-patterned substrate mounted onto gold plated live cell carriers .
NanoSIMS – structures in the third dimension
Bacteria analysis is performed using NanoSIMS (Nano Secondary Ion Mass Spectrometry) coupled with LMD and AFM (Atomic Force Microscopy) to obtain a three-dimensional image of element and isotopic compositions of a sample with a spatial resolution <50 nm. The LMD method proves advantageous here, too: the laser produces a grid pattern on the surface of the filters in use to mark structures of interest (Figure 2). This method is used in marine biology, for example, to analyze carbon and nitrogen fixation in the Pacific [2, 3] and the Baltic Sea .
Fig. 2: Filter labeling with a laser microdissection (LMD) system for catalyzed reporter deposition (CARD)-FISH and nanometer-scale secondary ion MS (NanoSIMS) analyses on the identical filter location. The small box photo shows a NanoSIMS image of 13C-incorporated cells (red) overlaid on a CARD-FISH image for the detection of archaeal cells. The size of each grid is about 50 × 50 µm marked by the LMD system .
LMD in live cell research – a method for "deliberate destruction"
The Leica LMD is also especially useful in the live cell sector for the deliberate mechanical destruction of cellular structures such as centrosomes, microtubules and membranes. For instance, cell membranes can be perforated with the laser to allow permeation of substances that were previously completely blocked by the membrane, according to Prof. Monica Gotta from the Department of Genetic Medicine and Development of the University of Geneva. Most importantly, it is possible to manipulate the spindle apparatus of cells during cell division (Figure 3).
Fig. 3: Mitotic spindle ablation in a wild type one-cell embryo. A C. elegans one-cell embryo expressing α-tubulin fused to the GFP. The Mitotic spindle just before (a) and after laser ablation (b). The two spindle poles separate after the ablation .
- Spiegelhalter C, Tosch V, Hentsch D, Koch M, Kessler P, Schwab Y and Laporte J: From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy. PLoS ONE 5 (2): e9014 (2010); doi: 10.1371/journal.pone.0009014.
- Morono Y, Terada T, Nishizawa M, Ito M, Hillion F, Takahata N, Sano Y and Inagaki F: Carbon and nitrogen assimilation in deep subseafloor microbial cells. PNAS 108 (45): 18295–300 (2011).
- Thompson AW, Foster RA, Krupke A, Carter BJ, Musat N, Vaulot D, Kuypers MMM and Zehr JP: Unicellular Cyanobacterium Symbiotic with a Single-celled Eukaryotic Alga. Science 337 (6101): 1546-50 (2012); doi: 10.1126/science.1222700.
- Polerecky L, Adam B, Milucka J, Musat N, Vagner T and Kuypers MMM: Look@NanoSIMS – a tool for the analysis of nanoSIMS data in environmental microbiology. Environmental Microbiology 14 (4): 1009–23 (2012).
- Grill SW, Gönczy P, Stelzer EHK and Hyman AA: Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409: 630–33 (2001); doi: 10.1038/35054572.
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