Laser capture microdissection: Big data from small samples

Abstract

Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions.

Fig. 1. Mechanism of tissue capture using the PALM and Arcturus LCM systems

When isolating cells with the Arcturus LCM system (A1–A4), tissue sections are prepared on a conventional microscope slide (A1) and a ‘cap’ that contains an infrared-sensitive transfer film is placed physically onto cells of interest (A2). An infrared laser is fired through the cap over the cells of interest, activating the membrane, which infiltrates into the underlying tissue (A3). When the cap is removed, captured tissue is removed, leaving behind unwanted cells (A4). When isolating cells with the PALM–LCM system (B1–B4), tissue sections are firstly prepared on membrane-coated microscope slides and collecting caps are filled with lysis buffer (B1). In this system there is no contact between film and tissue sections (B2). A UV laser is focused onto the focal plane of the section and used to cut around cells of interest by laser ablation (B3), physically detaching the cells of interest and the underlying membrane from the surrounding tissue. The laser is then focused directly below the cells of interest, and a single laser pulse is fired, catapulting them vertically into the overlying eppendorf cap (B4).

Fig. 2

Schematic diagram of sample sources and applications for LCM

Fig. 3. Applications of LCM on ‘omics’ study

LCM physically isolates the cells of interest, which then can be analyzed further. The remaining tissues on the slide are undisturbed, and other cell types can be isolated subsequently by LCM. (ESI MS, electrospray ionization mass spectrometry; FISH, fluorescence in situ hybridization; MALBAC, multiple annealing and looping-based amplification cycle; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MDA, multiple displacement amplification; MSI, mass spectrometry imaging; STRT, single-cell tagged reverse transcription; WGA, whole-genome amplification.)