Development of a sequential laser microdissection tissue cuts workflow for the spatial and quantitative analysis of drugs in fresh frozen tissue sections

Abstract

Mass spectrometry imaging (MSI) is a well-established technique that allows to determine the distribution of small molecules, such as lipids, metabolites, and drugs, as well as large molecules in tissue sections. Because of the tissue heterogeneity, resulting in different matrix effects, and to the fact that the measured compounds are not entirely "extracted" from the tissue during the measurement, the absolute quantitative aspect of MSI is limited. To combine compound quantification with spatial information on fresh frozen unstained tissue sections, laser (capture) microdissection has been used to isolate tissue sections for compound extraction and LC-MS/MS quantification. Although this method relying on manual ROIs selection is rather sensitive compared to traditional MSI methods, it lacks the throughput needed to screen entire tissue sections. To apply a higher throughput tissue screening approach, we propose herein a workflow for performing indiscriminate and sequential LMD tissue section cuts that can cover up to 96 cuts collected in a 96 well plate on Leica LMD systems, for further extractions and LC-MS/MS analysis. Our workflow relies on the creation and implementation of 96 squares microgrid templates for the LMD cut of different area sizes (30x30 μm2, 50x50 μm2, 100x100 μm2, 200x200 μm2, 270x270 μm2 and 500x500 μm2) using 5 different magnifications (5x, 10x, 20x, 40x and 63x), on fresh frozen tissue sections. The method was applied on 20μm mouse brain and liver tissue sections. The tissue cut collection yields were evaluated visually and by the detection of the sprayed standards on the tissue sections, and found to be ranging from 78% to 91%, and the throughput of the LMD cuts and collection in a 96 well format, was measured to be from 19 to 37 minutes per tissue section, depending on the 96 squares microgrid template and the corresponding magnification lens used. Further extraction and LC-MS/MS analysis of 3 different compounds previously sprayed on a mouse liver tissue section allowed to determine the LLOQ the workflow allows to achieve when using the different templates.

Fig 1. Workflow for the development of an importable 96 squares microgrid format for the LMD7 software, proposed by Leica microsystems.

Fig 2. Images of fresh frozen liver tissue sections of varying thicknesses ranging from 12 to 200 μm, mounted on PEN membrane slides, to evaluate the tissue homogeneity and morphology.

Captured with a x2.5 objective.

Fig 3. Overview of a partly dissected tissue section using the 270×270 μm 96 squares microgrid template on a fresh frozen mouse liver tissue section (x10 magnification).

Fig 4. Dissected 96-well formats for all templates (30, 50, 100, 200, 270, 500 μm) with optimized laser and microscope settings.

(A) Fresh frozen mouse liver section. (B) Fresh frozen liver section after H&E staining. (C) Fresh frozen mouse brain tissue section. (D) Fresh frozen right brain section after H&E staining. Abbreviations used are as follows: CER = cerebellum; CerB = cerebrum; HP = hippocampus; HT = hypothalamus; LV = lateral ventricle; MB = midbrain; MO = medulla oblongata; OB = olfactory bulb; OT = olfactory tubercle and TH = thalamus [22].