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Detection of Individual Cells in Tissue using MALDI-TOF Imaging at 10 µm Pixel Size

ASMS 2013, WP09-142

Introduction

References

Results Eckhard Belau1, Jane-Marie Kowalski2, Janine Rattke1, Alice Ly3, Soeren Oliver Deininger1, Detlev Suckau1, Axel Walch3, Marius Ueffing3, Michael Becker1

1Bruker Daltonik GmbH, Bremen, Germany 2Bruker Daltonics Inc., Billerica, MA, USA 3Helmholtz Zentrum München (GmbH), Neuherberg, Germany

Spatial resolution is currently one of the key parameters in MALDI imaging method development. It is influenced by a number of key factors:

-  Possible raster width / Pixel size (affected by laser spot size and sample positioning)

-  Ion yield per Pixel (affected by sample preparation, analyte abundance and instrument sensitivity)

-  Crystal size / compound delocalization (affected by sample preparation)

In a realistic experiment (i.e. biological tissue section) the influence of these factors can not be accessed independently.

In practice, raster width (an instrument parameter) is often used synonymously with spatial resolution. Resolution however, should be defined as the minimum distance between two structures that can be visualized separately. A suitable model sample must have defined structures of relevant size.

Sample preparation by matrix sublimation produces small crystals with minimal delocalization at the cost of efficient extraction. For (phospho)lipids (which are abundant and readily ionized) the ion yield is sufficient to evaluate instrumental parameters (such as laser spot size)

For lipid imaging 10µm sections of fresh-frozen tissue (rat cerebellum and testis, pig retina) were obtained on a cryostat (CM 1950 UV, Leica) and thaw-mounted on conductive glass slides. After tissue desiccation, 2,5-dihydroxybenzoic acid matrix (DHB) was applied using a custom-built device using a 152mm inner diameter sublimation apparatus (Ace Glass, NJ, USA) according to [1]. MALDI image acquisitions were conducted on a autoflex Speed MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen Germany) in reflector mode, employing a beam profile diameter of ~5µm. A 1 kHz laser frequency was used and 100 shots were accumulated per pixel with a 10 µm raster width.

For protein imaging, cerebellum sections were first washed in ethanol and then desiccated. After sublimation with Sinapinc Acid (SA) matrix, the sample was recrystallized in a custom-built chamber according to [2]. MALDI imaging acquisition was conducted on an ultraflextreme MALDI-TOF mass spectrometer (Bruker Daltonik) in linear mode with a raster width of 30µm, summing up 300 shots per pixel.

Post-acquisition H&E staining followed standard protocols; the resulting microscopic images were co-registered with the MALDI images using flexImaging 4.0 (Bruker Daltonik).

[1] Hankin JA et al.; J Am Soc Mass Spectrom 18(9); 2007

[2] Deutskens F et al.; J Mass Spectrom (46), 2011

[3] Alexandrov T et al.; J Proteome Res 9(12), 2010

[4] Schober Y et al.; Anal Chem 84(15), 2012

Conclusions •  Individual neurons resolved in tissue

by MSI with 10 µm raster width

•  Raster width ≠ Spatial resolution

•  Biological model systems with small, repetitive structures are required to evaluate resolution

•  Statistical analysis can improve feature detection

•  Proteins analyzed at high spatial resolution

MALDI-TOF Imaging

Fig. 3: MALDI Imaging of individual cells in rat cerebellum. (A)  Overview of specimen and measurement area (55,272 spectra at 10 µm raster width). (B) Image of three m/z-values in the lipid mass range (C) Enlarged view of inset in (B), distribution of m/z 878.9 (D) Scheme of cerebellar anatomy GL = granular layer, ML = molecular layer, PC = Purkinje cells (E) Zoomed in view of (C). An individual Purkinje neuron is resolved as a ~ 3×3 pixel object with high intensities for m/z 878.9 (F) Post-MSI H&E of the area shown in (E). At 10 µm raster width, the Purkinje neuron soma (~30 µm ∅) is clearly resolved. Also note the burn pattern of the laser (~5 µm ∅)

B

Fig. 4: Improved feature detection in high resolution MSI using unsupervised multivariate statistics (A)  Anatomy of the porcine retina. Highly organized layers of 10-70 µm thickness represent an excellent model system to evaluate spatial resolution. (B) Individual m/z-values fail to separate all layers because of the complex distribution pattern of individual molecules. (C) Mass spectra where sorted by similarity using a hierarchical clustering approach as in [3]. (D) In contrast to individual images, the resulting segmentation map (superimposed on the H&E image) clearly highlights 7 distinct morphological layers

C

D

Fig. 5: Protein imaging at high resolution (A)  Post-acquisition H&E stain (also see Fig. 2D) (B)-(E) Individual m/z images acquired at 30 µm raster width. The Purkinje cell layer is clearly discriminated in (C), although 30 µm raster width is not sufficient to resolve individual cells as in Fig. 2C (F) Compound image of four different m/z-values.

B C

D E F

A

Fig. 2 Lipid imaging in rat testis. Morphological features of appropriate size are a key requirement to demonstrate actual resolution in any image. A cross section of the seminiferous tubules presents numerous, repetitive structures of 20-200 µm size. Distribution of three different phospholipids (m/z of 801.6, 809.6 and 813.6) displayed at 10, 20, and 50 µm raster size shows these features with different clarity.

10 µm

20 µm 50 µm

100µm

Using sublimation sample preparation and optimized instrument settings, we were able to visualize individual neurons in rat cerebellum by MALDI-TOF imaging of a phospholipid signal (m/z 878.9, Fig. 3). Although MSI of individual cells in culture has been previously reported [4], detection against a complex cellular background represents are more challenging task. Using the cerebellum as a model system, we also demonstrate the discrepancy between raster width and spatial resolution: 10µm raster width is insufficient to resolve granule cells with 10µm diameter against a complex tissue background.

In Pig retina (Fig. 4), we demonstrate how statistical analysis improves the detection of small structures by MSI. Dry sample preparation and small spot size limit the detection to abundant compounds, most of which are not exclusive to one structure. Multivariate analysis allows to visualize complex relationships between expression patterns and highlights individual structures more clearly.

Detection of less abundant/higher MW compounds at high spatial resolution without delocalization is notoriously difficult as it requires washing and more efficient extraction during sample prep. Combining sublimation and recrystallization, we show some promising first results (Fig. 5) for protein imaging close to cellular resolution.

Methods

A B

C

D

Fig. 1: Custom-built setup for matrix preparation (A)  Vacuum-control unit (B) Sublimation apparatus (C) Heated plate & sand bath (D) Cold trap

A