doi.org/10.26434/chemrxiv.12275507.v1

An Integrated Microfluidic Probe for Mass Spectrometry Imaging ofBiological SamplesXiangtang Li, ruichuan yin, hang hu, Yingju Li, Xiaofei Sun, Sudhansu Dey, Julia Laskin

Submitted date: 08/05/2020 • Posted date: 13/05/2020Licence: CC BY-NC-ND 4.0Citation information: Li, Xiangtang; yin, ruichuan; hu, hang; Li, Yingju; Sun, Xiaofei; Dey, Sudhansu; et al.(2020): An Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Samples. ChemRxiv.Preprint. https://doi.org/10.26434/chemrxiv.12275507.v1

Ambient ionization based on liquid extraction is widely used in mass spectrometryimaging (MSI) of molecules in biological samples. The development of nanospray desorption electrosprayionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution.However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broaderscientific community. Herein, we describe the design and performance of an integrated microfluidic probe(iMFP) for nano-DESI MSI. The glass iMFP fabricated using photolithography, wet etching, and polishingshows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; thespatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP iseasy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquidextraction-based MSI in biological research, drug discovery, and clinical studies.

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An Integrated Microfluidic Probe for Mass Spectrometry Imaging

of Biological Samples

Xiangtang Li[a], Ruichuan Yin[a], Hang Hu[a], Yingju Li[b], Xiaofei Sun[b], Sudhansu K. Dey[b], and Julia

Laskin[a]*

[a] Xiangtang Li, Ruichuan Yin, Hang Hu, Prof. Julia Laskin Department of Chemistry, Purdue University 560 Oval Drive, West Lafayette, IN 47907 (USA) E-mail: jlaskin@purdue.edu [b] Yingju Li, Prof. Xiaofei Sun, Prof. Sudhansu K. Dey.

Division of Reproductive Sciences, Cincinnati Children’s Hospital Medical Centre and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229, (USA)E-mail: xiaofei.sun@cchmc.org and sk.dey@cchmc.org

Supporting information for this article is given via a link at the end of the document.

Abstract: Ambient ionization based on liquid extraction is widely

used in mass spectrometry imaging (MSI) of molecules in biological

samples. The development of nanospray desorption electrospray

ionization (nano-DESI) has enabled the robust imaging of tissue

sections with high spatial resolution. However, the fabrication of the

nano-DESI probe is challenging, which limits its dissemination to the

broader scientific community. Herein, we describe the design and

performance of an integrated microfluidic probe (iMFP) for nano-

DESI MSI. The glass iMFP fabricated using photolithography, wet

etching, and polishing shows comparable performance to the

capillary-based nano-DESI MSI in terms of stability and sensitivity;

the spatial resolution of better than 25 µm was obtained in these first

proof-of-principle experiments. The iMFP is easy to operate and

align in front of a mass spectrometer, which will facilitate broader use

of liquid extraction-based MSI in biological research, drug discovery,

and clinical studies.

Mass spectrometry imaging (MSI)[1] is a powerful analytical tool,which enables both targeted and untargeted label-free detectionof molecules in biological samples with high sensitivity andchemical specificity. Although matrix-assisted laser desorptionionization (MALDI) MSI[1a,2] is by far the most widely usedtechnique, substantial effort has been dedicated to thedevelopment of ambient MSI approaches.[1c,3] Ambient ionizationtechniques alleviate the need for sample pre-treatment prior toanalysis and enable imaging of biological systems in their nativestate.[1c] Several of these approaches rely on localized liquidextraction.[3c] Among others, these include desorptionelectrospray ionization (DESI),[1b,4] liquid micro-junction surfacesampling probe (LMJ-SSP),[5] nanospray desorption electrosprayionization (nano-DESI),[6] and single probe,[7] amongst others.Liquid extraction provides the advantages of gentle removal ofmolecules from specific locations on the surface, flexibleselection of the extraction solvent for the efficient extraction ofspecific classes of analytes,[8] quantification of the extractedanalytes by adding standards to the solvent, [3,6a] and efficientcompensation for matrix effects.[9] Nano-DESI MSI developed byour group uses two fused silica capillaries in a “V-shaped”configuration referred to as a nano-DESI probe. The probe formsa liquid bridge on the sample surface, into which analytemolecules are extracted and subsequently ionized at a massspectrometer inlet. High spatial resolution is achieved usingfinely pulled capillaries and a shear force probe, which controlsthe distance between the probe and sample surface. [10] This

configuration generates high-quality ion images with a spatialresolution of better than 10 µm.[11] Despite the advances in thedevelopment of this technique, the fabrication and alignment ofthe finely pulled capillaries are still challenging. The ability tofabricate an integrated probe for the robust nano-DESI imagingwith high spatial resolution will allow the broader scientificcommunity to adapt this technique to a wide range ofapplications.

Figure 1. Design and assembly of the integrated microfluidic probe for nano-DESI MSI. (A) Schematic drawing of the iMFP; (B) Photograph of the iMFP.(C) Schematic illustration of sampling port and liquid bridge between the probeand sample. (D) Photograph of the iMFP in front of a mass spectrometer: 1,instrument mounting flange; 2, XYZ inline micro-positioner; 3,iMFP; 4,capillary supplying the extraction solvent; 5, sample holder; 6, Dino-Litemicroscope; 7, syringe pump.

1

Microfluidic technology is a powerful tool for themanipulation of sub-nanoliter liquid volumes, which facilitates theanalysis of small samples The ability to process small samplevolumes makes the coupling of microfluidic devices with massspectrometry (MS) particularly advantageous.[12]. Belder et al.developed a glass microfluidic chip with a monolithic nanosprayemitter, which greatly enhancing the ionization efficiency. [13]

Alternatively, ESI has been performed directly from a corner of arectangular glass microchip used for coupling electrophoreticseparations with ESI-MS.[14] The dual-probe microfluidic chip has been used for sampling of analytes fromsurfaces of dry-spot samples and nanoliter droplets.[15] Thesestudies have demonstrated the power of microfluidics coupled toMS for the analysis of liquid samples. In order to extend thesecapabilities to MSI, it is important to design a device, which willbe able to extract analytes from a well-defined location on asurface and transfer them to a mass spectrometer.

Herein, we introduce an integrated microfluidic probe (iMFP)for nano-DESI MSI and demonstrate its capabilities for imagingof tissue sections. The design of the iMFP and a photograph ofthe probe are shown in Figures 1A and 1B, respectively. Theprobe comprises the solvent and spray channels and integratesthe sampling port (Figure 1C) and nanospray emitter into asingle chip. Figure 1D shows a photograph of the probe installedon a Q-Exactive HFX mass spectrometer (Thermo). Theextraction solvent is propelled through the solvent channel by asyringe pump; analyte molecules are extracted into the liquidbridge formed at the sampling port and transferred to a massspectrometer through the spray channel. Ionization occurs at thefinely polished monolithic spray emitter with the high voltageapplied to the syringe needle.

The iMFP is fabricated using the procedure described indetail in the experimental section of the supporting information.Briefly, photolithography and wet etching are used to generatechannels with a final depth of ∼25 μm and a width of ∼40 μm(Figure 1A). A glass wafer containing the microfluidic channels isbonded with a blank glass wafer at 590˚C for 3 hrs. Subsequentmultistep grinding is used to fabricate a finely polished sprayemitter and sampling port. The sharp spray emitter determinessignal stability. The design of the sampling port is critical to thesize and stability of the liquid bridge, which determines theanalyte sampling efficiency and the spatial resolution of theprobe. The optimized geometry of the sampling port, whichprovides stable signals and enables sensitive detection ofanalytes on the sample surface is shown in Figure 1C. Thedistance between the apex (Figure 1C) to the edge of the port is~40 µm; the angle between the solvent and spray channels of30° provides a stable flow and helps maintain a small size of theliquid bridge on the surface.

The stability of the probe evaluated using a 9:1(v/v)methanol/water solution containing 320 nM oflysophosphatidylcholine (LPC) 19:0 standard is shown in FigureS1A. After one hour of continuous signal recording, the relativestandard deviation of the signal of the internal standard is ~4 %.The signal-to-noise ratio of ~90 was obtained for the mostabundant lipid peak in the single-pixel mass spectrum of themouse uterine tissue section (Figure S1B), which is comparableto the results obtained using a conventional capillary-basednano-DESI probe.

Mouse uterine tissue is an excellent model system, whichcontains several distinct cell types distributed over a small cross-

sectional area of around 2 mm. These include luminal epithelium(LE), glandular epithelium (GE), and stroma (S) highlighted inFigure 2A. A detailed description of the experimental parametersis provided in the supporting information. Imaging experimentswere performed using the “three-point-plane” approachdescribed in our previous study.[6b] The approach compensatesfor the tilt of the sample plane and is the simplest way to controlthe distance between the sampling port of the iMFP and thesample surface. At least fifty-six phospholipids were identified inthe sample based on accurate m/z (Supporting Information,Table S1) and tandem mass spectrometry data (MS2). Ionimages obtained using iMFP MSI are shown in Figure 2A andFigure S2. Select images in Figure 2A correspond tosphingomyelin(SM) 42:2, phosphatidylcholine (PC) 32:0,PC36:2, PC34:1, and SM34:1 and highlight the characteristicspatial profiles of phospholipids observed in mouse uterinetissue sections. We observe distinct patterns of phospholipidlocalization to the heterogeneous cell types (LE, GE, andstroma) of the mouse uterine tissue. Specifically, SM34:1 isenhanced in both LE and GE whereas SM 42:2 is only enhancedin LE. In contrast to SM species, PC species show distinctlydifferent distributions depending on the length of the fatty acylchains and degree of unsaturation. For example, PC32:0 isenhanced in stroma, i.e. depressed in LE and GE. PC 34:1 isevenly distributed across the section, and PC 36:2 is enhancedin LE. Positive mode ion images were also obtained for a similarmouse uterine tissue section using high-resolution capillary-based nano-DESI MSI for comparison with iMFP (Figure 2B).This comparison indicates that iMFP provides ion images, whichare in good agreement with the best-performing capillary-basednano-DESI probe.[11]

Figure 2. Representative positive ion images of [M+Na]+ ions of molecules inmouse uterine tissues obtained using iMFP (A) and capillary-based nano-DESI probe (B). Scale bar: 1 mm; the intensity scale: black (low), yellow(high).

Spatial resolution is another important parameter describingthe performance of MSI techniques. In this study, we used the“80-20” rule[16] to estimate the upper limit of the spatialresolution. In this approach, the spatial resolution is calculatedfrom the distance, across which the abundance of the sharpestfeatures in the image changes between 20% and 80%. Accuratemeasurement of the spatial resolution requires the presence ofsteep chemical gradients in the sample. We used the ion imageof SM 34:1 (Figure 3A), which shows distinct localization in the

2

tissue. Figure 3B shows a line profile for SM 34:1 extractedalong the direction indicated by the white line in Figure 3A. Theline profile crosses the boundaries of different cell types andcontains multiple peaks. We estimate the spatial resolution fromthe transition regions between LE (or GE) and stroma (Figure3C) to be in a range of 22 to 25 µm as shown in Figure 3D. Weconservatively estimate that the upper limit of the spatialresolution obtained in this study is 25 µm.

To further verify the robustness and stability of the iMFP forMSI experiments, we acquired ion images for a fairly largemouse brain tissue section (7.7mm × 5.5mm). The results areshown in Figure S3. In this experiment, we used the sameconditions as in Figure 2 but increased the scan rate to 40 µm/s,which allowed us to acquire the image in 4 hrs (80 lines × 3minutes/line). Representative ion images of sodium adducts([M+Na]+) of phospholipids in mouse brain tissue are shown inFigure S3 (B-F). Consistent with our previous study,[6b, 9] weobserved that matrix effects play an important role in the imagingof brain tissue sections. Ion suppression results in a non-uniformdistribution of the LPC 19:0 internal standard used in thisexperiment (Figure S3-B). Good-quality ion images ofphospholipids (Figure S3 C-F) confirm the stability of the probeover the course of a 4 hr-long experiment.

Figure 3. Estimating the spatial resolution of the iMFP: (A) An ion image of SM34:1 in mouse uterine tissue section; a white line indicates the location of theline profile shown in panel B. (B) A representative line profile of SM34:1 alongthe white line in panel A. The ion signal is normalized to the TIC. (C) Anexpanded view of the boundary region between GE, LE, and stroma. (D) Apartial line profile extracted along the white line shown in panel C. The spatialresolution ranges from 22 to 25 µm. Blue arrows indicate the maximum(100%) and minimum (0%) values; red dashed lines indicate the positions atwhich the SM 34:1 signal is at 20% and 80% of its minimum and maximumvalue, respectively, for a specific region.

Our first proof-of-concept experiments provide evidence thatthe incorporation of the iMFP into nano-DESI MSI is a promisingstrategy for making this imaging technique broadly accessible.We demonstrate the sensitivity and robust operation of the iMFPfor imaging of biological tissues. Similar to the capillary-basednano-DESI MSI, the composition of the extraction solvent usedin the iMFP can be adjusted to facilitate the detection of differentclasses of molecules.[8b] Furthermore, the use of solventscontaining internal standards is advantageous for evaluating andcompensating for matrix effects in iMFP MSI. [9] The integrateddevice is easy to align in front of a mass spectrometer and easyto operate making it attractive for commercialization.

Experiments performed over the course of several monthsindicate that the same iMFP device can be re-used many times.

In summary, we have developed a new integratedmicrofluidic nano-DESI MSI probe, iMFP, and evaluated itsperformance for imaging of biological tissues. We optimized thegeometry of the device to enable efficient extraction ofmolecules from the sample and transfer to a mass spectrometerand provide stable ion signals. We demonstrate a comparableperformance of the iMFP and the best capillary-based nano-DESI MSI and a spatial resolution of better than 25 µm. Thedevice is compatible with any mass spectrometer making itbroadly applicable to different types of MSI experiments. Weenvision that with further improvement in the fabrication process,the probe will become an inexpensive “consumable”, which willadvance its dissemination to the broad scientific community.Future developments will focus on improving the spatialresolution and coupling of the iMFP to high-performance massspectrometers capable of operating at high repetition rates,which will speed up the image acquisition process. The iMFP willadvance the capabilities of MSI in biological and clinicalresearch.

Acknowledgments

The authors gratefully acknowledge the financial support fromthe National Institutes of Health (NIH) Common Fund, throughthe Office of Strategic Coordination/Office of the NIH Directorunder award UG3HL145593(HuBMAP Program, JL). The workon mouse uterus was supported in part by NIH grants(HD068524 and DA006668). We would like to thank JoonHyeong Park (the Birck Nanotechnology Center, PurdueUniversity) for helpful discussions and technical assistance withthe development of the photomask.

Keywords: mass spectrometry imaging • integrated microfluidic

probe • lipids • mouse brain • mouse uterine sections

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Entry for the Table of Contents

The integrated microfluidic probe ( iMFP) is easy to operate and align in front of a mass spectrometer which will facilitate broader use of liquid extraction-based MSI in biological research, drug discovery, and clinical studies. The incorporation of the iMFP into nano-DESI MSI is a promising strategy for making this imaging technique accessible to the broad scientific community.

Institute and/or researcher Twitter usernames: @lablaskin

download fileview on ChemRxivManuscript-An Integrated Microfluidic Probe-ChemRxiv.... (733.76 KiB)

1

An Integrated Microfluidic Probe for Mass Spectrometry Imaging

of Biological Samples

Xiangtang Li[a], Ruichuan Yin[a], Hang Hu[a], Yingju Li[b], Xiaofei Sun[b], Sudhansu K. Dey[b], and Julia

Laskin[a]*

[a] Xiangtang Li, Ruichuan Yin, Hang Hu, Prof. Julia Laskin

Department of Chemistry, Purdue University

560 Oval Drive, West Lafayette, IN 47907 (USA)

E-mail: jlaskin@purdue.edu

[b] Yingju Li, Prof. Xiaofei Sun, Prof. Sudhansu K. Dey.

Division of Reproductive Sciences, Cincinnati Children’s Hospital Medical Centre and Department of Pediatrics, University of Cincinnati College of

Medicine, Cincinnati, Ohio, 45229, (USA)

E-mail: xiaofei.sun@cchmc.org and sk.dey@cchmc.org

Supporting information for this article is given via a link at the end of the document.

Abstract: Ambient ionization based on liquid extraction is widely used

in mass spectrometry imaging (MSI) of molecules in biological

samples. The development of nanospray desorption electrospray

ionization (nano-DESI) has enabled the robust imaging of tissue

sections with high spatial resolution. However, the fabrication of the

nano-DESI probe is challenging, which limits its dissemination to the

broader scientific community. Herein, we describe the design and

performance of an integrated microfluidic probe (iMFP) for nano-DESI

MSI. The glass iMFP fabricated using photolithography, wet etching,

and polishing shows comparable performance to the capillary-based

nano-DESI MSI in terms of stability and sensitivity; the spatial

resolution of better than 25 µm was obtained in these first proof-of-

principle experiments. The iMFP is easy to operate and align in front

of a mass spectrometer, which will facilitate broader use of liquid

extraction-based MSI in biological research, drug discovery, and

clinical studies.

Mass spectrometry imaging (MSI)[1] is a powerful analytical tool,

which enables both targeted and untargeted label-free detection

of molecules in biological samples with high sensitivity and

chemical specificity. Although matrix-assisted laser desorption

ionization (MALDI) MSI[1a,2] is by far the most widely used

technique, substantial effort has been dedicated to the

development of ambient MSI approaches.[1c,3] Ambient ionization

techniques alleviate the need for sample pre-treatment prior to

analysis and enable imaging of biological systems in their native

state.[1c] Several of these approaches rely on localized liquid

extraction.[3c] Among others, these include desorption

electrospray ionization (DESI),[1b,4] liquid micro-junction surface

sampling probe (LMJ-SSP),[5] nanospray desorption electrospray

ionization (nano-DESI),[6] and single probe,[7] amongst others.

Liquid extraction provides the advantages of gentle removal of

molecules from specific locations on the surface, flexible selection

of the extraction solvent for the efficient extraction of specific

classes of analytes,[8] quantification of the extracted analytes by

adding standards to the solvent,[3,6a] and efficient compensation

for matrix effects.[9] Nano-DESI MSI developed by our group uses

two fused silica capillaries in a “V-shaped” configuration referred

to as a nano-DESI probe. The probe forms a liquid bridge on the

sample surface, into which analyte molecules are extracted and

subsequently ionized at a mass spectrometer inlet. High spatial

resolution is achieved using finely pulled capillaries and a shear

force probe, which controls the distance between the probe and

sample surface.[10] This configuration generates high-quality ion

images with a spatial resolution of better than 10 µm.[11] Despite

the advances in the development of this technique, the fabrication

and alignment of the finely pulled capillaries are still challenging.

The ability to fabricate an integrated probe for the robust nano-

DESI imaging with high spatial resolution will allow the broader

scientific community to adapt this technique to a wide range of

applications.

Figure 1. Design and assembly of the integrated microfluidic probe for nano-

DESI MSI. (A) Schematic drawing of the iMFP; (B) Photograph of the iMFP. (C)

Schematic illustration of sampling port and liquid bridge between the probe and

sample. (D) Photograph of the iMFP in front of a mass spectrometer: 1,

instrument mounting flange; 2, XYZ inline micro-positioner; 3,iMFP; 4, capillary

supplying the extraction solvent; 5, sample holder; 6, Dino-Lite microscope; 7,

syringe pump.

Microfluidic technology is a powerful tool for the manipulation

of sub-nanoliter liquid volumes, which facilitates the analysis of

small samples The ability to process small sample volumes

makes the coupling of microfluidic devices with mass

spectrometry (MS) particularly advantageous.[12]. Belder et al.

developed a glass microfluidic chip with a monolithic nanospray

emitter, which greatly enhancing the ionization efficiency.[13]

Alternatively, ESI has been performed directly from a corner of a

2

rectangular glass microchip used for coupling electrophoretic

separations with ESI-MS.[14] The dual-probe

microfluidic chip has been used for sampling of analytes from

surfaces of dry-spot samples and nanoliter droplets.[15] These

studies have demonstrated the power of microfluidics coupled to

MS for the analysis of liquid samples. In order to extend these

capabilities to MSI, it is important to design a device, which will be

able to extract analytes from a well-defined location on a surface

and transfer them to a mass spectrometer.

Herein, we introduce an integrated microfluidic probe (iMFP)

for nano-DESI MSI and demonstrate its capabilities for imaging of

tissue sections. The design of the iMFP and a photograph of the

probe are shown in Figures 1A and 1B, respectively. The probe

comprises the solvent and spray channels and integrates the

sampling port (Figure 1C) and nanospray emitter into a single chip.

Figure 1D shows a photograph of the probe installed on a Q-

Exactive HFX mass spectrometer (Thermo). The extraction

solvent is propelled through the solvent channel by a syringe

pump; analyte molecules are extracted into the liquid bridge

formed at the sampling port and transferred to a mass

spectrometer through the spray channel. Ionization occurs at the

finely polished monolithic spray emitter with the high voltage

applied to the syringe needle.

The iMFP is fabricated using the procedure described in detail

in the experimental section of the supporting information. Briefly,

photolithography and wet etching are used to generate channels

with a final depth of ∼25 μm and a width of ∼40 μm (Figure 1A).

A glass wafer containing the microfluidic channels is bonded with

a blank glass wafer at 590˚C for 3 hrs. Subsequent multistep

grinding is used to fabricate a finely polished spray emitter and

sampling port. The sharp spray emitter determines signal stability.

The design of the sampling port is critical to the size and stability

of the liquid bridge, which determines the analyte sampling

efficiency and the spatial resolution of the probe. The optimized

geometry of the sampling port, which provides stable signals and

enables sensitive detection of analytes on the sample surface is

shown in Figure 1C. The distance between the apex (Figure 1C)

to the edge of the port is ~40 µm; the angle between the solvent

and spray channels of 30° provides a stable flow and helps

maintain a small size of the liquid bridge on the surface.

The stability of the probe evaluated using a 9:1(v/v)

methanol/water solution containing 320 nM of

lysophosphatidylcholine (LPC) 19:0 standard is shown in Figure

S1A. After one hour of continuous signal recording, the relative

standard deviation of the signal of the internal standard is ~4 %.

The signal-to-noise ratio of ~90 was obtained for the most

abundant lipid peak in the single-pixel mass spectrum of the

mouse uterine tissue section (Figure S1B), which is comparable

to the results obtained using a conventional capillary-based nano-

DESI probe.

Mouse uterine tissue is an excellent model system, which

contains several distinct cell types distributed over a small cross-

sectional area of around 2 mm. These include luminal epithelium

(LE), glandular epithelium (GE), and stroma (S) highlighted in

Figure 2A. A detailed description of the experimental parameters

is provided in the supporting information. Imaging experiments

were performed using the “three-point-plane” approach described

in our previous study.[6b] The approach compensates for the tilt of

the sample plane and is the simplest way to control the distance

between the sampling port of the iMFP and the sample surface.

At least fifty-six phospholipids were identified in the sample based

on accurate m/z (Supporting Information, Table S1) and tandem

mass spectrometry data (MS2). Ion images obtained using iMFP

MSI are shown in Figure 2A and Figure S2. Select images in

Figure 2A correspond to sphingomyelin(SM) 42:2,

phosphatidylcholine (PC) 32:0, PC36:2, PC34:1, and SM34:1 and

highlight the characteristic spatial profiles of phospholipids

observed in mouse uterine tissue sections. We observe distinct

patterns of phospholipid localization to the heterogeneous cell

types (LE, GE, and stroma) of the mouse uterine tissue.

Specifically, SM34:1 is enhanced in both LE and GE whereas SM

42:2 is only enhanced in LE. In contrast to SM species, PC

species show distinctly different distributions depending on the

length of the fatty acyl chains and degree of unsaturation. For

example, PC32:0 is enhanced in stroma, i.e. depressed in LE and

GE. PC 34:1 is evenly distributed across the section, and PC 36:2

is enhanced in LE. Positive mode ion images were also obtained

for a similar mouse uterine tissue section using high-resolution

capillary-based nano-DESI MSI for comparison with iMFP (Figure

2B). This comparison indicates that iMFP provides ion images,

which are in good agreement with the best-performing capillary-

based nano-DESI probe.[11]

Figure 2. Representative positive ion images of [M+Na]+ ions of molecules in

mouse uterine tissues obtained using iMFP (A) and capillary-based nano-DESI

probe (B). Scale bar: 1 mm; the intensity scale: black (low), yellow (high).

Spatial resolution is another important parameter describing

the performance of MSI techniques. In this study, we used the

“80-20” rule[16] to estimate the upper limit of the spatial resolution.

In this approach, the spatial resolution is calculated from the

distance, across which the abundance of the sharpest features in

the image changes between 20% and 80%. Accurate

measurement of the spatial resolution requires the presence of

steep chemical gradients in the sample. We used the ion image

of SM 34:1 (Figure 3A), which shows distinct localization in the

tissue. Figure 3B shows a line profile for SM 34:1 extracted along

the direction indicated by the white line in Figure 3A. The line

profile crosses the boundaries of different cell types and contains

multiple peaks. We estimate the spatial resolution from the

transition regions between LE (or GE) and stroma (Figure 3C) to

be in a range of 22 to 25 µm as shown in Figure 3D. We

3

conservatively estimate that the upper limit of the spatial

resolution obtained in this study is 25 µm.

To further verify the robustness and stability of the iMFP for

MSI experiments, we acquired ion images for a fairly large mouse

brain tissue section (7.7mm × 5.5mm). The results are shown in

Figure S3. In this experiment, we used the same conditions as in

Figure 2 but increased the scan rate to 40 µm/s, which allowed us

to acquire the image in 4 hrs (80 lines × 3 minutes/line).

Representative ion images of sodium adducts ([M+Na]+) of

phospholipids in mouse brain tissue are shown in Figure S3 (B-

F). Consistent with our previous study,[6b, 9] we observed that

matrix effects play an important role in the imaging of brain tissue

sections. Ion suppression results in a non-uniform distribution of

the LPC 19:0 internal standard used in this experiment (Figure

S3-B). Good-quality ion images of phospholipids (Figure S3 C-F)

confirm the stability of the probe over the course of a 4 hr-long

experiment.

Figure 3. Estimating the spatial resolution of the iMFP: (A) An ion image of SM

34:1 in mouse uterine tissue section; a white line indicates the location of the

line profile shown in panel B. (B) A representative line profile of SM34:1 along

the white line in panel A. The ion signal is normalized to the TIC. (C) An

expanded view of the boundary region between GE, LE, and stroma. (D) A

partial line profile extracted along the white line shown in panel C. The spatial

resolution ranges from 22 to 25 µm. Blue arrows indicate the maximum (100%)

and minimum (0%) values; red dashed lines indicate the positions at which the

SM 34:1 signal is at 20% and 80% of its minimum and maximum value,

respectively, for a specific region.

Our first proof-of-concept experiments provide evidence that

the incorporation of the iMFP into nano-DESI MSI is a promising

strategy for making this imaging technique broadly accessible.

We demonstrate the sensitivity and robust operation of the iMFP

for imaging of biological tissues. Similar to the capillary-based

nano-DESI MSI, the composition of the extraction solvent used in

the iMFP can be adjusted to facilitate the detection of different

classes of molecules.[8b] Furthermore, the use of solvents

containing internal standards is advantageous for evaluating and

compensating for matrix effects in iMFP MSI.[9] The integrated

device is easy to align in front of a mass spectrometer and easy

to operate making it attractive for commercialization. Experiments

performed over the course of several months indicate that the

same iMFP device can be re-used many times.

In summary, we have developed a new integrated microfluidic

nano-DESI MSI probe, iMFP, and evaluated its performance for

imaging of biological tissues. We optimized the geometry of the

device to enable efficient extraction of molecules from the sample

and transfer to a mass spectrometer and provide stable ion

signals. We demonstrate a comparable performance of the iMFP

and the best capillary-based nano-DESI MSI and a spatial

resolution of better than 25 µm. The device is compatible with any

mass spectrometer making it broadly applicable to different types

of MSI experiments. We envision that with further improvement in

the fabrication process, the probe will become an inexpensive

“consumable”, which will advance its dissemination to the broad

scientific community. Future developments will focus on

improving the spatial resolution and coupling of the iMFP to high-

performance mass spectrometers capable of operating at high

repetition rates, which will speed up the image acquisition process.

The iMFP will advance the capabilities of MSI in biological and

clinical research.

Acknowledgments

The authors gratefully acknowledge the financial support from the

National Institutes of Health (NIH) Common Fund, through the

Office of Strategic Coordination/Office of the NIH Director under

award UG3HL145593(HuBMAP Program, JL). The work on

mouse uterus was supported in part by NIH grants (HD068524

and DA006668). We would like to thank Joon Hyeong Park (the

Birck Nanotechnology Center, Purdue University) for helpful

discussions and technical assistance with the development of the

photomask.

Keywords: mass spectrometry imaging • integrated microfluidic

probe • lipids • mouse brain • mouse uterine sections

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5

Entry for the Table of Contents

The integrated microfluidic probe ( iMFP) is easy to operate and align in front of a mass spectrometer which will facilitate broader use

of liquid extraction-based MSI in biological research, drug discovery, and clinical studies. The incorporation of the iMFP into nano-

DESI MSI is a promising strategy for making this imaging technique accessible to the broad scientific community.

Institute and/or researcher Twitter usernames: @lablaskin

download fileview on ChemRxivManuscript-An Integrated Microfluidic Probe-ChemRxiv.... (466.01 KiB)

Supporting Information

An Integrated Microfluidic Probe for Mass Spectrometry

Imaging of Biological Samples

Xiangtang Li[a], Ruichuan Yin[a], Hang Hu[a], Yingju Li[b], Xiaofei Sun[b], Sudhansu K. Dey[b], and Julia Laskin[a]*

[a] Department of Chemistry, Purdue University, West Lafayette, IN 47907.

[b] Division of Reproductive Sciences, Cincinnati Children’s Hospital Medical Centre and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229

Table of Contents

Experimental Section……………………………………………………. S 2-5

Figure S1 – S3…………………………………………………………….S 6-8

Table S1……………………………………………………………………S 9

Experimental Section

S-1

I. Reagents and Materials

Methanol and Omnisolv LC-MS grade water were purchased from Millipore Sigma(Burlington, MA). LPC19:0 was purchased from Avanti Polar Lipids, (cat. No. 855776P) andused as an internal standard. Fused silica capillaries (50µm ID, 150µm OD) were purchasedfrom Polymicro Technologies (L.L.C., Phoenix, AZ). Blank photomasks with 500nm thickAZ1518 positive photoresist and 100nm thick chrome on soda-lime glass plates (4" × 4",0.090" thick) are from Nanofilm (Nanofilm.com, Westlake Village, CA); AZ1518 positivephotoresist is from Clariant Corp (Somerville, NJ); Megaposit MF26A is from DOW; CapitolScientific and CR-16 chrome etchant are from VWR, Hydrofluoric acid (HF), buffered oxideetch (BOE) and other chemicals are from J. T. Baker (Phillipsburg, NJ). Corning® soda-limemicroscope slides (L×W 75 mm × 50 mm, thick 0.9-1.1mm) were used as a substrate andcover wafer. Steel-reinforced epoxy resin (J-B Weld Company, LLC, Sulphur Springs, TX )and Dent Light Cured Dental Block Out Resin (Bargin dental, San Dimas, CA) were used forbonding of fused silica capillaries to the microfluidic chips; deionized (DI) water (18.2 MU,Milli-Q, Millipore) was used in the fabrication of the chip.

II. Equipment

An E-Beam Evaporator from CHA Industries (Fremont, CA) was used to deposit Chromelayers, an MJB3 mask aligner (Suss Microtech, Waterbury, VT) was used to perform the UVphotolithography, and a 6808P Spin Coater (Specialty Coating Systems, IN 46278 USA) wasused for spin coating of the photoresist. A model P-7 Profilometer (KLA Corporation,Milpitas, California) was used to measure the depth and width of the microfluidic channelsand a programmable furnace (The Mellen Company, Concord, NH 03301, USA) was used toperform the high-temperature bonding of glass microfluidic chips. Other equipment used inthis experiment includes a syringe pump (Legato 180, KD Scientific), a 2.5mL syringe(Model 1002 LTN SYR, Hamilton, cat. No. 81416), Dino-Lite digital microscopes (Dino-LiteDigital Microscope, cat. No. AM4515T8), and a Q Exactive HF-X mass spectrometer(Thermo Fisher Scientific, Waltham, MA).

III. Solvent Preparation

5 mL of 9:1 (v/v) methanol/water (Omnisolv LC-MS grade) mixture was first prepared in a20 mL scintillation vial. 10 µL of 200 µM LPC 19:0 was then added to the vial and thesolution was vortexed vigorously. The final concentration of LPC 19:0 is 400 nM. Thesolvent can be stored for a week at room temperature or several months at -20 °C.

IV. Biological Tissues

S-2

Tissue sections were prepared according to the previously described methods.[1] Briefly, oneuterine horn was collected from a 4 days pregnant mouse, frozen in freezing media (SuperFriendly Freeze`ItTM, Fisher), and sliced to a series of sections with a thickness of 12 µmusing a Leica CM 3050 cryostat. Brain tissue was collected from a healthy adult mouse. Thetissue was flash-frozen and sectioned into 10 µm-thick sections. The uterine and brain tissuesections were thaw-mounted onto regular microscope glass slides and stored at – 80 priorto imaging.

V. Fabrication of the Glass Microfluidic Chip and the Integrated Microfluidic Probe( iMFP).

Standard photolithography, chemical wet etching, and high-temperature bonding techniqueswere used to fabricate glass microfluidic chips. We used the fabrication procedures describedin previous publications[2] with some optimization of the composition of the etching solutionand etching time. The specific fabrication procedure is as follows:

(i) Fabrication of the photomask. The pattern designed in KLayout with GDSII format wastransferred to the blank photomasks using a 405nm wavelength laser in a HeidelbergMLA150 maskless aligner. The photomasks were developed in a megaposit MF26A andetched in the CR-16 chrome etchant.

(ii) Fabrication of the glass microfluidic chips. Corning® soda-lime microscope slides weresequentially washed in an ultrasonic cleaner with toluene, acetone, isopropanol, methanol,and DI water for 5 minutes in each washing step, and then dried with N2. The substrate waferwas soaked into the piranha solution (a mixture of concentrated sulfuric acid with hydrogenperoxide, in a ratio of 3:1) for 30 minutes, then rinsed with DI water, and dried by N 2.[Warning: the piranha solution is extremely energetic and may result in severe chemical andthermal burns. It must be handled with extreme care in the hood and using acid-resistantPPE]. A 150 nm-thick chrome layer was deposited onto the glass substrate using E-BeamEvaporator and subsequently, the photoresist was spin-coated to a thickness of ~500nm ontothe Cr surface using 6808P Spin Coater. The substrate wafer with the chrome and photoresistlayer was baked on a hot plate at 110 °C for 15 min, and then the pattern was generated onthe glass substrate with a conventional UV photolithography using MJB3 mask aligner. Theexposed areas were developed by immersing the substrate into a Megaposit MF26A for 2min, and the exposed chrome layer was removed with chrome etchant. Glass etching wasperformed in a vigorously stirred hydrofluoric acid solution (the BOE solution diluted threetimes with water) at room temperature. The 15 μm-wide microchannels patterned on the glasswere etched for 35 min to generate a depth of ∼25 μm and final width of ∼40 µm asconfirmed using the KLA P7 stylus profiler. After the photoresist and chrome layer on thesurface of the substrate were removed, the substrate wafer with the microfluidic channels andthe cover wafer were immersed into the piranha solution for 30 min. Finally, the high-temperature bonding was performed using an increasing temperature gradient of 10˚C/minfrom 25 to 590 ˚C. The device was maintained at 590 ˚C for 3 hrs and subsequently cooleddown to room temperature using a decreasing temperature gradient of -10˚C/min.

S-3

(iii) Fabrication of the iMFP. Subsequent multistep grinding and polishing to fabricate theintegrated nanospray emitter and sampling port were performed with electric polishing toolsand different grit sandpaper (from 800-grit to 1500-grit). Nanospray emitter and samplingport were inspected throughout the process using a microscope. The final thickness of theiMFP was ~1.0mm, the diameter of the nanospray emitter tip is ~50µm, and the width of thesampling port is ~50µm. The solvent channel was connected to a fused silica capillary usingsteel-reinforced epoxy resin and a block-out resin.

VI.The iMFP-Based Nano-DESI Imaging Platform

The integrated microfluidic nano-DESI MS imaging platform is comprised of a syringe pumpwith a 2.5mL syringe for solvent delivery, a micro-positioner, XYZ motorized stages, asample holder, and two Dino-Lite digital microscopes for monitoring the nano-DESI probeduring imaging experiments. One of the microscopes is focused on the sample extraction portand the other monitored the nanospray emitter tip and MS inlet. The iMFP is mounted onto amicropositioner and positioned in front of a mass spectrometer inlet. The distance betweenthe nanospray emitter tip and the MS inlet orifice is ~0.5mm. A 10 cm long fused-silicacapillary (50µm ID, 150µm OD) connected to the syringe pump supplies the solvent to theiMFP. The spray voltage of +3.0 kV is applied to the syringe needle through a high-voltagecable. A 10-MΩ resistor is integrated into the high-voltage cable to avoid potential electricshock induced by a high spray voltage. A microscope glass slide containing tissue sections ismounted onto the sample holder. The sample positioning XYZ stage is controlled by acustom Labview program. The extraction solvent is propelled through the iMFP to thesampling port brought in close proximity to the sample and the extracted analyte moleculesare delivered to the mass spectrometer through the spray channel.

VII. The Setup of the Parameters for Mass Spectrometry Imaging Experiments

All the experiments with mouse uterine tissue and mouse brain tissue sections wereperformed on a Q Exactive HF-X mass spectrometer. A high voltage of +3.0 kV and an RFFunnel voltage of +100 V were applied in positive mode; mass spectra were acquired in therange of m/z 133−2000 with a mass resolution of 60,000 at m/z 200; AGC was set at 1×106

and the maximum injection time was 200 ms, the heated capillary was held at 250 °C. MSIdata were acquired in lines by scanning the sample under the iMFP using a motorized XYZstage (Zaber, Technologies, Vancouver, BC) and stepping between the lines. The scan rate of20 µm/s and step of 40 µm was used for imaging mouse uterine tissue sections with a totalacquisition time of ~2 hrs; for mouse brain tissue sections, the scan rate was 40 µm/s, thedistance between the line was 40 µm, and the total acquisition time was ~4 hrs. Mass spectrawere acquired in positive ion mode.

VIII. Visualization of the Raw Files Using MSI QuickView

A custom-designed MSI QuickView program[3] was used to convert mass spectrometry datainto ion images. A detailed description of the program can be found in our previous work. The

S-4

steps are briefly summarized as follows: i) loading the raw files into MSI QuickView; ii)defining the aspect ratio of the sampled area; iii) uploading a mass list to be visualized; iv)generating ion images for each m/z; v) saving ion images into a folder. Ion images of lipidsare normalized either to the TIC or signal of the internal standard.

References:

[1] R. Yin, K. E. Burnum-Johnson, X. Sun, S. K. Dey, J. Laskin, Nat. Protoc. 2019, 14, 3445-3470.

[2] a) C. Iliescu, H. Taylor, M. Avram, J. Miao, S. Franssila, Biomicrofluidics 2012, 6, 016505(1-16); b) W. Göpel, J. Hesse, J. N. Zemel, Sensors: a comprehensive survey, 1989; c) M. Stjernström, J. Roeraade, J. Micromech. Microeng.1998, 8, 33-38.

[3] I. Lanekoff, B. S. Heath, A. Liyu, M. Thomas, J. P. Carson, J. Laskin, Anal. Chem.2012, 84, 8351-8356.

S-5

Figure S1. Performance evaluation of the iMFP, (A) Ion chronogram of the internal standard(LPC 19:0) signal from continuous monitoring for around one hour, the signal is normalizedto the total ion current (TIC). In this experiment, the iMFP is brought in contact with thesurface of a glass slide and the signal of the standard at m/z 560.37 is measured as a functionof time; (B) A single-pixel positive mode nano-DESI spectrum of a mouse uterine tissueshowing S/N of ~90 for the most abundant lipid peak.

S-6

Figure S2. Positive ion images of phospholipids in mouse uterine tissue sections obtainedusing the iMFP. The experimental conditions are as follows: scan rate of 20 µm/s, solventflow rate of 1.0 µL/min, spray voltage of 3000 V, and a distance from the emitter tip to themass spectrometer inlet of ~0.5 mm.

S-7

Figure S3. Representative positive ion images of [M+Na]+ ions of phospholipids in mousebrain tissue acquired using the iMFP. (A) Optical image of the mouse brain. Ion images of(B) the internal standard, LPC19:0, at m/z 560.3525; (C) PC36:2 at m/z 808.5812; (D)LPC16:0 at m/z 518.3205; (E) PC 34:0 at m/z 784.5747; (F) PC34:1 at m/z 782.5657. Scalebar: 2 mm; total area analyzed in this experiment: 7.7 mm × 5.5 mm.

S-8

Table S1 Positive mode peak assignments of molecules observed in mouse uterine tissuesections using iMFP MSI.

Molecule Exactmass

Assignmentof the adduct

Molecule Exactmass

Assignmentof the adduct

1 FA 20:4 327.2299 [M+Na]+ 29 PC 36:2 808.584 [M+Na]+

2 MG 18:2 377.2668 [M+Na]+ 30 PC 36:1 810.6006 [M+Na]+

3 MG 18:1 379.2821 [M+Na]+ 31 PS 36:1 812.541 [M+Na]+

4 LPA 18:1 459.2488 [M+Na]+ 32 PC 36:4 820.5267 [M+K]+

5 LPC 16:0 518.3221 [M+Na]+ 33 PC 36:3 822.5432 [M+K]+

6 LPE 20:5 522.2577 [M+Na]+ 34 PC 36:2 824.5566 [M+K]+

7 LPC 18:1 544.3377 [M+Na]+ 35 PC 36:1 826.5744 [M+K]+

8 LPC 18:0 546.3534 [M+Na]+ 36 PC 38:6 828.5523 [M+Na]+

9 LPS 20:0 576.2703 [M+Na]+ 37 PC 38:5 830.5679 [M+Na]+

10 SM 34:1 725.5579 [M+Na]+ 38 PC 38:4 832.5839 [M+Na]+

11 PC 30:0 728.5218 [M+Na]+ 39 PC 40:6 834.6008 [M+H]+

12 SM 34:1 741.5301 [M+K]+ 40 SM 42:2 835.6046 [M+Na]+

13 PE 36:5 746.5108 [M+Na]+ 41 PC 38:4 848.558 [M+K]+

14 PC 32:1 754.537 (M+Na)+ 42 PC 40:6 856.5843 [M+Na]+

15 PC 32:0 756.5525 [M+Na]+ 43 PC 40:5 858.599 [M+Na]+

16 PC 34:1 760.5864 [M+H]+ 44 PI 40:7 931.5295 [M+Na]+

17 PE 36:2 766.5371 [M+Na]+ 45 DG(32:0) 591.4958 [M+Na]+

18 PC 32:1 770.511 [M+K]+ 46 DG(34:2) 615.4958 [M+Na]+

19 PC 32:0 772.5266 [M+K]+ 47 DG(34:1) 617.5115 [M+Na]+

20 PE 38:5 774.5419 [M+Na]+ 48 DG(36:4) 639.4958 [M+Na]+

21 PC 34:2 780.5525 [M+Na]+ 49 DG(36:3) 641.5115 [M+Na]+

22 PC 34:1 782.5682 [M+Na]+ 50 DG(36:2) 643.5271 [M+Na]+

23 PC 36:2 786.6024 [M+H]+ 51 DG(38:6) 663.4958 [M+Na]+

24 PE 38:4 790.5368 [M+Na]+ 52 PE(34:0) 742.5357 [M+Na]+

25 PC 34:2 796.5253 [M+K]+ 53 PC(O-32:0) 742.5721 [M+Na]+

26 PC 34:1 798.5423 [M+K]+ 54 PC(O-34:1) 768.5878 [M+Na]+

27 PC 36:4 804.5525 [M+Na]+ 55 TG(46:8) 785.5690 [M+Na]+

28 PC 36:3 806.5697 [M+Na]+ 56 PE(42:6) 826.5721 [M+Na]+

S-9

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