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ISBN: 978-0-12-818998-6
Preface xi
v
vi Contents
Index 255
Preface
The idea about an introductory book on imaging mass spectrometry (MS) ger-
minated when I first talked to Kathryn Eryilmaz from Elsevier at the annual
American Society for Mass Spectrometry Conference. I mainly told her about
what I wanted to see in such a book from the perspective of many scientists who
are interested in bringing the imaging MS to their labs to benefit their research.
Many of my suggestions were based on what I noticed in my job as a subject
matter expert for imaging MS working with other scientists, from experts to
newly curious. Most of them needed a simple guide that encompassed various
aspects of imaging MS.
Later, when I was approached with writing or editing one, I took some
time to think mainly because my last book on Single Cell Metabolism took
more effort than anticipated and took quite a long time to finish. I wrote a book
proposal on my initial thoughts on the book. The proposal was sent to sev-
eral reviewers. Thank you for the constructive suggestions that prompted me to
reframe the focus of my book. The book got a slow start, mostly because I kept
revising the style and type of content. Additionally, as a new dad of twin boys,
time was scarce. However, when not distracted, I got a lot done on the weekends
and super early mornings before the world around me slowly woke up.
As COVID-19 hit and life around me changed, I had more time during the
weekend. I was on temporary leave for a few months, which turned out to be
the most productive time for writing this book. Most of the book was finished
during that time. Now, I see the value of a sabbatical in an academic setting.
One of the simple routines of meeting with the Elsevier project manager, Lena
Sparks, periodically helped in pushing the book along. I made a Gantt chart
with a progress bar for each chapter—a trick I learned when I was a Research
Director for a DARPA project.
I am delighted with the final result. The aim of the book is to give a com-
prehensive introduction to many aspects of imaging MS for biomedical appli-
cations. The book is not an encyclopedia for everything on imaging MS or a
compilation of detailed step-by-step protocols. I encourage readers to consult
other reviews published each year on various aspects of imaging MS and, if
more interested, dive deep into the original publication. The imaging MS com-
munity is still small, and most of us, when you ask for help, more than willing
to exchange ideas and collaborate. I have aimed to include fundamental topics
that are used for biomedical imaging applications and wrote in the aspect of a
brief review and perspective on the topic.
xi
xii Preface
One of my favorite outcomes of this book was that I got to learn a lot also
while working on the book. Some topics, such as ionization, I was extremely
familiar with, but many others I learned as I went along. It was a rewarding
experience.
My parents, Govinda Ram and Sharda, and wife Lauren have been very
supportive during the writing of the book. Thank you for your support and love.
Also, thank you for your love, my daughter, Munchie, who is a happy pug, and
my toddler twin boys, Kai and Neel.
Bindesh Shrestha
Swampscott, MA
Chapter 1
Imaging mass spectrometry (MS) is an analytical technique that can visualize the
distribution of molecules using a tool called a mass spectrometer. In the case of bio-
logical samples, the visualized molecules include any species that can be detected
by the mass spectrometer, such as metabolites, drugs, lipids, peptides, and proteins.
A mass spectrometer is an analytical tool that can measure the mass-to-charge
ratio (m/z) of ions of molecules detected in a sample. The molecular analysis
of bulk biological samples is done after the sample is homogenized, extracted,
or processed according to the molecule-of-interest. The processed aliquot is
injected into liquid chromatography for separation. The separated molecules are
ionized using an electrospray mechanism and detected by a mass spectrometer.
The detected m/z ion intensities are assigned to molecules and examined for the
presence of one or multiple molecules or their up/downregulation. This general
bulk analysis workflow template is widely used in all MS-based omics analyses,
such as metabolomics, proteomics, as well as in quantitative and qualitative
molecular analyses. Such bulk tissue analysis can provide information on the
identity and the concentration of molecule-of-interest in a bulk sample but does
not provide insight into the location of a molecule within the sample. In the
imaging MS workflow, a thin slice of tissue is sectioned instead of homogeniza-
tion of the chunk of tissue. The sectioning is followed by sample preparation
steps such as matrix application, and finally, the sample is analyzed by desorp-
tion/sampling and ionization using a mass spectrometer. A comparison of work-
flow between bulk analysis of tissue and imaging MS is illustrated in Fig. 1.1.
FIG. 1.1 A comparison between bulk tissue analysis workflow using electrospray liquid chroma-
tography–mass spectrometer and tissue imaging using matrix-assisted laser desorption/ionization
mass spectrometer.
mass spectrum
pixel
ion images
FIG. 1.2 General representation of imaging MS workflow, where a tissue section such as mouse
brain section is interrogated pixel-by-pixel producing mass spectrum for each pixel, and the ion
intensity distribution for each ion in each pixel is plotted as a false-color image.
Fundamentals of imaging mass spectrometry Chapter | 1 3
mass
spectrometer
desorption
source α
movement in x
α (x pixel size)
ize y
TYPEWRITER SERPENTINE
l s in
)
xe nt
pi e
(y vem
o
m
FIG. 1.3 During the imaging MS acquisition, the molecules on the tissue sections are sampled
by a focused desorption mechanism. By moving the sample stage in a prescribed manner, such as
in typewriter or serpentine modes, spatial information of molecules on each pixel can be obtained.
FIG. 1.4 A conceptual framework of imaging MS data is illustrated by a four-pixel image consist-
ing of three ions. Mass spectra are generated for each pixel can be converted to ion intensities of all
the ions at each pixel. Finally, MS images for all ions are obtained by correlating their ion intensities
on each pixel with a colormap definition.
4 Introduction to spatial mapping of biomolecules by imaging mass spectrometry
each ion in mass spectra plots denote their intensities. This plot can also be rep-
resented as an intensity table. MS images are created by assigning a false-color
intensity for each ion at each pixel, as shown in the figure. The pixel-by-pixel
imaging MS workflow is most commonly used and also sometimes referred to
as microprobe mode. In an alternative instrumental setup, called microscope
mode, the ions are imaged using a position-sensitive detector. In theory, micro-
probe imaging instruments should have higher spatial because the resolution
is not dictated by the optical properties of the laser beam but the ion optics of
the mass spectrometer with the ability to image below the diffraction limit.28
Microscope mode can potentially have higher throughput but often lower mass
resolving power.29–30 Almost all of the imaging MS studies discussed in this
book and the literature is done pixel-by-pixel or in microprobe mode.
In principle, each pixel of imaging MS data can be considered as an inde-
pendent MS experiment. Like any MS experiments, we can either aim to detect
all the ions in the acquired mass range, called here as profiling MS imaging, or
aim a selected mass or fragment, named here as targeted MS imaging. In pro-
filing MS imaging experiments, all the ions above a threshold detection limit
in that biological microenvironment are detected. In this manner, the spatial
distribution of hundreds to thousands of molecules can be obtained without a
strict selection criterion, labeling, or a priori knowledge. It should be noted that
there is a degree of inherent selection due to the choice of sample preparation
workflow, experimental parameters or conditions, or type of instrumentation.
MIN
2.50mm
FIG. 1.5 Visualization of three molecular lipid ions using desorption electrospray ionization
(DESI) imaging mass sagittal rodent brain section without any labeling is shown on top. The bot-
tom shows an ion overlay image of the same three ions showing contextual distribution.
A
Brain Lung Kidney
Stomach
Liver contents
10 mm
B
0 100
Brain C
Lung Kidney
0 100
FIG. 1.6 A comparison of desorption electrospray ionization (DESI) mass spectrometry image (B)
with whole-body autoradiography (WBA) image is shown with their respective optical image at (A) and
(C). Adapted with permission from Kertesz V, Van Berkel GJ, Vavrek M, Koeplinger KA, Schneider
BB, Covey TR. Anal Chem. 2008;80:5168–5177. Copyright 2007 American Chemical Society.
A MARCELINE DESBORDES-VALMORE,
A vous, fille de la Flandre, et qui en êtes une des gloires
modernes, cette naïve tradition des Flandres.
De Balzac.