Bruker ImagePrep User manual

Revision 4 (April 2013)
ImagePrep
User Manual
(for Software Version 2.0)

Copyright Bruker Daltonik GmbH
Page 2 of 77 ImagePrep User Manual Revision 4
Copyright
© Copyright 2013
Bruker Daltonik GmbH
All Rights Reserved.
Reproduction, adaptation, or
translation without prior written
permission is prohibited, except
as allowed under the copyright
laws.
Document History
See Table of Changes
First edition: Aug 2007
Printed in Germany
Warranty
The information contained in this
document is subject to change
without notice.
Bruker Daltonik GmbH makes no
warranty of any kind with regard
to this material, including, but not
limited to, the implied warranties
of merchantability and fitness for
a particular purpose.
Bruker Daltonik GmbH shall not
be liable for errors contained
herein or for incidental or
consequential damages in
connec
tion with the furnishing,
performance or use of this
material.
Bruker Daltonik GmbH assumes
no responsibility for the use or
reliability of its software on
equipment that is not furnished
by Bruker Daltonik GmbH.
Safety Information
Safety class
The ins
trument is a Safety
Class I instrument and has
been designed and tested in
accordance with IEC
Publication 1010: Safety
Requirements for Electrical
Equipment for Measurement,
Control, and Laboratory Use.
WARNING
Connecting an instrument to a
power source
which has not
equipped with a protective
earth contact creates a shock
hazard for the operator and can
damage the instrument.
Likewise, interrupting the
protective conductor inside or
outside the instrument or
disconnecting the protective
earth terminal c
reates a shock
hazard for the operator and can
damage the instrument.
WARNING
The instrument has to be
disconnected from its power
source before the inner cover is
removed or
the instrument is
otherwise opened
All connections of the
instrument have to be used in
correct way.
It is only permitted to use wires
and cables provided
by the
manufacturer.
Instrument Identification
Each instrument is identified by
a unique serial number. This
serial numbers is located on a
label at
the rear of the
instrument.
When corresponding with
Bruker Daltonik GmbH about
your instrument, be sure to
include the full serial number.
Write the serial number of your
instrument here for reference:
Serial #:
Manual Conventions
Cautions
Cautions call attention to
procedures which, if not correctly
performed or adhered to, could
result in
damage to the
instrument.
Warnings
Warnings call attention to
procedures which, if not correctly
performed or adhered to, could
result in personal injury.
Part Numbers
In this manual
, Bruker Daltonik
GmbH part numbers are generally
listed in parentheses after the
name of the part or in tables in the
parts section.
A few tools and supplies listed
have no part numbers and are not
available from Bruker Daltonik
GmbH. Most of these can be
obtained from laboratory supply
companies.
ImagePrep User Manual
# 8261654
ImagePrep User Manual, Revision 4 (April 2013)
Copyright: Bruker Daltonik GmbH
Fahrenheitstr. 4
D-28359 Bremen
Germany
Phone: [phone]
FAX: [phone]
Email: [e-mail]
Internet: http://www.bruker.com

Bruker Daltonik GmbH Safety Symbols
ImagePrep User Manual Revision 4 Page 3 of 77
Safety Symbols
The following symbols appear in this manual where special attention is necessary.
NOTE
This symbol is used to draw attention to points that may affect
the operation or performance of the equipment.
WARNING
This symbol is used to indicate processes and procedures that
involve harmful substances ( e.g. Organic solvents).
Take appropriate care and refer to the Material Safety data Sheets
for the substances.
Equipment Disposal
Under Europe
an Directive 2002/96/EC, this label applies to electrical
and electronic equipment installed and sold in the European Union.
The consumer is committed by law to dispose of such equipment at the
end of its lifetime. When disposing of the equipment or parts thereof, it
must not be disposed of in household waste. To do so can contaminate
and pollute the environment.
To dispose of the equipment responsibly, contact your community
waste disposal authority. The details are regulated in national law.

Technical Support Bruker Daltonik GmbH
Page 4 of 77 ImagePrep User Manual Revision 4
Technical Support
If you need any assistance with this product please call or write to:
Address: BRUKER Daltonik GmbH,
Fahrenheitstr. 4,
28359 Bremen,
Germany
Phone: [phone] Technical / Software Support
[phone] Sales Department
Fax: [phone] Technical / Software Support
E-mail: [e-mail] Technical / Software Support
[e-mail] Sales Department
Internet: http://www.bruker.com
Table of Changes
Revision
Date Changes Remarks
1 Aug 2007 First edition -
1.1 Oct 2007 Editorial update -
2 Aug 2009 Updated hardware and ImagePrep software
Version 2.2 -
3 Jun 2010 Procedural changes
4 Apr 2013 WEEE, SAP renumbering and web address
update, added figure and table numbering

Bruker Daltonik GmbH Getting Started
ImagePrep User Manual Revision 4 Page 5 of 77
Getting Started
•Read the manual carefully!
•Sections written in black letters in the Table of Contents contain important
information for all users.
•Sections written in orange (light-grey) letters in the Table of Contents contain
important information for expert users.
•Place the instrument in a ventilated fume hood.
•Connect the instrument nitrogen source with a pressure with about
to 2-8 bar.
•Set the local date and time (see section 6.7).
•The instrument was delivered with a pre-adjusted spray head.
Prior to first use check the spray performance with methanol as described in
section 6.2.
What’s New in Version 2.0
•New spray head is supported section 6.1
•Simplified Spray Head Adjustment procedure section 6.2
•Improved sample loading procedure section 3.3
•New Test Spray buttons section 3.5.3
•Improved clean method section 3.5.5
•Diverse bug fixes

Table of Contents Bruker Daltonik GmbH
Page 6 of 77 ImagePrep User Manual Revision 4
Table of Contents
all users / experts
COPYRIGHT .................................................................................................................II
SAFETY SYMBOLS........................................................................................................III
TECHNICAL SUPPORT..................................................................................................IV
Table of Changes.................................................................................................. iv
GETTING STARTED.......................................................................................................V
TABLE OF CONTENTS..................................................................................................VI
1SAFETY AND SITE REQUIREMENT ISSUES................................................................8
1.1 Safety Precautions........................................................................................8
1.2 Installation and Site Requirements................................................................9
1.3 List of Approved Chemicals ..........................................................................9
2GENERAL INTRODUCTION ....................................................................................10
3SAMPLE PREPARATION WORKFLOW DESCRIPTION ...............................................12
3.1 Switching-on / Rebooting............................................................................12
3.2 Preparing the Tissue Sections ....................................................................13
3.3 Loading the Sample....................................................................................14
3.4 Loading the Matrix Solution.........................................................................15
3.5 Automatic Sample Preparation....................................................................16
3.5.1 Select Method........................................................................................17
3.5.2 Customize Method .................................................................................19
3.5.3 Test Spray and Start Automatic Preparation...........................................21
3.5.4 Mounting the Glass Slide into the Adapter..............................................22
3.5.5 Cleaning the Spray Chamber and the Spray Head.................................23
3.6 Manual Sample Preparation with ImagePrep..............................................25

Bruker Daltonik GmbH Table of Contents
ImagePrep User Manual Revision 4 Page 7 of 77
all users / experts
4 Method Development.......................................................................................26
4.1 Hardware and Software Requirements....................................................26
4.2 Interpretation of the Scattered Light Curves ..........................................27
4.3 The Method Development GUI.................................................................29
4.3.1 Method Access window.......................................................................29
4.3.2 Starting Methods for Test Purposes...................................................31
4.3.3 Method Editor: Phase Overview Level................................................32
4.3.4 Editing a Phase.....................................................................................35
4.3.4.1 The Matrix Thickness Window......................................................36
4.3.4.2 The Nebulize Window....................................................................39
4.3.4.3 The Incubation Window ................................................................45
4.3.4.4 The Dry Window ............................................................................47
4.4 General Method Development Rules.......................................................50
5TROUBLESHOOTING ............................................................................................52
5.1 Error Messages ..........................................................................................52
5.2 Other Problems...........................................................................................55
5.3 FAQ (Frequently Asked Questions) ............................................................59
6MAINTENANCE ....................................................................................................62
6.1 Spray Generator Replacement ...................................................................62
6.2 Global Spray Power Adjustment .................................................................64
6.3 Dismount the Spray Head...........................................................................66
6.4 Installation of Software and Method Updates..............................................66
6.5 Generate a Status Report...........................................................................67
6.6 System Reboot (see section 3.1)................................................................69
6.7 Date / Time Setting.....................................................................................69
6.8 Touch Panel Stylus.....................................................................................69
6.9 Filter Replacement......................................................................................69
6.10 Fuse Replacement......................................................................................69
6.11 List of Spare Parts ......................................................................................70
Appendix 1:.............................................................................................................71
Appendix 2:.............................................................................................................73

Bruker Daltonik GmbH Safety and Site Requirement Issues
Page 8 of 77 ImagePrep User Manual Revision 4
1 Safety and Site Requirement Issues
1.1 Safety Precautions
WARNING
Operate instrument
in ventilated fume hood only
This instrument vaporizes chemicals
and emits organic vapors.
NOTE
Only use manufacturer’s approved chemicals!
See list in section 1.3
NOTE
Never disable or bypass any of the safety features.
Figure
1
Instrument in
ventilated
fume hood

Bruker Daltonik GmbH Safety and Site Requirement Issues
ImagePrep User Manual Revision 4 Page 9 of 77
1.2 Installation and Site Requirements
•Fume hood mandatory
•Power 110-240 VAC,
50/60 Hz
200 VA
•Nitrogen - Quality 4.0 (99.99%)
or better
- 2-8 bar (29 – 116 PSI)
- 4 mm tubing
(or 1/8’’ with adapter)
•Temperature 18-24°C (64–75°F)
for best analytical performance
For details please refer to Site Preparation
Specifications document.
1.3 List of Approved Chemicals
•Select the solvents from the list
(or mixtures thereof) (see Figure 3).
•Cleaning solvent: methanol.
Do not use other solvents for cleaning!
•All common MALDI matrices can be used.
The matrix concentration should be at least 20%
below saturation.
•Optionally you can acidify the solution with up to
0.5% TFA.
NOTE
Other solvents or chemicals can be used with
manufacturer’s agreement.
Water
Ethanol
Methanol
2-Propanol
Acetonitrile
Figure
2 Connections
Figure
3
List of
solvents

General Introduction Bruker Daltonik GmbH
Page 10 of 77 ImagePrep User Manual Revision 4
2 General Introduction
Sample preparation is crucial for the quality of MALDI-tissue-imaging data.
Unfortunately, the current matrix application protocols have significant
disadvantages:
While pneumatic spray preparations provide good homogeneity and spatial
resolution of the images, the process is manual and highly irreproducible.
Depending of the degree of tissue wetting either the analyte molecules are badly
incorporated into the matrix (too dry) or the spatial resolution is lost (too wet).
Nano-spotting on the other hand provides quality spectra but as a sequential
process it is slow, spatial resolution is limited by the spot raster (typical >200 µm)
and perfect alignment with the mass spectrometer is critical.
ImagePrep is a new preparation device for MALDI Imaging that reproducibly
provides high resolution images at high speed and high spectra quality at the same
time.
On tissue, a lateral resolution of 50 µm can be achieved.
In the ImagePrep device, matrix
aerosol is created by vibrational
vaporization (see Figure 4), that is
gently deposited onto tissue
sections. During the whole
preparation process the spray
chamber is filled with nitrogen in
order to prevent sample oxidation
and to obtain reproducible
experimental conditions
independent of the humidity in the
ambient air.
An average droplet size of 20μm is generated with all droplet diameters being
smaller than 50μm, which corresponds well to the final matrix crystal size.
Multiple sections on a microscope slide can be homogeneously matrix-coated,
typically with 30-100 cycles within about one hour. Each cycle consists of three
phases:
1. deposit droplet layer,
2. incubate in controlled atmosphere, and
3. allow partial/complete drying.
Figure
4 Vibrational vaporization

Bruker Daltonik GmbH General Introduction
ImagePrep User Manual Revision 4 Page 11 of 77
An optical sensor monitors scattered light from the matrix-layer that allows control
of all relevant preparation parameters in real-time: deposition periods, intervals,
matrix-layer-thickness, wetness, drying rate. This QC-process built into the
preparation process provides highly reproducible preparations over several
slides and on different days.
ImagePrep is a push-button system: The user just selects a method, adapts it to
the respective tissue to be coated with three intuitive parameters (matrix thickness,
incubation time and wetness) and starts the sample preparation. The tissue section
is coated automatically without further user intervention. You will find a detailed
description of the whole workflow in section 3.
If you want to develop your own dedicated methods you can use the ImagePrep
method editor. This is a kind of expert mode which gives you full access to all
preparation parameters. Section 4 describes how to use the method editor.

Sample Preparation Workflow Description Bruker Daltonik GmbH
Page 12 of 77 ImagePrep User Manual Revision 4
3 Sample Preparation Workflow Description
3.1 Switching-on / Rebooting
The instrument is switched on / off with
the main switch, which is located on the
left side (see Figure 5). This main switch
is used also for rebooting the system.
The program starts automatically and the
auto-start procedure takes about two
minutes. Please wait until you see the
ImagePrep main menu.
NOTE
Make sure that no USB device is plugged into the
instrument when starting the boot process.
main switch
Figure
5 Main switch

Bruker Daltonik GmbH Sample Preparation Workflow Description
ImagePrep User Manual Revision 4 Page 13 of 77
3.2 Preparing the Tissue Sections
1. For optimal MALDI results we recommend preparing 10-15 µm cryosections
of native snap-frozen tissue.
Note: Tissue fixation, staining and embedding in resins affects the spectra
quality!
2. Mount tissue sections on ITO coated microscope glass slides (75x25 mm,
Bruker # 8237001). These transparent slides are perfectly suited to light
microscopy and for the optical sensor of ImagePrep. Furthermore their
electrical conductivity fulfills a prerequisite for successful MALDI TOF analysis.
Note: Make sure that the tissue is mounted on the side coated with
conductive material. Use ohmmeter to test it!
3. If you want to analyze proteins, wash sections in Petri dish twicewith
70% EtOH for 1-2 minutes followed by one wash in 95% EtOH for 1-2 minutes.
Note: Small molecules imaging (m < 3 kDa) requires a shorter washing in
order to avoid analyte delocalization.
4. Dehydrate sections for at least 15 minutes in a
vacuum desiccator (see Figure 6).
5. Scan slide in order to obtain an optical image.
For details please refer to the flexImaging manuals.
6. For best results, matrix should be applied and
data acquired directly after sectioning.
Note: If this is not possible, you can store the
sections on the glass slides in a vacuum
desiccator or under nitrogen atmosphere for
a few days before applying matrix. Do not store matrix-coated slides for
more than a day.
For further details on tissue sample preparation please refer to:
Schwartz S., Reyzer M., Caprioli R.
Direct tissue analysis using MALDI MS: Practical aspects of sample preparation
J. Mass Spectrom. 2003; 38: 699-708
Figure
6 Vacuum
desiccator

Sample Preparation Workflow Description Bruker Daltonik GmbH
Page 14 of 77 ImagePrep User Manual Revision 4
3.3 Loading the Sample
Position slide on the elevated rectangular area on the bottom of the ImagePrep
spray chamber.
Note: Wear gloves when handling the cover slip to prevent contamination of the
cover slip.
* There are two reasons for this:
a) In contrast to the underlying ITO coated slide, the cover slip is hydrophilic and does not show
the inverted scattered light signal problem described in section 4.4.
b) The cover slip is clean and is not coated with smear from tissue washing.
Do not position the tissue on the
sensor window (see Figure 7). The
scattering behavior of the sample is
unpredictable.
Ensure that the sensor “sees” a free
slide area, i.e. an area without sample
material. The most reproducible results
are obtained if you place a coverslip on
the tissue-free slide area* on top of the
glass slide over the sensor window (see
Figure 8-1).
We recommend that narrow size-
optimized
coverslips are used (see
Figure 8-2), Bruker # 8267942) .
Figure 8
Correctly positioned
tissue and coverslip
Figure
7 Badly positioned tissue

Bruker Daltonik GmbH Sample Preparation Workflow Description
ImagePrep User Manual Revision 4 Page 15 of 77
3.4 Loading the Matrix Solution
1. Prepare the matrix solution.
Note: If you use ImagePrep standard methods, you will find the matrix
recipe in the information window which appears when you open this
method.
If you want to spray other matrix solutions with ImagePrep,
please observe the following general rules:
a) Select the solvents from the following list (or mixtures thereof):
water, ethanol, methanol, 2-propanol, acetonitrile.
Other solvents can be used with manufacturer’s agreement.
b) The matrix concentration should be at least 20% below
saturation.
c) Optionally you can acidify the solution with up to 0.5% TFA
2. a) Open side door (see Figure 9).
b) Tip the matrix bottle to the vertical position. (see Figure 10-b)
c) Remove the glass bottle (see Figure 10-c).
3. Fill the 10 mL bottle with approximately 5 mL matrix solution and
remount it into to the ImagePrep by reversing the removal process.
Note: 1. Typical matrix consumption per slide: 1-2 mL
2. For special applications (e.g. tryptic on-tissue digestion) fill at least
≥200 µL liquid in the matrix bottle.
a
Figure
9 Open side door
Figure
10
Remove
glass
bottle

Sample Preparation Workflow Description Bruker Daltonik GmbH
Page 16 of 77 ImagePrep User Manual Revision 4
4. Close side door and lid.
5. Start preparation right after matrix loading.
Long delays lead to spray head clogging.
In order to ensure high quality sample preparations,
Bruker recommends that the spray generator (#
8
261614) is replaced after every 30 preparations. An
internal counter will inform you when the replacement
is necessary. The replacement procedure is described
in detail in section 6.1.
Figure 11 Spray
generator

Bruker Daltonik GmbH Sample Preparation Workflow Description
ImagePrep User Manual Revision 4 Page 17 of 77
3.5 Automatic Sample Preparation
3.5.1 Select Method
Switch on the ImagePrep device with the main switch (see section 3.1).
Select AutoPrep in the main menu once the software has opened (see Figure 12).
Depending on the matrix used and on the analyte molecules under scrutiny,
different preparation methods have to be used. These methods contain all relevant
information about the preparation procedure and the respective parameters.
Select a methodfrom the drop-down box (see Figure 13). Information on the
application and the matrix recipe of the selected method can be displayed by
opening the Information… window (see Figure 14).
A number of standard methods are preinstalled on ImagePrep covering the most
common applications (see table below). To access more methods and updates
(e.g. methods for on-tissue enzymatic digestion or for TLC-MALDI), see
Appendix 3.
Section 6.4 describes how to install additional methods on ImagePrep.
User methods can be created and edited with the method editor (see section 4).
Customization (see section 3.5.2) allows the methods to be adapted to specific
requirements, e. g. tissue specific.
Figure
14 Information
window
Figure
13 Select
preparation
method
Figure
12 ImagePrep
main menu
SA_Proteins
Default
SA_Proteins
Default
SA_Proteins
Default
SA_Proteins
Default
Name:
SA_Proteins
Information:
Proteins with Sinapinic Acid
10g/l SA in 60% ACN / 0.2% TFA
SA_Proteins
Default
SA_Proteins
Default
Name:
SA_Proteins
Information:
Proteins with Sinapinic Acid
10g/l SA in 60% ACN / 0.2% TFA

Sample Preparation Workflow Description Bruker Daltonik GmbH
Page 18 of 77 ImagePrep User Manual Revision 4
Table
1 Preinstalled methods on ImagePrep; * xy is
the version number, e.g.
SA_nsh01 or DHB_nsh05
Methods for
old spray head
Figure 15 Old
spray
head
Methods for
new spray head (nsh)
Figure 16 New
spray
head
Scope Matrix Recipe
SA_V xy * SA_nsh xy * Protein measurement with
sinapinic acid matrix The matrix recipe is
given in the
Information…
window
(see below in this
section).
CCA_V xy * CCA_nsh xy * Drug and small molecule imaging
with HCCA matrix
DHB_V xy * DHB_nsh xy * Protein, peptide and drug analysis with
2,5-DHB matrix
Instrument_Test Dedicated method to
test the instrument

Bruker Daltonik GmbH Sample Preparation Workflow Description
ImagePrep User Manual Revision 4 Page 19 of 77
3.5.2 Customize Method
Method customization allows the user
to tune the selected method via
three intuitive parameters (Matrix
Thickness, Incubation Time, and
Wetness). Customization is therefore
a valuable tool to adapt methods to
specific tissue types.
All three parameters are set with
sliders and the settings can be saved
individually (e. g., "brain“, "liver“,
"Paul’s special preparation“) and later
recalled from the dropdown box.
In the Default customization all three sliders are set to the middle position (see
Figure 17). This instrument setting was tested by Bruker on rat brain tissue and is
also widely applicable to other tissues.
Therefore, Bruker recommends trying the default settings first before adjusting the
sliders for optimization.
SA_Proteins
Default
SA_Proteins
Default
SA_Proteins
Default
SA_Proteins
Tissue 1
SA_Proteins
Tissue 1
SA_Proteins
Tissue 2
SA_Proteins
Tissue 2
Figure
17 Method customization
window
Figure
18
Different customizations adapt the methods to different tissue
types

Sample Preparation Workflow Description Bruker Daltonik GmbH
Page 20 of 77 ImagePrep User Manual Revision 4
The table below gives you some hints on how to use the customization
parameters.
Customization
Parameter Explanation General Rules
Matrix
Thickness Total amount of matrix applied Matrix amount is not very critical above a certain limit.
Incubation Time Waiting period after each spray cycle (no active drying
with gas flow)
Incubation time ↑:
better analyte incorporation into
matrix crystals, but longer prep time
needed
Wetness Residual wetness on tissue
when next spray cycle starts
Wetness ↑:better spectra, but more
analyte delocalization
Wetness ↓:less analyte delocalization
but worse spectra
The setting ranges for the sliders are defined in the method in a practical way:
Even extreme settings do not cause the preparation to crash, although they might
not necessarily show good mass spectrometric results in every case.
The settings of the sliders operate in each of the different phases of the method
individually; therefore no numbers can be set for the scale of the sliders but rather
qualitative indications, like wet, dry, short, long.
Example: In a method consisting of two phases, the incubation time in the first
phase is set to 10 s ± 8 s and in the second phase to 20 s ± 10 s.
When the user now sets at the customization slider of the incubation
time to Short / Default / Long, this would result in an incubation time
of 2 s / 10 s / 18 s in the first phase and of 10 s / 20 s / 30 s in the
second phase.
If a customization is not allowed in any of the phases, then the slider for the
respective parameter is grayed-out and inactive.
Table
2 Customization Parameters