Page 1
Revision 4 (April 2013)
ImagePrep
User Manual
(for Software Version 2.0)
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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 His tory
See Table of Changes
First edition: Aug 2007
Printed in Germany
Warranty
The information con tained in thi s
document is subject to change
without notice.
Bruker Daltonik GmbH makes n o
warranty of an y kind with regard
to this material, i n clu di ng , bu t not
limited to, the im plied warranti es
of merchantabil ity and fi tness fo r
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 Informa ti on
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 o perat or an d 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 o perat or an d 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 i s 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 n umber of your
instrument here for reference:
Serial #:
Manual Conventions
Cautions
Cautions call attention to
procedures whi ch, if not correctl y
performed or adhered to, could
result in
damage to the
instrument.
Warnings
Warnings call attention to
procedures whi ch, if not correctl y
performed or adhered to, could
result in personal injury.
Part Numbers
In this manual
, Bruker Daltonik
GmbH part numb ers a re 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 n um be rs 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: +49 (421) 2205-345
FAX: +49 (421) 2205-103
Email: maldi.sw.support@bdal.de
Internet: http://www.bruker.com
Page 3
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.
Page 4
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: +49 (0)421 2205-345 Technical / Software Suppo rt
+49 (0)421 2205-200 Sales Department
Fax: +49 (0)421 2205-103 Technical / Software Suppor t
E-mail: maldi.sw.support@bdal.de Technical / Software Suppo rt
sales@bdal.de 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
Page 5
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
Page 6
Table of Contents Bruker Daltonik GmbH
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Table of Contents
all users / experts
C
OPYRIGHT ................................................................................................................. II
S
AFETY SYMBOLS ........................................................................................................III
T
ECHNICAL SUPPORT .................................................................................................. IV
Table of Changes .................................................................................................. iv
G
ETTING STARTED ....................................................................................................... V
T
ABLE OF CONTENTS .................................................................................................. VI
1 S
AFETY AN D SITE REQUIREMENT ISSUES................................................................ 8
1.1 Safety Precautions ........................................................................................ 8
1.2 Installation and Site Requirements ................................................................ 9
1.3 List of Approved Chemicals .......................................................................... 9
2 G
ENERAL INTRODUCTION .................................................................................... 10
3 S
AMPLE 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
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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
5 T
ROUBLESHOOTING ............................................................................................ 52
5.1 Er r or Messages .......................................................................................... 52
5.2 Other Problems ........................................................................................... 55
5.3 FAQ (Frequently Asked Questions) ............................................................ 59
6 M
AINTENANCE .................................................................................................... 62
6.1 Spr ay Gener ator Replacement ................................................................... 62
6.2 Global Spray Power Adjustment ................................................................. 64
6.3 Dism ount 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 Dat e / Time Setting ..................................................................................... 69
6.8 Touch Panel Stylus ..................................................................................... 69
6.9 F ilter Replacement ...................................................................................... 69
6.10 Fuse Replacement ...................................................................................... 69
6.11 List of Spare Parts ...................................................................................... 70
Appendix 1: ............................................................................................................. 71
Appendix 2: ............................................................................................................. 73
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Bruker Daltonik GmbH Safety and Site Requirement Issues
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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
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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
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General Introduction Bruker Daltonik GmbH
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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
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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 intuit ive 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.
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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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
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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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 twice with
70% EtOH for 1-2 minutes followed by one wash in 95% EtOH for 1-2 minute s.
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
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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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 sig nal 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 sizeoptimized
coverslips are used (see
Figure 8-2), Bruker # 8267942) .
Figure 8
Correctly positioned
tissue and coverslip
Figure
7 Badly positioned tissue
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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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
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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4. Close side door and lid.
5. Star t prepar ation 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
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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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 method from 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 ImagePr ep 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
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Sample Preparation Workflow Description Bruker Daltonik Gm bH
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Table
1 Prei nstalled 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
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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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 set tings 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
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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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 incorporati on 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 f or the sliders ar e 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 t he 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 Custom i z ation Parameters
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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3.5.3 Test Spray and Start Automatic Preparation
After selecting a method, make sure that the lid and side door of the instrument are
closed. Always check the spray before starting t he preparat ion: Tap the Test button
to switch on the spray for 3 seconds (see Figure 19). The spray should cover the
glass slide completely (see Figure 20). If it is too weak / too strong you can tune t he
power by max. +/- 5%. If the spr ay still doesn’t look ok , t he power off set adjustment
is wrong (please refer to section 6.2).
After checking the spray power, tap the Start button to start the sam ple preparation
(see Figure 21). Fir st, the system will be flushed with nitrogen in order to achieve
Figure
21 Start
Button
Figure
23 Layer
Thickness
gauge
Figure
22 Automatic
preparation
workflow
Figure
19 Test spray
button
Figure
20 Adjust spray power
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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reproducible experimental conditions and to prevent sample oxidation. This flushing
can take up to three minutes.
The automatic preparation then starts. During this workflow a sensor-controlled
cycle of spraying, incubation and active drying is repeated many times until the
required thickness of the matrix layer is achieved (see Figure 22). During the
preparation, the Layer Thickness gauge gives the user information about the
actual matrix layer thickness and the estimated time remaining (see Figure 23). The
preparation can be interrupted / cancelled at any time by tapping Pause/Abort. If
you pause the preparation, make sure that the sample is dried completely before
restarting.
3.5.4 Mount ing the Glass Slide into the Adapter
Wipe off the matrix at the edges of the
slides (see red stripes in Figure 24) before
inserting the slides into the adapter target
(Bruker # 8235380). These regions on the
slide ensure electrical contact with the
MALDI adapter target.
Figure
24
Wipe slide before
inserting
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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3.5.5 Clea ning the Spray Chamber and the Spra y Head
Cleaning the Spray Chamber
After the preparation has been finished, the Clean
Instrument window opens automatically to guide
you through the cleaning procedure (see Figure 25).
This window is also accessible via the main menu.
1. Remove sample slide from the chamber.
2. Replace residual matrix in the glass bottle with
10 mL methanol.
3. Tap Start. Cleaning takes only a few minutes.
Note: Do not use solvents other than methanol for cleaning.
Afterwards wipe out the spr ay chamber with the wet wadded paper tissue. The metal
sheet of the spray head should be clean after t he cleaning procedure is complete. If it
is not clean, restart the cleaning with fresh methanol.
Note: T o disinfect the spray chamber restart the cleaning with 70% EtOH.
Cleaning the Spray Head
Whereas the metal sheet and the spray chamber have to be cleaned after every
preparation, the spray head needs only to be washed from time to time when massive
matrix crystallization is observed on the yellow protective film.
To wash, dismount the metal sheet from the
spray cap and (1) sonicate the spray cap in
alcohol (EtOH or MeOH, NOT water) for
approx. one minute (see Figure 26-1). After the
washing procedure, (2) carefully dry the device
with pressurized air or nitrogen (see Figure 26-
2). Blow air into the two openings next to the
clamp for at least 30 seconds each in order to
dry the interior parts of the spray head.
Figure
25 Clean
Instrument
window
Figure
26
Spray head cleaning
procedure
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Sample Preparation Workflow Description Bruker Daltonik GmbH
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Never wash with water or aqueous solvents because water does not evaporate
completely from inside the spray cap. This would result in partial internal short-circuits
and in a reduced spray power.
Note: Never try to clean the spray cap when it is mounted on the spray head.
The wetness inside would immediately decrease the spray power!
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Bruker Daltonik GmbH Sample Preparation Workflow Description
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3.6 Manual Sample Preparation with ImagePrep
The Manual Operation mode allows operating the instrument for a defined number
of cycles at a given spray power with a fixed time scheme (spray time incubation
time dry time). All settings in the Manual Operation window (see Figure 27) are
self-explanatory, with the exception of the spray power modulation which is
described in detail in section 4.3.4.2.
Always check the spray before starting the preparation: Tap the Test Spray button
to switch on the spray for 3 seconds.
Note: The sensor control is disabled in the manual workflow (see Figure 28).
Therefore the manual mode does not give reproducible results for matrix
solutions due to the fact that the spray head becomes partially clogged.
NO
sensor
control
Figure
27 Manual Operation mode window
Figure
28 Manual
preparation
workflow
Figure
29
Preparation in
Progress
window
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4 Method Development
An expert mode for method development is available on I magePrep. This enables
experienced users to create their own methods. Below you will find a detailed
description for configuration of the system for this purpose.
4.1 Hardware and Software Requirements
The following items are needed for method development
on ImagePrep:
1. Laptop computer
displays the scattered light curves during preparation.
2. ADC (Analog Digital Converter) (see Figure 30)
The ADC converts the analog signal (0–5 V) of the scattered light sensor to a
digital signal in order to display the scattered light curve on the laptop computer.
An ADC (National Instruments NI USB-6008) is included with the instrument.
Connect the ADC to your computer (USB) and to ImagePrep (Signal Out, BNC
connector on the rear of the instrument). A detailed description of ADC software
(Data Logger) installation is given in Appendix 2.
3. Password
An instrument specific password is needed to enable method development
access on ImagePrep software. Bruker provides customers with this password on
request: Send the controller ID (see menu Other About) to
maldi.sw.support@bdal.de
. The password must be re-entered with every reboot.
Figure 30 Method development by means of
Analog Digital Converter (ADC)
ADC
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4.2 Interpretation of the Scattered Light Curves
An optical sensor monitors light scattered from the crystalline matrix layer to
control all the relevant preparation parameters in real-time: wetness, matrix
layer thickness, drying rate, deposition periods and intervals (see Figure 31 ).
During the spray period the scattered light intensity quickly fades due to
refraction index matching. This signal drop is proportional to the amount of
liquid applied.
Note: If the sensor signal first drops and then increases during the spray
period, the sample is too wet and too much liquid has been applied. In this case
ImagePrep will apply additional drying time to ensure that the sample is
completely dry before the next spray is applied.
During the incubation phase the sensor signal increases slowly. This is
caused by the almost saturated vapor atmosphere in the chamber.
Drying the wet sample with a stream of nitrogen causes a rapid i ncrease
of the scattered light signal. Once the sample has dried completely the
sensor signal levels off at a higher level compared with the sensor signal before
the spraying cycle. This indicates the growth in matrix layer thickness.
Note: The matrix layer thickness can be determined at complete dryness only.
time
scattered light
intensity
time
scattered light
intensity
Figure
31
Scattered light intensity of a typical spray cycle
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In a typical sample preparation workflow (see Figure 32) the sample is kept
wet for most of the time and dried completely only every 5
th
to 20th cyc le. The
challenge of method development is to keep the sample wet enough, but not
too wet to avoid analyte delocalization.
dry wet
0 10 20 30 40 50 60 70 80
Cycle No.
0 15 30
Time [min]
thick thin
scattered light intensity
Sample
completely
dry
matrix
layer
growth
Sample
too
wet
dry wet
0 10 20 30 40 50 60 70 80
Cycle No.
0 15 30
Time [min]
thick thin
scattered light intensity
Sample
completely
dry
matrix
layer
growth
Sample
too
wet
Figure
32 Scattered light intensity of a complex workflow
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4.3 The Method Development GUI
4.3.1 Method Access windo w
To open the method development GUI, tap Other… in the main menu and
then Method Editor Open… (see Figure 33).
An instrument specific password is needed to enable method development on
the ImagePrep. Bruker provides customers with this password on request: Send
the controller ID (see menu Other About) to maldi.sw.support@bdal.de
. The
password must be re-entered with every reboot.
Figure
33 Opening the Method Access window
Figure
34 Actions in Method Access window
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The Method Access window allows the following actions (see Figure 34):
• Select Method Select a specific method
• Edit method Described in section 4.3.3
• Save method Save method changes
• Start Select the phase number you want to start with and tap
the Start… button
Note: Even a method which has been modified but not saved can be started.
In this case Not yet saved is the method name.
Note: If you want to delete a method, please follow the steps described below:
1. Tap Select… to open the file system.
2. Press on the method you want to delete for more than 2s
to open context menu (equivalent to right mouse button)
3. Tap Delete and confirm.
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4.3.2 Starting Methods for Test Purposes
During the iterative process of method development you may want to test a
method which you have edited. In this case, start the sample preparation directly
from the Method Access window (see Figure 35) without leaving the method
development GUI:
Figure 36
Start sample
preparation
1. Select method.
2. Enter the phase you want to start
with (1 to start from the beginning
of the method).
3. Tap Start…
4. Define allowed customization
(see section 3.5.2).
5. Test and tune the spray power
(see section 3.5.3)
6. Tap Start (see Figure 36).
Note: A method which has been modified but not saved can be started. In this
case Not yet saved will appear in the method name field.
You can pause or abort t he pr epar ation at any tim e. If you pause the preparation
it is possible to switch on the drying gas flow for faster drying.
Note: It is recommended that the preparation is dried completely before
proceeding with the run. This is, in general, a good practice but critical for
proper operation in sensor control mode.
Figure
35 Define
customization and
test spray power
SA_Proteins
Default
SA_Proteins
Default
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The Preparation in Progress window (see
Figure 37) gives valuable process information:
• Current phase (number and name)
• Spray cycle in current phase
• State: What is ImagePrep doing right now?
Detection: Errors & events (See Appendix 1)
• Estimated time remaining
• Matrix layer thickness
(100% corresponds to goal of preparation)
• Automated spray power boost if weak spray is
detected.
4.3.3 Method Editor: Phase Overview Level
Methods consist of one or more phases each with individual spraying,
incubating and drying conditions (see Figure 38). Different phases allow you to
adapt the parameters of the ImagePrep during a matrix preparat ion, i.e. at the
beginning of a preparation it may be desirable to choose more gentle spray
conditions than towards the end of the matrix coating process.
Example:
1
2
3
4
0 10 20 30 40 50 60
Time [min]
Sensor Output [V]
phase 1
phase 2
phase 3
phase 4
phase 5
1
2
3
4
0 10 20 30 40 50 60
Time [min]
Sensor Output [V]
phase 1
phase 2
phase 3
phase 4
phase 5
Figure
37 Information in
Preparation in Progress
window
Figure
38 Individual preparation phases of a method
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Select a method and tap Edit…, the Phases overview window opens (see
Figure 39):
Figure
39 Edit phases in Phases overview window
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This window allows the following actions:
Edit (Method Information)
The info text describes the method as a whole.
The info text usually contains a short description of the application in the first
line (e.g. “SA, Proteins”) and specifies the composition of the recommended
matrix solution (e.g. “10 g/L SA in 60%ACN / 0.2%TFA”) in the second line.
Edit (Phase)
Select a phase from the list. Press Edit Phase… to edit the parameters of the
respective phase. See detailed description in section 4.3.4.
Duplicate (Phase)
Duplicate a phase by selecting a phase from the list and then tapping
Duplicate.
Delete (Phase)
Delete a phase by selecting a phase from the list and then tapping Delete.
Up / Dwn (Phase)
Change the list order by selecting a phase from list and then shifting its position
by tapping Up / Dwn.
Name (Phase)
Edit a phase name by selecting a phase from list and then tapping Name…
Note: There is no “new phase” button! To create a new phase: duplicate an
existing phase, rename this phase and edit it.
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4.3.4 Edit ing a Phase
1. Select the phase to edit in the
Phases overview window.
2. Tap Edit Phase (see Figure 40).
When editing a phase you:
1. Define the final matrix layer thickness desired in that phase
2. Define the spray duration and strength
3. Define an incubation time (without drying gas)
4. Define drying conditions (with drying gas)
The phase editor consists of four windows (see Figu re 41 ) which are described
in detail below.
At the bottom of all four windows you will find:
1
2
3
4
1
2
3
4
Figure
40 Edit Phase button
Figure
41 Actions of the Phase editor
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• Navigation buttons to navigate to the next or previous window,
• An OK button to leave the phase editor and to keep your changes,
• A Cancel button to leave the phase editor and abandon changes.
4.3.4.1 The Matr ix Thickness Window
This window (see Figure 42) specifies the
desired amount of matrix to be applied
within the selected phase (not in the
preparation run as a whole). The amount
of matrix applied can be specified in two
different ways:
• You can either apply a defined number of spray cycles.
• Or use the recommended sensor control mode to set a range (minimum /
maximum number) of cycles and a Final Voltage Difference for the sensor
output. In this case the optical sensor measures the amount of matrix (sensor
voltage) which has been applied since the beginning of the phase. The phase
ends when the sensor signal has increased by the given Final Voltage
Difference, provided that the number of spray cycles is in the defined range.
The phase will also end if the maximum number of spray cycles is reached
even if the Final Voltage Difference criterion is not met.
Figure
42
Matrix Thickness
window
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Figure 43 illustrates the Final Voltage Difference ΔU in the different phases:
Matrix thickness customization
1
2
3
4
0 10 20 30 40 50 60
Time [min]
Sensor Output [V]
phase 1
phase 2
phase 3
phase 4
phase 5
ΔU
1
ΔU
3
ΔU
4
ΔU
5
ΔU
2
1
2
3
4
0 10 20 30 40 50 60
Time [min]
Sensor Output [V]
phase 1
phase 2
phase 3
phase 4
phase 5
ΔU
1
ΔU
3
ΔU
4
ΔU
5
ΔU
2
SA_Proteins
Default
SA_Proteins
Default
0.2
0.5
0.8
SA_Proteins
Default
SA_Proteins
Default
0.2
0.5
0.8
SA_Proteins
Default
SA_Proteins
Default
0.20.2
0.50.5
0.80.8
Figure
43 Final Voltage Difference in different phases
Figu
re 44
Influence of the customize values in a special phase on the
matrix thickness slider in the general customization window
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Matrix thickness customization permits controlling of the thickness of a coating
directly from the AutoPrep window (see Figure 44). If you later want to allow the
customization of the matrix thickness of this phase by using the slider in the
Automatic Matrix Preparation window (see section 3.5.2) you can input a
Customize value for the Final Voltage Difference. The Customize value
directly defines the range for the Matrix Thickness slider in the Automatic
Matrix Preparation window. In the example below the Final Voltage Difference
is set to 0.5 ± 0.3 V which corresponds to the following slider positions: Thin =
0.2 V, Default = 0.5 V, Thick = 0.8 V.
If you do not want matrix thickness variation of this particular phase by chang ing
the general slider position, enter a Customize value of zero.
Please keep in mind that you are able to define the customize value for every
phase. Thus the slider in the Automatic Matrix Preparation window affects all
phases according to the individual customization values. When the slider position
is set to e.g. Thick, ImagePrep applies the individual maximum am ount of m atr ix
in every phase of the preparation. If the customize value for any phase is set to
zero, the slider position will not influence the matrix thickness of these phases.
Table
3 Typical values i n the Matrix Thickness window
Parameter Typical values Comment
Final Voltage Difference
0.05 – 0.6 V
In workflows with multiple phas es .
For reference:
≈1 V: sensor output for empty slide
3-4 V: output for dense matrix layer
Therefore 2-3 V total voltage difference
(sum over all phases) is typical.
Customize
Smaller number than
final voltage difference
Influences the Matrix Thickness slider.
Zero means no customization.
min. / max. number of
cycles
Application dependent
Restrict number of cycles.
Note:
The smaller the range the more
precise the run-time estimation.
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4.3.4.2 The Nebulize Window
The spray parameters to be applied
in a selected phase are set in the
Nebulize window (see Figure 45).
How strong?
How long?
How strong?
How long?
Test the spray.
Switches on the
spray for
3 seconds
Figure
45 Nebulize window parameters
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You have to answer three essential questions:
• What is the desired spray strength?
• What is the desired spray duration?
• Is a special spray sequence required?
How strong? ↔ Sp ray Power
The spray power can be adjusted on a scale from 0 to 100% of the maximum
available spray power.
Furthermore, you can define a power modulation which is a 1.5 Hz-oscillation
of the power in order to get a more homogeneous matrix coverage of the slide.
There is a direct correlation between the range x of the spray and the spray
power P (see Figure 46).
How long? ↔ Spray on-time
• You can either apply a defined spray on-time in this phase.
• Or use the recommended sensor control mode, where the amount of matrix
applied per cycle is controlled by the voltage drop (U
drop
) from the light
scattering sensor. The underlying physical effect is that scattered light
intensity decreases with increasing wetness. Sensor control can therefore
measure actively the wetness of the preparation and control the strength of
sample
sprayhead
<x>
Power
Wobble
±
Δx
+ ΔP
- Δ
P
<P>
sample
sprayhead
<x>
Power
Wobble
±
Δx
+ ΔP
- Δ
P
<P>
Power Modulation
Example:
Power = 50% and Modulation = 20%
means that the spray power varies from 30% to 70%
with a 1.5 Hz frequency.
Figure
46
Correlation between spray range and
spray power
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the spray to achieve the desired wetness of the sample. Matrix application is
stopped when the desired wetness (U
drop
) is reached.
Note: The reference level for the voltage drop is always the preceding dry
level.
Figure 47 below illustrates the sensor controlled mode for the dry workflow
(sample is dried completely after every spray cycle) and the semi-dry workflow
where the sample is kept wet for a number of cycles before complete drying of
the preparation.
As you can see from the figure the lower signal level jitters slightly. This can be
explained by the delay between sensor reading and spray generation. As the
spray process is stopped, remaining aerosol settles onto the preparation
resulting in the observed signal fluctuation (“overspray phenomenon”).
Figure
47 I
llustration of different workflows in
sensor controlled mode
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Spray Sequence EXPERT MODE ONLY
The Spray Sequence sub-
window allows for fine tuning of
the matrix spraying process for
special purposes. Matrix can
either be applied in one
continuous application
(common case) or in a
sequence consisting of
defined on-times and break
times (see Figure 48). In both
cases the spray stops when the
spray on-time criterion is met:
either fixed time or sensor
controlled voltage drop. You
can set three parameters in this
window:
• Wait Before Start
This is the waiting time with no gas flow after drying of the sample has been
performed using drying gas. Keep in mind that drying of the sample causes
turbulences in the chamber that can affect the direction and quality of the
spray. Therefore, a short wait period can be specified allowing the
turbulences in the chamber to calm down. Typical wait time settings are in
the order of 0-2 s.
Note: If you apply a non-zero value for the waiting period in Semidry
Workflows, the drying process continues by slightly exceeding the
residual wetness point defined in the Drying window.
• On-time List
This list contains the sequence of on-times to be used in the spray cycle. For
standard workflows with continuous spraying, the list should have only one
entry which is the maximum allowed spray on-time (20 s).
You can edit the list by selecting entries and changing their values (to enter,
click onto one entry in the list). You can also duplicate entries.
Note: Usually not all entries from the list are used; the spray simply stops
when the spray on-time criterion is met. In the event where the end of
the list is reached and the selected on-time criterion is not met, the
spray will be stopped.
• Break Time: This is the interruption period between two on-times.
Stop
Start
Incubation
Drying
Wait
Spray on
Break
Stop
Start
Drying
Wait
Spray on
Continuous Spray
(Standard)
Special Spray
Sequence
Figure
48
Spray Sequence window
options
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Table
4 Typical values i n the Nebulize window
Parameter Typical values Comment
Spray Power 20-80%
Test spray power and modulation for
homogeneous matrix coverage on slide.
After any exchange of the spray generator, run
the spray head adjustment procedure (see
section 6.1 and section 6.2)
Modulation ≤ Spray Power
Test spray power and Modulation for
homogeneous matrix coverage on slide.
Fixed on-time 1-3 s
Use fixed on-time only in initialization phase
where the sensor control does not work
reliably due to no or low matrix amount on the
slide.
Sensor Control 0.05 -0.5 V
If this value is set too high, you risk flooding
the sample.
General rule:
Lower values for early phases with low matrix
amount on the slide.
Higher values when more matrix is on the
slide.
Spray Sequence:
Wait before start
0-3 s
Try 1s first and choose a waiting time of >1 s
only in cases where spray is deflected b y
drying gas turbulence which might result in
inhomogeneous matrix coverage of the slide.
Spray Sequence:
Spray on-time table
20 s
This should be the only entry for standard
applications with continuous spray.
Note: Short times (<0.1 s) are usually
correlated with shorter spray distance!
Break Time 0.1-0.2 s
Of no importance if spray on-time table
contains one entry only.
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Note: If ImagePrep detects that the spray is too weak, it automatically increases the
spray power. The spray power boost is shown in the Preparation in Progress
window.
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4.3.4.3 The Incuba t ion Window
In this window you can define the incubation time (see Figure 49). During t he
incubation period there is no spray and no active drying with gas flow (see
Figure 50). At this point of the proces s, the spray chamber is almost saturated
with solvent vapor and the sprayed droplets are sitting on top of the tissue.
These conditions provide ideal conditions for extraction of analyte and
incorporation into the growing matrix crystal.
Figure 49 Incubation window Figure 50 Incubati on period
workflow
Incubation time customization
Incubation time can be customized. The Customize value directly defines the
range for the Incubation Time slider in the Automatic Matrix Preparation
window (see section 3.5.2). In the example below the Incubation Time is set to
30 ± 30 s which corresponds to the following slider positions: Short = 0 s,
Default = 30 s, Long = 60 s.
In case you do not want user-selectable incubation time variation, simply select
a Customization value of zero.
Please keep in mind that you are able to define the customize value for every
phase individually. Thus the slider in the Automatic Matrix Preparation
window affects all phases according to their specific Customize values. If the
slider position is set to e.g. Long, ImagePrep applies the individual maximum
incubation time in every phase of the preparation with exception of the phases
where the customize value is set to zero.
wait
spray on
incubation
drying
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Parameter Typical values
Incubation Time 0 – 60 s
Customize < Incubation Time
Table
5 Typical values i n the Incubation window
SA_Proteins
Default
SA_Proteins
Default
0s
30s
60s
Figure
51
Influence of the customize values in a special phase on the
incubation time slider of the general customization window
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4.3.4.4 The Dry Window
The ImagePrep permits active drying of samples using dry Nitrogen. In this step,
remaining liquid present on the surface of the preparation is evaporated. An onboard pressure regulator supplies a constant gas flow of about 3L/min into the
spray chamber. Note that the gas flow does not depend on the external gas
pressure as long as the external pressure is within the specified range (2-5 bar).
wait
spray on
incubation
drying
The drying conditions to be applied in the selected phase are defined in this
window (see Figure 52 ):
• You can either apply a fixed drying time.
• Or use the recommended sensor control mode, where the drying is
stopped when a predefined residual wetness grade is reached. The
wetness grade refers to the voltage drop from the preceding dry level (see
Figure 54).
Choose 0% residual wetness if the sample should dry completely in every
cycle of the selected phase (Dry Workflow).
Choose 1–100% if the next spray should be applied before complete dryness
is reached. In this Semidry Workflow you have the additional option to
insert steps with complete drying of t he sample at a given frequency. In this
case define Complete drying after n
th
cycle.
Note: The matrix layer thickness can only be measured at complete dryness.
Note: Fixed spray on-time is not compatible with the semidry workflow.
Figure
53 Drying phase workflow
Figure
52 Dry window
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Safe Dry is an optional, additional drying period which is subsequently
applied whenever the sensor detects complete dryness of the sample (i.e.
saturation of the scattered light signal). This Safe Drying time ensures that
the total sample dries completely and not only the small area above the
sensor.
Wetness customization
Customization of the residual wetness can be activated using the Customize
value. The Customize value defines the range for the Wetness slider in the
Automatic Matrix Preparation window. In the example below, the Residual
Wetness Grade is set to 40 ± 20% which corresponds to the following slider
positions: Wet = 60%, Default = 40%, Dry= 20% (see Fig ure 55).
If you do not want residual wetness variation influenced by the user, enter a
Customize value of zero.
Please keep in mind that customization can be specified for every phase in a
method. The slider affects all phases, therefore if the slider posit ion is set to Wet,
ImagePrep applies the maximum wetness grade in every phase of the
preparation that the customize value is set to a value other than zero.
Figure
54 Illustration of different drying conditions
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Table
6 Typical values i n the Dry window
Parameter Typical values Comment
Fixed Dry Time 0 – 120 s
Fixed Drying time is not recommended in
automated preparation workflows.
0 if you want no drying.
”Flooded” samples dry within about 5 min.
Residual Wetness
Grade
0-70% 0 for complete drying (dry workflow).
Customize 0-20%
Value must be smaller than residual
wetness RW and 100-RW.
Complete drying
every n cycle
2-15
We recommend drying the sample to
complete dryness at least every 10-15
th
cycles. This allows easy monitoring of the
matrix thickness.
Safe Dry 20-40 s
Figure
55
Influence of the customize values in a special phase on the
wetness slider of the general customization window
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4.4 General Method Development Rules
• If you want to develop a new method, start with an existing method for a
similar matrix solvent (volatility, organic solvent content).
• Initialization phase: At the beginning of a preparation there is no matrix on the
slide. Therefore the sensor control doesn’t work properly: For this reason we
recommend to start each preparation with a dedicated Initialization phase.
During that phase, a sufficient amount of matrix (about 0.4 V Final Voltage
Difference in the Matrix Thickness window) is applied in a “blind mode” (no
sensor control!) with fixed spray time and dry time. Typical parameters can be
found in existing pre-installed methods.
• Hydrophobic surfaces like the ITO coated glass slides can create an inverted
sensor signal for the first few spray cycles, i.e. the scattered light intensity will
increase rather than decrease during spray application (see Figure 58). In order
to prevent this problem, we recommend that a cover slip is placed on the free
slide area on top of the glass slide and over t he sensor window (see Figure 59
and section 3.3). The hydrophilic cover slip surface generates regular sensor
signals from the beginning of the preparation process.
1
2
2
0 5 10 15
Time [min]
Detector Output [V]
inverted
indifferent
regular
Figure
56 Inverted signal during the initialization phase
Figure
57
Inverted signal problem is prevented by using a
cover slip (see section 3.3)
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• After defining the initialization phase, add a number of main phases
(1-5) with an increasing amount of matrix applied per cycle. General rul e: The
more matrix that is on the slide the more matrix can (and should) be applied
without the risk of “flooding” the sample.
• In general, method development is an iterative process:
a) Change parameters of a phase
b) Test the phase by starting the method at this phase
Note: Proper testing requires that the matrix layer has a thickness, which is
typical for this phase, e.g. don’t test the initialization phase on slides
which are already covered with plenty of matrix.
c) Monitor sensor output and sample appearance in parallel (tissue should look
glossy, not too dry, not too wet!).
d) Repeat a-c, if necessary.
e) After having optimized all phases, check MS results for spectra quality (many
peaks?) and analyte dislocation (look at the tissue border). Spectra quality
can simply be judged by comparing the ImagePrep preparation with manual
dried droplet preparation, which usually gives the best results (but, of course,
very poor spatial resolution).
Note: Dried droplet preparation for tissue: Spot 2 x 0.4 µL matrix using a
mechanical pipette, allow drying between both depositions.
• Finally optimize (= minimize!) cycle number range and define
customization values in your new method.
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5 Troubleshooting
5.1 Error Messages
Message The chamber is open.
Cause Side door or glass lid not closed.
Remedy Close side door and glass lid.
If this does not help, please reboot the instrument.
Message The spray intensity is too weak.
Cause This error can only occur if spray on-time is sensor controlled: The scattered
light signal does not decrease fast enough when the spray is switched on. In
this case the software increases the spray power stepwise up to 100%. Only
if this power boost is not sufficient, the preparation will be aborted with this
message. This usually happens if the spray is too weak, because:
a) matrix reservoir is empty
b) air bubble inside flow tube
c) spray generator is clogged (massive crystal formation!)
Remedy a) Check and refill the reservoir.
b) Dismount and remount matrix bottle.
c) Try to decrease matrix concentration and/or increase higher organic
solvent concentration.
Message The oxygen sensor cannot detec t ' n o rmal' O2 concentration.
Cause The oxygen sensor is probably malfunctioning.
Remedy Please contact Bruker service.
Message The nitrogen flow is too low.
Cause a) The nitrogen inlet is not connected properly.
b) The nitrogen pressure is below 2 bar (29 PSI).
Remedy Check both options. If this does not help, please contact Bruker service.
Table
7 Error Messages
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Message The nitrogen flow is too high.
Cause The nitrogen pressure is higher than 8 bar (116 PSI).
Remedy Set pressure between 2-8 bar (29-116 PSI) .
If this does not help, please contact Bruker service.
Message Could not prepare a sufficient amount of matrix in phase xy.
Cause The Final Voltage Difference defined in the Matrix Thickness window
cannot be reached in the respective phase, because
a) The number of spray cycles is too low
b) The spray power is too weak
c) The matrix concentration is too low
Remedy a) Increase the maximum number of cycles in the Matrix Thickness window.
b) Increase the spray power either of the respective phase in the Nebulize
window or increase the Global Spray Power in the Spray Head
Adjustment window. Note: in the latter case, please consider that this
change will be applied to all phases of all methods.
c) Carefully increase matrix concentration, but stay well below saturation.
Message Filling the chamber timed out.
Cause This error occurs if the oxygen concentration in the chamber does not
decrease sufficiently / fast enough during flushing the system with nitrogen.
Potential reasons:
a) The gas inlet is connected to an oxygen source (e.g. pressurized air).
b) There is a leak in the lid or side door seals.
c) The filter is clogged.
Remedy a) Use nitrogen.
b) Check O-ring seals on the lid and the side door.
c) Replace filter (see section 6.9)
Message Could not initialize di g ital I/O ports: Get configuration pin 0 failed
Cause The ImagePrep software has been started twice.
Remedy Reboot the system.
Message Sample too wet (detected during spraying)
Cause This error occurs if the scattered light signal first decreases quickly and then
increases while the spray is still switched on. This happens if the voltage drop
per spray cycle defined in the Nebulize window is too high compared to the
current matrix thickness (see table with typical values in section 4.3.4.2). In
this case the software stops the spray automatically and adds an additional
drying time of 5 minutes.
Remedy Decrease the voltage drop defined in the Nebulize window.
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Message The sample is too wet (detected du ring drying).
Cause This error occurs in semidry workflows if the scattered light signal does not
increase quickly enough du r ing drying the sample. In this case the software
automatically adds up to 5 times 2 minutes of additional drying time. If this is
not sufficient the preparation will be stopped.
Remedy Decrease the voltage drop defined in the Nebulize window and/or the
number of cycles (in the Drying window) after which the sample is dried
completely.
Message Could not create directory: \USB Removable Disk …
Cause Not all folders and files can be written during the creation of a status report
because the USB device has insufficient free space left.
Remedy Make sure that there is enough space on the USB device and repeat the
status report generation.
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5.2 Other Problems
The system does not boot.
• Ensure that no USB device is connected to the instrument; otherwise the ImagePrep
software will not start automatically.
• Check the fuse and replace it, if necessary (see section 6.10).
The spray is too weak.
• Ensure that the spray head has been cleaned properly prior to the preparation.
• Perform the 10% test with methanol (see section 6.2).
If the spray looks similar to that shown in the 10% figure, the global power offset is
correct. In this case increase the spray power in the relevant method (see section
4.3.4.2).
If the spray looks weaker than the pattern shown in the 10%
figure, inner parts of the spray head might have become wet
and a short-circuit has reduced the spray power. This can be
confirmed by measuring the resistance between the two gold
contacts on the spray head (see
Figure 58): An internal
short-circuit would give <1 MΩ resistance. In this case,
please clean and dry the spray head according to the
instructions given in section 3.5.5. After remounting the
spray generator readjust the global power offset (see section
6.2).
There is no spray visible, even at 100% power
• Mechanically check the gold spring contacts (see Figure 58). If one of the spring contacts
gets stuck, try to fix the problem with a drop of oil.
• It may also be that the spring contacts are ok, but they cannot reach their counterparts on
the spray chamber. Check if the o-ring on the spray head opening of the chamber is
properly seated in the slot.
• If this doesn‘t help, contact Bruker service.
Table
8 O t her Problems
Figure
58 Measuring
resistance
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Draining of matrix liquid / spr ay h ead leaking.
• Don’t worry if a few droplets of matrix liquid drain right after mounting a matrix bottle to the
ImagePrep.
• If larger volumes drain from the spray head or if you observe permanent droplet release
please make sure that the matrix bottle is securely fitted to the sealing cone and that the
spray generator is pr oper l y adjusted (s ee section 6.1).
The matrix consumption is too high.
• Usually the matrix consumption per typical preparation is well below 10 mL.
• If the matrix consumption is excessively high, please check for draining of matrix liquid
and follow the suggestions given in the preceding section.
If you intend to prepare a very thick matrix layer exceeding the 10 mL bottle volume, split the
method into two sub methods and refill the matrix reservoir in between.
The coating does not look homogeneous.
• General rule: Larger crystals on the left (right) side of the slide indicate that the spray has
been too weak (strong).
• Check global spray power offset and readjust value, if necessary (see section 6.2).
• If the global spray power offset is ok, readjust the spray power and modulation settings in
the method. Generally, increasing modulation gives better coverage.
• Cause: The sample is too wet errors (see section 5.1) occur.
• Make sure that the spray head is not clogged.
If this does not help, please contact Bruker service.
The sample looks too wet / shows wet sub areas.
• Caution! Analyte molecules can delocalize if the sample becomes too wet.
• Thoroughly observe the sample preparation process and identify the phase(s) when this
occurs in the method development mode.
• Adapt the parameters of the relevant phase: Decrease the voltage drop defined in the
Nebulize window and/or the number of cycles (in the Drying window) after which the
sample is dried completely.
If the area directly above the sensor looks ok but other sub areas on the slide are too wet,
increase the Safe Dry time (see section 4.3.4.4).
The sample is too wet during initi alization phase.
• Usual ly the spra y on-time is not sensor controlled in the initialization phase. Therefore it is
important to adjust the spray on-time and spray power carefully. If the sample is too wet,
decrease the spray on-time.
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A matrix puddle is on the table at the left side of the instrument.
• This is caused by residual liquid in the leaking off the flow channel after having
dismounted the matrix bottle. Tipping the bottle up and down twice before dismounting it
usually solves this problem.
The spectra look nice, but the analyte is delocalized.
• In this case the sample preparation was too wet in one or more phases.
The matrix crystals are too large.
• Over-large matrix crystals usually correspond with preparations that are too wet!
• If the crystals are too large on the left side of the slide, the spray is not strong enough and
the power needs to be increased.
• If the crystals are too large on the right side of the slide, the spray is too strong.
Generally, water-soluble matrices like DHB give larger crystals than water-insoluble matrices.
The spray head clogs.
• Ensure that the spray generator is mounted and adjusted correctly
(section 6.1 and section 6.2).
• Note: If the spray generator is mounted too tightly, it will not vibrate properly and, as a
consequence, it tends to clog.
• Ensure that the spray head has been cleaned properly.
• If the spray head clogs during the initialization phase, the spray power might be too low.
Therefore increase the spray power and decrease the spray on-time.
Note: Strong and short spray pulses are better than weak and long pulses!
• Check whether the matrix solution composition is close to saturation. Generally you can
prevent clogging by decreasing the matrix concentration and by increasing the organic
solvent concentration. Note: In this case you need more spray cycles to reach the same
matrix layer thickness.
The spectra show very few peaks.
• Check manual dried droplet preparation on that tissue: Spot 2 x 0.4 µL matrix using a
mechanical pipette, allow drying between both depositions. If the results improve
significantly, this indicates that the ImagePrep workflow was too dry, i.e. the analyte
extraction was insufficient. If the spectra quality is still poor, this is pointing to the tissue
quality. Try to improve the washing step of the tissue or the tissue preparation protocol in
general (see section 3.2).
Wrong timestamp for the log files.
• Adjust date and time as described in section 6.7.
• In rare cases the internal battery might be dead. In this case, please contact the Bruker
service.
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The drying phase is skipped.
• This is normal if the sample has reached the residual wetness level defined in the relevant
phase during incubation. In this case the drying phase will be skipped automatically.
It can be caused by incompatible sensor controlled drying with fixed spray on-time workflow
(see section 4.3.4.2 and section 4.3.4.4
).
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5.3 FAQ (Frequently Asked Q uesti ons )
Can I perform enzymatic on-tissue digestion with my ImagePrep?
• Yes.
To access detailed instructions and a dedicated method, see Appendix 3.
Can I prepare TLC MALDI plates with my Ima g ePrep?
• Yes.
To access detailed instructions and a dedicated method, see Appendix 3.
What has to be considered if I do not use my ImagePrep for a long period of time?
• Switch off the instrument.
• Dismount the matrix bottle and remove the collecting pan from the spray chamber.
• Clean the spray chamber thoroughly and make sure that everything is completely dry
afterwards.
• Open the side door slightly to allow air circulation.
Can I also cover opaque targets (e.g. metal plates, TLC plates) with matrix in my
ImagePrep?
• Yes, but in this case you must not activate sensor controlling because the sensor is
covered by the opaque targets. Instead, use methods with fixed times for spraying and
drying (e.g. use manual preparation as described in section 3.6)
Can I recrystallize a matrix layer with my ImagePrep?
• Yes. If you want to recrystallize a matrix layer fill the matrix bottle with dedicated
recrystallization solvent and create a method according to your needs. This method
consists of one phase only where you define the fixed number of recrystallization cycles
to be applied, the sensor controlled spray on-time and drying conditions. The method
editor is described in detail in section 4.3.
Table
9 Frequently Asked Questions
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How can I control the atmosphere in the spray chamber?
• The spray chamber is completely decoupled from the outside humidity.
• Zero humidity is reached by drying the samples with nitrogen.
• You can also achieve almost saturated atmosphere which is favorable in workflows where
very long liquid/tissue interaction time periods are needed (e.g. enzymatic on-tissue
digestion). Saturated atmosphere can be realized under conditions where the sample
dries during the incubation time without the need for active drying with nitrogen. Please
choose the following settings:
1. In the Matrix Thickness window:
- fixed number of cycles (> 10)
2. In the Nebulize window:
- sensor controlled spray time
3. In the Incubation window:
- long incubation time (> 5 min)
4. In the Drying window:
- Sensor controlled drying with residual wetness grade of 20-80%
- Complete drying after > 8 cycles.
- Safety dry time of 5 min.
When you look at the scattering light curve you will see that the drying time of the sample
during the incubation will become longer and longer from spray cycle to spray cycle
indicating that the atmos phere is approaching saturation.
Note: In this workflow the time remaining will be substantially overestimated during the
preparation.
How can I ensure that my ImagePrep works well?
• Coat a blank ITO glass slide on its conductive side (Bruker # 8237001) with sinapinic acid
using the ‘Instrument_Test’ method (included in the delivery). Use the matrix recipe given
in the Information window of that method.
The ImagePrep works well if:
(a) no error message shows up during the preparation,
(b) the preparation is successfully finished (messages will show up), and
(c) the matrix coating on the slide looks homogeneous.
My ‘animal’ does not seem to fit into the s p ray chamber. What can I do?
• ImagePrep is a device optimized for matrix coating on standard format microscopic glass
slides (75 x 25 mm).
• For larger tissue samples we recommend that double-size ITO coated glass slides
(75 x 50 mm, BigSlides, 100 pcs, Bruker # 8259387) are used along with the appropriate
adapter target (MTP TLC Adapter, Bruker # 8255595).
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Can I save/archive the scattered light curves and all relevant parameters of my matrix
preparations?
• Yes.
• If you connect the ImagePrep to a PC (see section 4.1) and display the scattered light
curves using the Data Logger software included in delivery, the curves can be archived
with this software.
Note: The Data Logger software cannot archive any ImagePrep methods and
parameters.
• Even if ImagePrep is used as a stand-alone system without connection to a computer, the
scattered light curves and all relevant methods and preparation parameters of the
previous 20 preparations are saved internally and can be copied to a USB storage device
for archiving purposes. For details please refer to section 6.5.
I did not use VI Logging. Can I still get the layer thickness curve displayed?
• Yes. Even if ImagePrep is used as a stand-alone system without connection to a
computer, the scattered light curves and all relevant methods and preparation parameters
for the previous 20 preparations are saved internally and can be copied to a USB storage
device for archiving purposes. For details please refer to section 6.5.
What can I do if the tissue or substra te shows abnormal scattering behavior?
• Blank, hydrophobic substrates show abnormal scattering behavior, i.e. the scattered light
signal will increase rather than decrease upon droplet deposition (see Figure 58 in section
4.4). This is especially true for ITO glass slides before they get coated with matrix. To
prevent this problem we recommend that a cover slip is placed on the free slide area on
top of the glass slide over the sensor window (see section 3.3). The hydrophilic cover slip
surface generates regular sensor signals from the beginning of the preparation process.
Can I “seed” matrix with my ImagePrep?
• Yes, if you use 100% organic solvent and the following parameters the matrix droplets dry
almost completely on the fly and solid matrix flakes are deposited as seed crystals.
1. In the Matrix Thickness window: - fixed number of seed cycles
2. In the Nebulize window: - fixed, very short spray on-time (e.g. 0.2 s)
3. In the Incubation window: - zero incubation time
4. In the Drying window: - fixed drying time (10 s)
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6 Maintenance
6.1 Spray Generator Replacement
Note: If your instrument is equipped with the older version of the spray
head, we recommend that it is upgraded using the Spray Head
Upgrade Kit (# 8261619)
In order to ensure high quality sample preparations, Bruker recommends that
the spray generator (# 8261614) is replaced after every 30 preparations. This
thin perforated metal sheet wears mechanically and chemically, resulting in
larger pinhole diameters and uneven spray. An internal count er will inform you
when the replacement is necessary.
Note: Handle this fragile part with care!
The procedure below shows how to dismount and reassemble the spray
generator (see Figure 59, Figure 60 and Figur e 61).
After completion of the mechanical adjustment, proceed with the global power
adjustment of the new spray generator (see next section 6.2).
A) Dismount the spray cap and the spray generator
1. Unscrew thumbscrew
2. Remove spray cap
3. Open clamp
4. Pull spray generator out of the cap
Figure
59 Dismounting the spray generator
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B) Reassemble the spray generator
1. Position metal sheet on jig.
Note: Correct orientation is mandatory (sharp / rounded corner).
2. Open Clamp.
3. Locate cap on jig, fix metal sheet with finger and slide cap towards the sheet
till it hits the shoulder on the jig.
4. Close Clamp.
5. Finished!
C) Remount the spray cap
Figure
60 Reassembling the spray generator
1. Press cap fully onto spray head.
No further adjustment is necessary!
2. Lock cap position with thumbscrew.
Figure
61
Remounting the
spray cap
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6.2 Global Spray Power Adjustment
The global power adjustment corrects the slight differences between individual
spray generators. Adjust global power adjustment every time the spray generator
is replaced.
The power offset setting is a global correction which is automat ically applied to
all power settings in all methods. If the power offset is chosen correctly, no
changes in previously defined methods are needed.
A global spray power offset can be defined in the spray head adjustment window
(see Figure 62). Open the window by tapping Other… in the main menu and then
Spray Head Adjust….
Follow the instructions given below.
Figure
62 Spray Head Adjustment window
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Step by step instructions to determine the correct offset value
1. Fill matrix reservoir with methanol.
2. Perform 100% test
3. Perform 10% test and adjust offset
4. Reset the counter after mounting a new spray generator.
5. Press OK to apply the offset value to all spray power settings in all
methods.
Click on 100% radio button (Figure 63). The spray can be activated and
deactivated by tapping Spray On.
Verify that the 100% spray reaches the lamp on the opposite side of the chamber.
Verify that there is no matrix liquid dripping from the spray head during this test. If
this is the case, the mechanical adjustment of the spray head is not correct (see
section 6.1).
Click on the 10% radio button (Figure 64).
The spray should look roughly the same as shown in the 10% picture above.
If the spray is stronger or weaker shift the offset until the spray looks ok (Figure 64).
If 25% offset is not enough, contact Bruker service.
F
igure 63 Test Spray 100% radio button
Figure
64 Test Spray 10% and Offset buttons
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6.3 Dismount the Spray Head
The spray head has a sliding lock and can be easily removed for cleaning
purposes without any tools (see Figure 65).
6.4 Installation of Software and Method Updates
Software updates can be performed by copying the relevant files to a USB device
(follow the instructions provided with the update), connecting the device to
ImagePrep and starting the updat e from the Updates window. Open that window
by selecting Other… in the m ain menu and then Updates Install… (see Figure
66).
Methods can be copied to ImagePrep in the same way.
To access the latest methods and software updates, see Appendix 3.
Figure
65 Dismounting the spray head
Figure
66 Software Updates window
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6.5 Generate a Status Report
Status reports contain all relevant information about the current instrument
status. A status report is a directory (named with a time stamp) consisting of:
SysInfo.txt
file
Contains instrument ID, program version …
Logs
directory
Log files (*.log)
… contain information on the most recently performed 20 matrix
preparations (m ethod, c ustom ization, o ptic al s ens or r eadou t, all events
that happened during the preparation …). Each log file represents a
single preparation and, therefore, has its own time stamp. T he optical
sensor readout (la yer thickness curve) ca n be visualized by importing
the respective log file into e.g. Microsoft Excel (Import filter settings:
delimited / space) and plotting column L.
Note: Each line in the table represents a 0.1 s time interval.
Error log files (*.erl)
… contain error events which did not happen during matrix
preparations and as a result will not appear in the log files (*.log).
Methods
directory
Contains all methods currently installed on the ImagePrep.
Settings
directory
Contains non user-accessible instrument settings.
If you want to contact our service via email, it is useful to attach the zipped
status report. Furthermore, you can use status reports for archiving purposes
and for visualization of the optical sensor readout.
Table 10 Information in Status Report
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Status reports can be saved on a USB device connected to the instrument by
starting Status Report… from the Other Functions menu (see Figure 67).
Figure
67 Status Report window
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6.6 System Reboot (see section 3.1)
Use the main switch located on the left side to reboot the system.
6.7 Date / Time Setting
In order to get a correct time stamp name for the log files, set the instrument time
by starting Time and Date Adjust… from the Other Functions menu.
6.8 Touch Panel Stylus
Use the stylus to operate the touch panel especially
to enter keypad characters. A stylus holder is
provided behind the front cover on the right hand side
(see Figure 68).
6.9 Filter Replacement
If the filter pad is clogged it should be replaced
(# 8249088) as shown in Figure 69 by removing the
magnetic ring.
6.10 Fuse Replacement
The AC power inlet at the back of the instrument is equipped with a fuse for
customer and instrument safety. If you have to exchange the fuse it is forbidden
to use fuse types other than Littelf use, type 215, rated 3.15 A, 250 VAC.
Figure
68 Position
of stylus
holder
Figure
69 Replacing
the filter
pad
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6.11 List of Spare Parts
Table
11 List of Spare Parts
Part No Spare part Quantity
# 8261614 Spray Generator II 10 x
# 8249088 Filter Pad 5 x
# 8246375 Matrix Bottle (10 mL, glass) 5 x
# 8237001 Glass Slides for MALDI Imaging 100 x
# 8267942 Coverslips for ImagePrep 200 x
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Appendix 1 Messages in the ‘Preparation in Progress’
Window
State
Initializing Spray power initialization at the beginning of a matrix preparation.
Nebulizing
Spray is switched on.
Incubating Spray and gas flow are switched off.
Drying Nitrogen gas flow is switched on.
Safety-Drying Additional drying time after sensor has detected complete dryness
(see section 4.3.4.4).
WaitingDry Waiting time with no gas flow before spr a y starts (s ee section 4.3.4.2).
Spray and gas flow are switched off.
WaitingNebulize Interruption period (‘break time’) between two spray on-
times (see
section 4.3.4.2). Spray and gas flow are switched off.
Paused Matrix preparation is p aused either manuall y by user or automatically
by software due to detected error.
Table
12 State messages
Figure
70 Preparation in Progress window
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Detection
OK Everything is fine.
NotEnoughMatrix Error ‘Could not prepare a suffic ient amount of m atrix in
phase xy’ detected.
WeakSpray ‘Spray too weak’ detected.
SprayQuantityInsufficient Spray is too weak even after maximum spray power
boost.
NebulizeTimeInsufficient Not eno ugh entries in the ’Spra y Sequence’ list to reac h
the voltage drop defined in the Nebulize window.
Occurs in sensor controlled mode only.
DryInIncubation During incubation the dry criterion has been reached.
The preparation will autom atically continue with the next
nebulize cycle by skipping the drying step.
TooWetNebulizing ‘Sample too wet (detecte d during spra ying)‘ detecte d for
the first time.
TooWetTwiceNebulizing ‘Sample too wet (detecte d during spra ying)‘ detec ted for
the second time.
TooWetDrying ‘Sample too wet (detected during drying)‘ detected.
CouldNotDry ‘Sample too we t (detec te d dur in g dr ying)‘ d etec te d. E v en
5 x 2min additional drying time was not sufficient.
Table
13 Detection messages
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Appendix 2 Software Setup Manual for ADC NI USB-
6008
The ADC converts the analog signal of the
scattered light sensor (0–5 V) to a digital signal
in order to display the scattered light curve on
the laptop computer (see section 4.1). An ADC
(National Instruments NI USB-6008) is
included in the delivery. Connect the ADC to
your computer (USB) and to ImagePrep
(Signal Out, BNC connector on the rear side of
the instrument). Follow the instructions below
to install, to configure and to start the ADC
software.
Software Installation
National Instruments has changed the software on the CDs delivered with the USB6008 device from VI Logger Lite to Signal Express.
Nevertheless, we recommend using VI Logger Lite in order to display the
ImagePrep sensor signal. The ImagePrep user manual refers to VI Logger Lite.
If your CDs are labeled “NI-DAQmx … Also includes: LabView Signal Express”
please follow the instructions given below (see Figure 72):
1. Insert CD1
2. Start Autorun from the FileManager (if necessary)
3. Select Install Software
4. Select all programs from the list for installation except
NI LabVIEW SignalExpress right click deselect)
5. Install software by following the instructions in the wizard
6. Get a free download of VI Logger Lite setup file from
https://lumen.ni.com/nicif/us/evalviloglitedownload/content.xhtml.
7. Inst all NI-DAQ / VI Logger (See the following screenshots. All default settings.)
ADC NI USB-6008
Figure
71
ADC (Analog
Digital Converter)
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Figure
72 Software installation procedure
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Software Configuration: Creating a task
Figure
73 Software configuration procedure
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Starting and Operating the VI Logger Software
Starting VI Logger software:
StartAll ProgramsNational InstrumentsVI LoggerVI Logger (NI-DAQmx)
Archive
New measurement can be started at this level only!
Help
Start / Stop
Figure
74 Overview of VI Logger Software interface
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Appendix 3 ImagePrep Downloads
To access all available ImagePrep downloads:
1. Log in or register and log in to www.bruker.com
.
2. On the www.bruker.com website, go to Service > Support & Upgrades >
Software Downloads.
3. On the Software Downloads page, click Mass Spectrometry.
4. On the Mass Spectrometry Software Support page, click ImagePrep.
Alternatively use this link;
http://www.bruker.com/service/support-upgrades/software-
downloads/mass-spectrometry/imageprep.html.
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