Olympus Fluoview FV1000 User manual

User’s Manual
FLUOVIEW
FV1000
CONFOCAL LASER SCANNING
BIOLOGICAL MICROSCOPE
[OPERATION] FV10-SW Ver 5.0c
This user’s manual is for the software to be run on Olympus FLUOVIEW FV1000 Confocal Laser
Scanning Biological Microscope. To ensure safety, obtain optimum performance and familiarize
yourself fully with this product, we recommend that you study this manual thoroughly before operation.
This user’s manual is composed of two volumes including “OPERATION INSTRUCTIONS” and
“MAINTENANCE”. Together with this manual, please also read the “SAFETY GUIDE”, “HARDWARE
GUIDE” of User’s manual FLUOVIEW FV1000 and the instruction manual of the microscope in order to
understand overall operation methods. To ensure the safety operation of laser system, we recommend
you to study the manual of each laser and the light source equipment besides this manual.
Retain this manual in an easily accessible place near a system for future reference.
Petition
A
X7274

CAUTION
1
Page
CAUTION
1. Reproduction, copying or duplication of a part or all of this software and manual is prohibited.
2. The information described in this manual may be subject to change without notice.
Registered Trademarks
Microsoft, Microsoft Windows, Excel for Windows are registered trademarks of Microsoft Corporation.
Other brand names and product names are trademarks or registered trademarks of their respective owners.

FLUOVIEW MANUAL CONFIGURATION
2Page
FLUOVIEW MANUAL CONFIGURATION
The FLUOVIEW system uses two manuals including this “User’s Manual” and the on-screen
manual built into the software (“Online Help”).
The User’s Manual is composed of the five following volumes and subject matter:
OPERATION INSTRUCTIONS
Describes the operation procedures of the FLUOVIEW system, for example, methods
for image acquisition and various image processing.
1 Getting Started FLUOVIEW................................................................................ 1-1
2 APPLIED OPERATIONS.................................................................................... 2-1
Appendix A List of Hot Keys.................................................................................. A-1
Appendix B Glossary ............................................................................................. B-1
Appendix C USER REGISTRATION OF FV1000 ................................................. C-1
Appendix D Change of Default Folder for [File I/O] Panel..................................... D-1
Appendix E List of Functions in the [Active Overlays] Dialog Box......................... E-1
Appendix F Hand Switch and Microscope Frame Function Allocation .................. F-1
MAINTENANCE
Describes maintenance of the FLUOVIEW system.
1 Software Setup................................................................................................... 1-1
2 Maintenance of Major System Units................................................................... 2-1
3 Setting the Confocal Aperture ............................................................................ 3-1
TROUBLESHOOTING
Describes countermeasures in case trouble occurs.
1 TROUBLESHOOTING GUIDE ........................................................................... 1-1
For Online Help, please see “1-3 Online Help” in “OPERATION INSTRUCTIONS” of this
manual.

NOTATIONS IN THIS MANUAL
3
Page
NOTATIONS IN THIS MANUAL
This manual complies with the following notations.
Notation of Caution, Notes and Tips
Notation Description
Caution to prevent injuries to the user or damage to the
product (including surrounding objects).
NOTE
Note for the user.
TIP
Hint or one-point advice for user reference.
Notation of panel, Command Buttons and Dialog Boxes
Notation Description
[Acquire] panel The name of a panel, dialog box, list box or check box is
enclosed inside square brackets ([ ]).
<OK> button
<Open File> button
The name of a button in a panel or dialog box is enclosed
inside angled brackets (< >).
Notation of Mouse Operations
Notation Description
clicking Action of pressing, then immediately releasing the mouse
button.
double-clicking Action of clicking the mouse button twice in quick succession.
dragging Action of moving the mouse while holding down the mouse
button, then releasing the mouse button at the desired
destination.
(Note) In this manual, clicking, double-clicking and dragging involves pressing the left button
of the mouse, unless otherwise specified.

NOTATIONS IN THIS MANUAL
4Page
Notation of key operations
Notation Description
Enter The name of a key is enclosed inside .
F1
+
Alt The positive sign (+) expresses the combination of more than
one key operation.
For example, F1
+
Alt refers to pressing the
F1 key while holding the Alt key down.
Direction keys Generic names given to the →, ←, ↑and
↓keys.
Notation of system-specific terms
Notation Description
XY observation
(Other observations)
Refers to observation with XY scanning.
(The same principle also applies to other observations such
as XZ, Xt, XYZ, XYt and XYZt.)
Note that some of the panels and dialog boxes shown in this manual are not the precise
reproductions of the originals. Some windows are resized to facilitate the reading and some
grayed-out characters are printed in readable characters.

Software Functional Configuration
5
Page
Software Functional Configuration
This software uses panel-type windows.
Usually, it is required to “select a menu then select the command to be executed” in order to
execute a function provided by software. With the panel system, a software function can be
executed easily by “selecting the panel page tab of the function to be executed”, just like
when using a system notebook or file folder.
Function Window and Image Window
The FLUOVIEW software is organized by two kinds of windows, the function windows and
the image .window.
The function windows include the [Acquire], [File I/O], [Tile], [Process], [Analyze] and
[Visualize] panels.
The image .window shows either the [Live] panel or the panel image loaded from a file
([(filename)] window).
In this manual, the function windows are referred to simply using their
page tabs.
Namely, the [File I/O] panel of function windows is referred to simply as the
[File I/O] panel.
NOTE
The function window
The image window

Software Functional Configuration
6Page
Panel Structure of the Software
This software cannot show the all function panels at a glance.Please use the following list of
the panels for reference in scrolling.
Acquire
Settings
Z Stage
Sets up the image acquisition and executes actual acquisition.
Sets the zooming ratio and observation mode for image acquisition.
Sets the microscope light path for image acquisition.
Sets the Z-direction scanning range for image acquisition.
Time Series Sets the interval period for image acquisition.
File I/O
Tile
Process
Math
Filters
Histogram
Experiment Editor
Analyze
Single
Saves, loads and deletes images.
Changes the image display method.
Processes the acquired images.
Performs mathematical and logical operations between images.
Filters images.
Changes the image contrast.
A
ppends two images, adds and extracts the channel.
A
nalyzes image data.
Obtains the intensity values, intensity distribution, length, area and
average intensity in images.
Orientation
Other Options
Series Obtains the change in the sum of intensity values in images.
Visualize Constructs an image from a different viewpoint or displays a 3D image.
Sets the image rotation angle, direction and number.
Sets the various 3D Rendering.
Dyes Sets the fluorescence dye method for image acquisition and reagent for
each channel.
Lasers Sets the intensity of laser and other option for image acquisition.

Software Functional Configuration
7
Page
Icons Executed by Dragging & Dropping
This software selects image files and observation methods (dye name) by means of
dragging & dropping. This allows simple selection based on an intuitive operation of
“selecting an icon (image file or observation method), dragging it to the desired position and
dropping it there”.

Software Functional Configuration
8Page
Identification of Images Depending on the Observation Methods
On many occasions, FLUOVIEW displays image
icons to allow identification of the observation
method used when each image is acquired. (See
table on the left.)
When the [File I/O], [Tile], [Process], [Analyze] or
[Visualize] panel is selected, the icon of the image
selected in the image window is displayed in a
frame at the top of the function panel. The image
icons are also displayed in the [Icon] field in the
[Files] list box in the [File I/O] panel or during
dragging of an image file.
Use these icons to identify the observation methods
used in image acquisition.
In all observation modes, the icons for 3
or more channels are identical.
Image Icon Significance
XZ observation
XZ observation, 2-channel mode
Xt observation
Xt observation, 2-channel mode
XZT observation
XZT observation, 2-channel
mode
XY observation
XY observation, 2-channel mode
XYt observation
XYt observation, 2-channel
mode
XYZ observation
XYZ observation, 2-channel
mode
XYZt observation
XYZt observation, 2-channel
mode
Point Scan
Animation image
Stereo 3D image: Image to be
viewed with color eyeglasses.
3 or more channels
TIP

O
OP PE ER RA AT TI IO ON N
I
IN NS ST TR RU UC CT TI IO ON NS S
This volume describes the operating procedures of the
FLUOVIEW FV1000 system.
“Getting Started FLUOVIEW” contains information on the basic
operation flow until acquisition of XY images.
“APPLIED OPERATIONS” provides detailed operating
procedures of the system.
Please read this volume so that you can understand the system
before use.
On This Volume

CONTENTS
1 Getting Started FLUOVIEW 1-1
1-1 Basic Operations .............................................................................1-1
1-1-1 Microscope .................................................................................................... 1-1
1-1-2 General Mouse Operation Procedures.......................................................... 1-7
1-1-3 Names of Major Panel and Window Controls and Their Functions............... 1-8
1-2 Outline of LSM Observation Procedures ......................................1-9
1-2-1 Turning Power On ....................................................................................... 1-11
1-2-2 Focusing on the Specimen.......................................................................... 1-12
1-2-2-1 Combination with BX .......................................................................................1-12
1-2-2-2 Combination with IX81 FVF .............................................................................1-14
1-2-3 Setting the LSM Light Path.......................................................................... 1-16
1-2-3-1 Combination with Upright Microscope (BX).....................................................1-16
1-2-3-2 Combination with Inverted Microscope (IX81 FVF) .........................................1-18
1-2-4 Selecting the Dyeing Method ...................................................................... 1-20
1-2-5 Selecting the Filters..................................................................................... 1-23
1-2-6 Setting the ND Filters .................................................................................. 1-26
1-2-7 Setting the Observation Condition............................................................... 1-27
1-2-7-1 Setting the Objective Magnification .................................................................1-27
1-2-7-2 Setting the Zoom Ratio to 1X...........................................................................1-28
1-2-7-3 Setting the Channels .......................................................................................1-28
1-2-7-4 Setting the Highest Scan Speed......................................................................1-29
1-2-7-5 Setting the XY Observation Mode ...................................................................1-30
1-2-7-6 Repeated Scanning Operation ........................................................................1-30
1-2-7-7 Setting the Cross-section to be Observed.......................................................1-31
1-2-7-8 Setting the Area to be Observed .....................................................................1-32
1-2-7-9 Setting a Lower Scan Speed ...........................................................................1-32
1-2-7-10 Stopping Repeated Scanning........................................................................1-33
1-2-8 Acquiring Image ..........................................................................................1-33
1-2-9 Saving Image .............................................................................................. 1-34
1-2-10 Exiting from the Software, Turning Power Off ........................................... 1-35

CONTENTS
1-3 Online Help..................................................................................... 1-36
1-3-1 Function Help ..............................................................................................1-36
1-3-2 Microscope Help..........................................................................................1-37
1-3-2-1 Configuring the Microscope ............................................................................ 1-40
1-3-2-2 Parfocality Correction and Jog Sensitivity Adjustment ................................... 1-46
1-3-2-3 Configuring the Filters (When using a filter system) ....................................... 1-52
1-3-2-4 Configuring the filters (When using a spectral detecting system)................... 1-56
1-3-2-5 Setting the C.A. Diameters ............................................................................. 1-59
2 APPLIED OPERATIONS 2-1
2-1 General Operation Procedure ........................................................ 2-1
2-1-1 Image Acquisition Procedure (Section (A))....................................................2-3
2-1-2 Image Acquisition Procedure in an Observation Mode (Section (B)) ............2-4
2-1-3 Examples of Operation Procedures...............................................................2-5
2-2 Image Acquisition............................................................................ 2-7
2-2-1 Image Acquisition in XY Observation Mode ..................................................2-8
2-2-1-1 Configuring the Microscope .............................................................................. 2-9
2-2-1-2 Setting the Filters ............................................................................................ 2-15
2-2-1-3 Setting the ND Filters ...................................................................................... 2-17
2-2-1-4 Setting the Observation Condition .................................................................. 2-19
2-2-1-5 Acquiring Image .............................................................................................. 2-28
2-2-1-6 Acquiring Image in Accumulation Mode.......................................................... 2-29
2-2-1-7 Saving the Acquired Image in File .................................................................. 2-33
2-2-2 Image Acquisition in Other Observation Modes ..........................................2-34
2-2-2-1 XZ Observation Mode ..................................................................................... 2-34
2-2-2-2 XT Observation Mode ..................................................................................... 2-38
2-2-2-3 XZT Observation Mode ................................................................................... 2-40
2-2-2-4 XYZ Observation Mode................................................................................... 2-46
2-2-2-5 XYT Observation Mode................................................................................... 2-51
2-2-2-6 XYZT Observation Mode ................................................................................ 2-55
2-2-3 Differences in Image Acquisition Method Between Fluorescent and Transmitted

CONTENTS
Images......................................................................................................... 2-61
2-2-3-1 Monochrome Image.........................................................................................2-61
2-2-3-2 Dual-Fluorochrome Image ...............................................................................2-64
2-2-3-3 Transmitted Image...........................................................................................2-67
2-2-4 Image Acquisition by Rotating It (Rotation Scan)........................................ 2-71
2-2-5 Image Acquisition of Only the Rectangular Position (Clip Scan)................. 2-72
2-2-6 Image Acquisition by Magnifying the Rectangular Position (Zoom-In Scan)2-75
2-2-7 High-Speed Image Acquisition .................................................................... 2-78
2-2-8 Image acquisition to prevent crosstalk between fluorescence (Sequential Scan)
.................................................................................................................... 2-79
2-2-8-1 Virtual Channel Function .................................................................................2-82
2-2-9 Image Acquisition of a Line at Desired Angle.............................................. 2-88
2-2-10 Display the change of image intensity (Point Scan) .................................. 2-89
2-2-11 Image Acquisition on Desired Line (XZ, XT or XZT Observation)............. 2-94
2-2-12 Image Acquisition in the Laser Excitation Mode........................................ 2-97
2-2-12-1 Making REX Mask File ..................................................................................2-98
2-2-12-2 Example of FRAP experiment .....................................................................2-103
2-2-13 Notes for image acquisition ..................................................................... 2-112
2-2-13-1 Memory of setting information for scanning region......................................2-112
2-3 Saving, Opening and Shredding Images...................................2-113
2-3-1 Saving Images........................................................................................... 2-116
2-3-1-1 Saving Images As a Series............................................................................2-116
2-3-1-2 Saving a Display ............................................................................................2-118
2-3-1-3 Saving Specified Area of Image ....................................................................2-120
2-3-1-4 Saving Animation Images ..............................................................................2-123
2-3-1-5 File Types Available for Save ........................................................................2-125
2-3-2 Opening Previously Saved Images ........................................................... 2-132
2-3-3 Shredding Images ..................................................................................... 2-133
2-3-4 Saving Comment Together with Image .....................................................2-135
2-3-5 Checking the Image Information/Acquisition Parameters.......................... 2-138
2-3-6 Saving the Image Information/Observation Condition............................... 2-142
2-3-7 Saving/Reading the Region File................................................................ 2-145

CONTENTS
2-3-7-1 Saving the Region File .................................................................................. 2-145
2-3-7-2 Reading the Region File ............................................................................... 2-147
2-4 Protocol processor...................................................................... 2-150
2-4-1 Starting the Protocol Processor.................................................................2-151
2-4-2 Editing the Protocol ...................................................................................2-152
2-4-2-1 Description of Setting Items .......................................................................... 2-153
2-4-2-2 Command List ............................................................................................... 2-160
2-4-2-3 Protocol Repetition Processing..................................................................... 2-165
2-4-2-4 Input supporting function............................................................................... 2-167
2-4-2-5 Protocol procedure supporting function ........................................................ 2-169
2-4-3 Saving the Protocol....................................................................................2-170
2-4-4 Executing the Protocol...............................................................................2-172
2-4-5 Loading a Protocol.....................................................................................2-174
2-4-6 Loading a protocol of previous format .......................................................2-175
2-4-7 COM Communication function...................................................................2-177
2-4-8 Example of assembling a protocol.............................................................2-178
2-4-8-1 Example of Setting Procedure of FRAP Observation ................................... 2-178
2-4-8-2 Example of Setting Procedure of XYT Observation for hours (Protocol using
repetition processing) ........................................................................................... 2-187
2-4-9 Pop-up Menu .............................................................................................2-195
2-4-10 Restrictions of [PAPP] setting..................................................................2-196
2-4-10-1 Restrictions with [Mode] and [Sub Mode] ................................................... 2-196
2-5 Changing the Image Display Method......................................... 2-198
2-5-1 Displaying an Image in Simulated Colors ..................................................2-198
2-5-2 Editing the LUT (Look Up Table) ...............................................................2-200
2-5-2-1 LUT Graph Editing According to Colors........................................................ 2-200
2-5-2-2 LUT Graph Editing by Gamma Correction.................................................... 2-202
2-5-3 Switching the Displayed Channels (Ch1 – Ch5)........................................2-203
2-5-4 Displaying Images of Multiple Channels Simultaneously (Side By Side Views,
Over And Under Views, Single View) ........................................................2-204
2-5-4-1 Displaying Images Separately Per Channel (Side By Side Views, Over And Under
Views) ................................................................................................................... 2-205

CONTENTS
2-5-4-2 Displaying Merged Image of Multiple Channels (Single View)......................2-206
2-5-5 Changing the Number of Divided Images ................................................. 2-208
2-5-5-1 Increasing the Number of Divided Images ....................................................2-208
2-5-5-2 Decreasing the Number of Divided Images...................................................2-210
2-5-6 Switching the Display Method of Multiple Images ..................................... 2-211
2-5-7 Displaying Multiple Image Slices Together ............................................... 2-213
2-5-7-1 Displaying Multiple Images Per Channel.......................................................2-214
2-5-7-2 Displaying Images of Two Channels Together..............................................2-216
2-5-7-3 Displaying Time-Lapse Images .....................................................................2-217
2-5-7-4 Displaying Multiple Multiple sections Images ................................................2-218
2-5-7-5 Displaying Same Images in Different Display Methods.................................2-218
2-5-7-6 Re-arranging Images Using the Same Display Method ................................2-221
2-5-7-7 Displaying Different Images Together ...........................................................2-221
2-5-8 Magnifying/Reducing an Image................................................................. 2-224
2-6 Image Processing ........................................................................2-225
2-6-1 Filtering...................................................................................................... 2-225
2-6-1-1 Contour Enhancement...................................................................................2-226
2-6-1-2 Noise Reduction ............................................................................................2-229
2-6-1-3 Image Sharpening .........................................................................................2-230
2-6-1-4 DIC Correcting DIC Level Irregularities .........................................................2-231
2-6-2 Contrast Conversion.................................................................................. 2-233
2-6-3 Mathematical Operations Between Images............................................... 2-236
2-6-3-1 Image Addition...............................................................................................2-236
2-6-3-2 Image Subtraction..........................................................................................2-239
2-6-3-3 Image Multiplication .......................................................................................2-239
2-6-3-4 Image Division ...............................................................................................2-240
2-6-3-5 NOT Image ....................................................................................................2-241
2-6-3-6 Image AND Image .........................................................................................2-243
2-6-3-7 Image OR Image ...........................................................................................2-244
2-6-3-8 Image XOR Image .........................................................................................2-244
2-6-4 Brightness overlap level between 2 channels (Colocalization).................. 2-246
2-6-4-1 Annotation Mode............................................................................................2-247

CONTENTS
2-6-4-2 Colocalization for series image data set ....................................................... 2-254
2-6-4-3 Image measurement ..................................................................................... 2-255
2-6-5 Appending image (Append).......................................................................2-258
2-6-5-1 Appending two images.................................................................................. 2-258
2-6-5-2 Appending image from several image data set ............................................ 2-262
2-6-6 Extract image (Crop)..................................................................................2-267
2-7 Image Analysis............................................................................. 2-274
2-7-1 Checking the Intensity of a Specific Part ...................................................2-275
2-7-1-1 Intensity Values on a Line (Line Profile) ....................................................... 2-275
2-7-1-2 Intensity Values on a Planar Region (Bird’s Eye View) ................................ 2-278
2-7-2 Checking the Intensity Distribution of a Specific Part ................................2-283
2-7-2-1 Intensity Distribution on a Line (Histogram) .................................................. 2-283
2-7-2-2 Intensity Distribution on a Planar Region (Histogram).................................. 2-284
2-7-3 Image Measurement..................................................................................2-286
2-7-3-1 Length Measurement .................................................................................... 2-286
2-7-3-2 Area Measurement........................................................................................ 2-286
2-7-3-3 Measuring the Change in Mean Value of Intensity ....................................... 2-287
2-7-3-4 Measuring the Change in Integrated Intensity .............................................. 2-293
2-8 Building an Image from a Different Viewpoint.......................... 2-295
2-8-1 Building Extended Focus Image from XYZ Image.....................................2-295
2-8-1-1 Display Switching to Built Image................................................................... 2-295
2-8-1-2 Turning Built Image into Single Image .......................................................... 2-298
2-8-1-3 Turning Built Image into time series image................................................... 2-300
2-8-2 Building line images to be viewed in Z direction........................................2-301
2-9 Viewing 3D Image ........................................................................ 2-305
2-9-1 Successive Display of Images...................................................................2-307
2-9-1-1 Changing the Successive Display Speed ..................................................... 2-308
2-9-1-2 Changing the successive image display position ......................................... 2-309
2-9-2 Animation...................................................................................................2-310
2-9-3 Building Stereo 3D Images........................................................................2-312
2-9-4 Building a 3D Image to be Viewed Through Color (Red/Green) Eyeglasses2-314
2-10 Viewing Images Following the Progress of Time................... 2-316

CONTENTS
2-10-1 Displaying Images Together.................................................................... 2-316
2-10-2 Displaying Images Successively ............................................................. 2-316
2-11 Transferring Data to Another Application...............................2-319
2-11-1 Transferring Analysis Data to Another Application.................................. 2-319
2-11-2 Transferring the Plot Image of Analysis Data to Another Application...... 2-322
2-11-3 Transferring Image Data to Another Application (Paint, etc.).................. 2-323
2-12 Entering Comment in Image .....................................................2-324
2-12-1 Writing Characters in Image.................................................................... 2-324
2-12-2 Displaying the Image Intensity ................................................................ 2-326
2-12-3 Displaying the X-coordinate/Y-coordinate of the Image .......................... 2-327
2-12-4 Drawing a Figure in Image ...................................................................... 2-328
2-12-5 Drawing a Scale in Image ....................................................................... 2-329
2-12-6 Drawing an Arrow in Image ..................................................................... 2-330
2-12-7 Drawing Color Bars in Image .................................................................. 2-331
2-12-8 Deleting Comment................................................................................... 2-332
2-12-9 Moving Comment .................................................................................... 2-333
2-12-10 Changing the Comment Size ................................................................ 2-334
2-12-11 Changing the Comment Color............................................................... 2-335
2-12-12 Changing the Comment Font ................................................................ 2-336
2-13 Image Output at Printer.............................................................2-337
2-14 Merger/Extraction of Image Channels .....................................2-338
2-14-1 Setting the Range of Multiple Image Slices............................................. 2-338
2-14-2 Merging Image Channels ........................................................................ 2-339
2-14-3 Extracting Channels from Image ............................................................. 2-344
2-15 Changing the Chart Display Method........................................2-348
2-15-1 [Chart] Panel ...........................................................................................2-348
2-15-2 [Series] Panel .......................................................................................... 2-356
2-16 Pop-up Menus ............................................................................2-359
Appendix A List of Hot Keys A-1

CONTENTS
Appendix B Glossary B-1
Appendix C USER REGISTRATION OF FV1000 C-1
Appendix C-1 User Registration.......................................................... C-1
Appendix C-2 Logging into the FV1000.............................................. C-4
Appendix C-3 Deleting a User ............................................................. C-5
Appendix D Change of Default Folder for [File I/O] Panel
D-1
Appendix E List of Functions in the [Active Overlays]
Dialog Box E-1
Appendix E-1 Coordinate Position Data..............................................E-1
Appendix E-1-1 X-Coordinate ................................................................................ E-1
Appendix E-1-2 Y-Coordinate ................................................................................ E-2
Appendix E-1-3 Other ............................................................................................ E-3
1 Z Position....................................................................................................................E-3
2 T Position....................................................................................................................E-3
3 Animation....................................................................................................................E-3
Appendix E-2 Intensity Data .................................................................E-4
Appendix E-3 Other ...............................................................................E-5
Appendix E-3-1 Channel Number .......................................................................... E-5
Appendix E-3-2 Objective Power ........................................................................... E-5
Appendix E-3-3 Date of Image Capturing .............................................................. E-5
Appendix E-3-4 Time of Image Capturing.............................................................. E-5
Appendix E-3-5 Image File Name .......................................................................... E-5
Appendix F Hand Switch and Microscope Frame Function