Instructions for Olympus-Upright Table of contents

Instructions for Olympus-Upright Table of contents
2017-06-21
INSTRUCTIONS OLYMPUS BX63 UPRIGHT 2017-05-24.DOCX
Instructions for Olympus-Upright
Table of contents
Start ................................................................................................................................................................. 2
Microscope: ...................................................................................................................................................... 3
Camera:............................................................................................................................................................ 3
Light path: ........................................................................................................................................................ 3
Objectives in nosepiece: .................................................................................................................................... 3
Mirrors (filtersets): ............................................................................................................................................ 4
Software........................................................................................................................................................... 7
Acquisition ....................................................................................................................................................... 7
RGB/monochrome. ........................................................................................................................................... 7
White balance ................................................................................................................................................... 8
Saturation/black level ........................................................................................................................................ 8
Line profile ....................................................................................................................................................... 8
Saving options................................................................................................................................................... 9
For single channel ............................................................................................................................................. 9
For multichannel/multidimensional images: ...................................................................................................... 10
Z sections ....................................................................................................................................................... 11
Time lapse ...................................................................................................................................................... 11
Tiling .............................................................................................................................................................. 12
Save/Export/Batch .......................................................................................................................................... 12
Convert to 8 bit flat TIFF .................................................................................................................................. 13
Histogram ....................................................................................................................................................... 15
Experiment Manager ....................................................................................................................................... 16
Scale bar......................................................................................................................................................... 18
Polarized light microscopy................................................................................................................................ 19
Backup files .................................................................................................................................................... 20
Shut down ...................................................................................................................................................... 20
Start
In this order:
(1) X-cite lamp
Ordinarily the computer, controller and touch pad are on in standby mode;
to start tap the touch pad if it is on.
Otherwise turn on:
(2) computer
(3) BX3-CBH (after power failure it turns itself on to standby)
(4) touch pad (after power failure it does not turn on)
Tap touchpad to activate, press on “full operation” after it becomes active

Open the X-cite lamp shutter when it becomes active

p. 2
Microscope:
Upright Olympus BX63.
Stage: manual xy
Nosepiece: mechanical z
Condensor NA
Objectives are changed manually.
Filterset cubes are changed mechanically.
Camera:
DP73 is a 17.28 megapixel color CCD (2 megapixel with pixel shifting), 12bit
1600x1200 pixel images at 15 fps, 800x600 at 27fps
Light path:
Halfway for ocular, all the way out for camera
Objectives in nosepiece:
Magnification
PlanApoN
UPlanFLN
UPlanFLN
UPlanFLN
UPlanSApo
UPlanSApo
2x
10x
20x
40x
60x
100x
Immersion
NA
Color correction
oil
oil
0.08
0.30
0.50
0.75
1.35
1.4
∞
∞
∞
∞
∞
∞
When working with the 2x objective, remove the conderser dry top lens from the path.
p. 3
Working
distance
0.17
0.17
0.17
0.17
On the Touchpad
To focus use the up and down arrows.
Range from 0-20,000, focus is at about 19,100.
On the microscope
Focus knob
Mirrors (filtersets):
Position
1
2
3
4
5
Button
DAPI
GFP
mCherry
Brightfield
Analyzer
Filter set
U-FUNA
U-FBNA
U-FGNA
-
Excitation
BP 360-370
BP 470-495
BP 540-550
p. 4
Dichroic
DM 410
DM 505
DM 570
Emission
BA 420-460
BA 510-550
BA 575-625
Fluorophore family
DAPI
GFP, Cy2, Alexa 488
Cy3, Alexa 555, mCherry
On the Touchpad
TRANSMITTED LIGHT
DIA tab (diascopic = transmitted)
On the microscope
DIABrightness tab:
Use the arrows to adjust lamp voltage:
Halogen brightness
DIAIris tab:
FS, field stop, use for Kohler
EPIFLUORESCENCE
EPI tab (epifluorescence)
Use the buttons to navigate the mirrors (filter cubes)
Change filter cubes
and open/close shutter
Open/close FL shutter
p. 5
To alternate transmitted/reflected use these buttons:
p. 6
Software
Use the Bookitlab dialogue to activate your reservation; this will open the Cell Sens Dimension software
In start page  Device list choose BX63
If you do not see tabs Camera control and Microscope control do
layout  reset layout
Settings (shutters, condenser, focus, turret filter cube, lamp voltage, aperture stops) can also be adjusted on
[Microscope control] panel (right panel)
[microscope manager] tab
Acquisition
top tab
In the left panel [Camera control]:
Pull out the light path rod
Use Live to start
RGB/monochrome.
Press to toggle.
(For fluorescence and fluorescence+transmitted choose monochrome)
When using brightfield in RGB mode, adjust
p. 7
White balance
Saturation/black level
Monitor by toggling <ctrl-H> or
Line profile
Or use line profile to monitor saturation
(appears below the image)
------------------------------------------------------------------------------------------------Set ISO to 100
For weak signal you can change to a higher ISO, however the noise will be higher
------------------------------------------------------------------------------------------------Set exposure time
-------------------------------------------------------------------------------------------------
p. 8
Set pixel size, for snapshot 4800 x 3600 (pixel shift) or 1600 x 1200
For movie 1600 x 1200
Saving options
In [Process Manager] tab
Set saving options, name and location of new images (USERS DATAYYYYMM-YYYYPI-name).
For single channel
Press Snapshot
Besides saving the file (FileSave) in the native VSI format you can also export the image to tiff:
do File  Save as  Tiff for further processing in image analysis programs.
p. 9
For multichannel/multidimensional images:
In the right panel - bottom:
Choose the tab [process manager]
------------------------------------------------------------------------------------------------Press automatic processes.
------------------------------------------------------------------------------------------------Automatic process modules:
Channels - Z sections - Time lapse - Autofocus
------------------------------------------------------------------------------------------------In Channels:
Check/uncheck “Aquire” for individual channels
For each channel, after setting all parameters in Camera Control (right panel), in the channels tab, press [Read
Settings].
-------------------------------------------------------------------------------------------------
p. 10
Z sections
In [Process manager] panel
Choose [Top and Bottom], focus to the desired top or bottom and press [Set].
Choose step size and consider automatically generating “Extended Focal Imaging”
------------------------------------------------------------------------------------------------Time lapse
In [Process manager] panel, set interval and number of cycles.
Press Start
-------------------------------------------------------------------------------------------------
p. 11
Tiling
In Process Manager, choose Manual Process
Choose Instant MIA and Overwrite pixels
Press start
Press start again
Move the stage slowly to cover the desired area. As long as the frame is turquoise you are doing fine. If you pass an
area twice the software will determine which iteration was better quality and overwrite.
If you go over an area with little data or too fast the frame will become fuchsia and you there is a chance you will not
be able to recover your path. You can always finish.
To finish press stop.
------------------------------------------------------------------------------------------------Save/Export/Batch
The multidimensional file you save will consist of a native VSI file and a folder of the same name. These should stay
in the same folder and their names should be identical and not changed later, even if to identical.
To open in FIJI download the Olympus Viewer Plugin
http://imagej.net/OlympusImageJPlugin
p. 12
Besides saving the file (FileSave) in the native VSI format you can also export the image to tiff:
do File  Export  Tiff for further processing in image analysis programs.
For batch export to tiff,
If files are RGB, 8 bit use the Save_as_TIFF macro:
ViewTool WindowsMacro Manager
Select batch mode:
Convert to 8 bit flat TIFF
If files are 12 bit channel stacks use the 12-bit-to-8-bit-flat-tiff macro:
Disregard warnings
Set input folder and output folders.
If the file names are the same, including subfolders will not help!!!
If file names are the same you will have to convert each folder separately!
p. 13
Set input folder:
Press Next
Set output folder and parameters:
You can define a new output folder on the fly.
Press Next
Press Start
p. 14
Disregard error messages
------------------------------------------------------------------------------------------------Histogram
select the tab [Adjustments]:
p. 15
Experiment Manager
(Intelligent way to save a multidimensional imaging configuration)
To make a new experiment:
Choose Experiment Manager tab (right panel, below)
Click on [New]
In the upper ruler click on Multichannel Group (
) and in the central panel draw a container (rectangle).
Click on Image Acquisition (
) and click inside the container.
Repeat for the number of remaining channels you need.


You can add Z-sections and Time Lapse by clicking on the appropriate sign on the ruler and drawing around the
channel container.
Click on Live either on the right panel or the left panel:
A new tab will appear behind the experiment in design with the Live window.
You can see both windows simultaneously by using WindownSplit/Unsplit
p. 16
From the microscope touchpad choose a channel
Set exposure, sensitivity, resolution…
Select a channel (yellow perimeter=selected), right click  [Get settings] to apply the current settings to the
selected channel.
p. 17
The option [Apply settings] will change the settings of the Live window to the settings stored on the channel.
A number of such experimental configurations can be set up consecutively.
Save the experimental setup to an .oex file
To start the experiment click on [Start]
Scale bar
To add scale bar: View scalebar
To burn scalebar, take snapshot and then image  burn info
p. 18
Polarized light microscopy
In the turret, choose the analyzer (no. 5) (EPI tab)
Turn the halogen lamp brightness to 5 (DIA tab  brightness)
Rotate the polarizer manually to exclude directly transmitted light
p. 19
Backup files
Move your files from the local USERS DATA folder to the server to access from any computer in the building
Please do not use a USB flash disk on this computer.
We have three computer in the Analysis room that have active virus protection for this purpose.
Shut down
(1) Quit CellSens
(2) Log off from Bookitlab
(3) On the touchpad press Off and again Off and then OK
(4) Turn off the X-cite lamp
(5) Cover microscope
p. 20
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