Preview unit; iSaCS
For technicians and
partly for sales managers!
To find the position of the entire tissue to be
scanned or parts of it, a preview of the slide is made. The tissue is defined as
a darker part of the slide in relation to its surrounding. Because dust, grease
or fingerprints (specks) fulfills often the same requirement as the tissue
itself; specks may be viewed as tissue also. Therefore, the surface of the
slide and the cover slip should be as clean as possible during capturing the
preview.
In the PCON, as in the other scanners also, the
preview unit is situated on the “Horseshoe” and the preview illumination unit.
The
unit is designed to fulfill the following 3 tasks:
- Create the brightfield preview
- Create the darkfield preview
- Reading barcodes
· The
unit is derived from the scanner P250
· Software relevant
explanations are based on the software version 1.19.
Contents
- Service program for Pannoramic scanners
and actual license file
- Pannoramic scanner software
and actual license file or dongle
- Pannoramic
Viewer software and actual license file or dongle
- The
test slide #2
- 0.9, 1.5, 2.5, 3 hex key wrenches
- Deeper knowledge of handling the Pannoramic scan
and Pannoramic Viewer software.
- Already installed
VRmagic driver.
The version numbers of the service program
“SlideScannerService.exe” and the scan program “SlideScanner.exe” are
important, because the preview calibration program uses program parts (.dll-files)
of the program “SlideScanner.exe”.
Attention: Do not mix the versions of SlideScanner.exe and SlideScannerService.exe!
Always use these programs with the same version number; otherwise the
SlideScannerService.exe program could produce unwanted results and
SlideScanner.exe does not work correctly or even freeze!
· The
path of the file MicroscopeConfiguration.ini, in the software version with the
operating system Windows® 7 is:
C:\ProgramData\3DHISTECH\SlideScanner\MicroscopeConfiguration.ini
[Microscope]; some settings are incorrect
SerialNumber=iSaCS_xxx
MicroscopeType=3DMic10
MicroscopeSubtype=Confocal
ScanCameraType=
PreviewCameraType=CVrmc_m8_pPro
BarcodeReaderType=PreviewCamera
LoaderType=SL_1Mag_12Slide_Sensor_Horizontal2
CameraChangerType=CC_none
ReflectorTurretType=RT_None
BrightfieldLightSourceType=RGBLedLight
ObjectiveChangerType=OC_2Pos
ObjectGuideXYZType=OGXYZ_FLASH4
FlashUnitType=NoFlashUnit
NDFilterType=NDType_None
PreviewLightType=PreviewLightUnitType_Type2
PowerSwitchBoardType=PowerSwitchBoard_Type1
Remark
Depending on the presence of the darkfield preview
illumination there are two parameter values possible in the section
[Microscope].
PreviewLightType=PreviewLightUnitType_Type1; if the darkfield preview illumination
unit is removed; this disables also the entire darkfield unit.
PreviewLightType=PreviewLightUnitType_Type2; if the darkfield preview illumination
unit is present; this enables the creation of the darkfield preview also.
· For
more detailed information please refer to the chapter “Section [Microscope]”
In the P250, as in the other scanners also, the entire
preview unit is situated on the “horseshoe” and the preview illumination unit;
but dimensions and the construction was modified, in relation to
the Pannoramic SCAN.
The entire preview
unit is a part of the scanner unit, and contains the following components:
- Horseshoe; it contains
the mountings for the preview unit
- Objective
and camera holder
- Preview
camera VRmc-8+ PRO
- Preview
objective, TAMRON
23FM16SP
- Preview mirror
- Barcode illumination
The preview mirror reflects
the image of the preview in an angle of 90 degrees; the mirror does not need
adjustments.
Preview objective and
camera holder
The
preview objective and camera holder allows the rotation of the preview camera
in a limited range; so the preview camera rotation angle will be adjusted.
To adjust the camera rotation angle:
- Hold the camera from beneath (the rear of the camera) and loosen
the position fixing bolt a little bit until the camera becomes barely
moveable.
- By loosening one of the “Rotation angle” bolts and tightening the
opposite bolt, the rotation angle will be modified.
- If the correct camera rotation angle is found, tighten the
“Position fixing” bolt; further information can be found in the chapter “Adjust the
preview camera rotation angle”.
In the
scanner P250 the preview camera VRmc-8+ PRO
is used to create the brightfield and darkfield preview and also
to capture the barcode.
Because
the camera has 3 different tasks, each kind of image is illuminated from
another illumination source and these are of a different kind also, the
adjustment of the aperture iris size and the focus position of the objective
are important.
- The distance ring has a thickness of 1.5 mm nominal.
- The shown focus value is for information only, the real value will
be found during the preview calibration process.
The barcode illumination consists of four LED’s and
these are situated on a small PCB.
To reach an evenly and bright illuminated barcode
area, the position of the LED’s can be modified by positioning the entire PCB
if the mounting bolts are loosened or by bending the LED’s carefully,
separately.
The brightfield illumination
is used to make the tissue visible for the brightfield scan
procedure during the darkfield illumination should be used to make
the preview for the fluorescent scan procedure.
- The darkfield preview can be used for
the brightfield scan operation any time also, but, because it is
a time-consuming operation, we recommend to using it only if the tissue is
very low in contrast.
Remark:
The darkfield illumination is not implemented if the
P250 is configured for brightfield scan only!
Construction, mountings and connections
The entire
preview illumination unit is mounted with two unit mounting bolts to the
edge of the scanner unit plate, above of the horseshoe mounting.
The brightfield illumination
creates a white illumination field in the size of the preview camera’s
field of view; the
position of the illumination field can be adjusted in a small range.- The brightfield illumination is
connected with the wire BGJ-1.
The darkfield illumination
unit is mounted in an angle
of nearly 45 degrees in relation to the slide bottom; only the focus
position of the illumination beam line have to be adjusted.- To adjust the focus position, loosen the focus position fixing bolt
of the darkfield illumination unit with a 0.9 mm hex key
wrench first, then drive the focus position adjustment bolt in or out;
detailed adjustment procedures are described later.
- The darkfield illumination is connected with the wires
DLJ-1 and DLJ-2, these connections are interchangeable to each other.
Remark:
The darkfield illumination is not implemented if the
P250 is configured for brightfield scan only!
Principle of preview creation
Brightfield illuminated
preview
The brightfield part
of the preview illumination unit illuminates the background of the entire
field of view of the preview camera, behind the slide.- Because the slide is much larger then the field of the preview
camera’s view, the scan area
of the slide is captured in 3 sections; the made 3 images are rotated, cut
and assembled by software.
- The required parameters are defined during the preview calibration
process and collected in the section [PreviewAndBarcodeScanning] of
the file MicroscopeConfiguration.ini.
- The assembled preview, the scan area of the slide, is shown in the
preview window of the slide scanner program.
“Scan
area” and “Barcode area”
If the
light beam line arrives as a thin line to the slide in an angle of nearly 45
degrees, the slide bottom and the slide top surfaces are working as a mirror
and many light of the light source will be reflected. The remaining low amount
of light illuminates the tissue, the exposure time of the preview camera rose
up to see the low amount of light and this is also the reason for the
sensitivity of stray light (ambient light).
The light
beam line illuminates one line of the slide bottom and the slide top also.- If there is a tissue (or dust), the light beam line will be white and
light arrives to the preview camera at the appropriate part. On the parts
where only glass is, the beam will be reflected; therefore these positions
are dark (black). In other words, the tissue (and the dust also) is shown
in white, all other parts of the slide are shown in black.
- To reach a black tissue part on a white background, black and white
(the gray scale) will be inverted by the software.
- The light beam illuminates the center of the preview camera’s field
of view; so many kind of distortion is minimized.
- The beam line is less than 0.5 mm in thickness and over
200 images are made to assemble the entire scan area and so the
tissue; this is the time-consuming procedure during capturing
the darkfield preview.
- During the darkfield calibration
procedure, the position and the rotation angle of the beam line is
defined; both parameters are situated in the section [PreviewAndBarcodeScanning]
of the file MicroscopeConfiguration.ini.
- The line-wise captured tissue (the entire scan area) is assembled
to a preview and shown in the preview window of the program
SlideScanner.exe.
When shell I use the brightfield or
the darkfield preview?
If the tissue
is reach in contrast, we recommend the use of the brightfield preview
image; it is made quickly and with a good resolution; it is less sensitive
for dust or specks in relation to the darkfield preview.
- The darkfield preview image is recommended for tissues
low in contrast, for brightfield or fluorescent scanning mode
likewise, but the surfaces of the slide and cover slip requires higher
cleanness then the brightfield preview and it is time-consuming during the
capturing process.
- The darkfield preview capturing process is very sensitive
in question of stray light (ambient light); it may be important if the
cover is opened or removed and the DF preview will be captured or
adjusted!
The scan area defines the part of the slide
on which the tissue, scanned by the scan camera, is expected to be. The
entire scan area is captured by the preview camera in three sections
and is shown in the preview area of the program SlideScanner.exe.
The scan area is limited by the mechanical
construction of the specimen holder and should be defined as large as possible;
it is not defined in relation to the cover slip.
In all cases, the specimen holder should never be
touched by the focus pin or the objective! In other words, the scan area
is that area of the slide, on which the focus pin and the objective can be
moved seemingly during scanning the tissues, without touching the specimen
holder.
PMIDI
The
slide is hold in the specimen holder with the help of three slide studs,
situated on the longer edges as shown.
The surrounding of the slide stud is left out
automatically from the scan process since the software version 1.14 and needs
not more to be excluded manually from the scan area (until software version
1.16).
·
During movements with the service program,
please do never touch the slide stud with the focus pin or the objective!
Since the software version 1.16
To
avoid, that the objective may touch the slide stud, the area of the slide studs
is defined explicitly; new parameters are implemented in the section
[HardwareLimits] of the file “MicroscopeConfiguration.ini”.
· Move
the stage to the positions “Knot#Left”, “Knot#Right”, “Knot#Top” and
“Knot#Bottom” just before the objective would touch the slide stud and
actualize the value of the appropriate parameter; the accuracy should be 100
steps.
· If the
objective was changed from 20x to 40x the slide stud area has to be defined
again.
· Because
the objective will never reach the slide stud “MidiKnot3”, these values should
be left unchanged.
· Please
take this into account, if software is upgraded from an earlier version to the
version 1.16 or higher!
The stud has a size of about
Top
to bottom: 12mm
Left
to right: 8mm
The
values are found at the end of the section [HardwareLimits] in the file
“MicroscopeConfiguration.ini”.
· The values, shown on the right are for demonstration
only and should not be used
· The defined areas are excluded from the scan area!
· Because
the objective will never reach the slide stud “MidiKnot3”, these values should
be left unchanged.
· The
path of the file “MicroscopeConfiguration.ini”, since the version 1.15 is:
C:\ProgramData\3DHISTECH\SlideScanner\MicroscopeConfiguration.ini
Remark
Because the values are always checked before executing
the number of steps to go in the scan program, the value of “-2000 steps” will be
cut to the value of the hardware limit!
The
barcode area is situated beside the scan area and contains stickers or labels
with information about the tissue.
If the label contains a barcode, the analyzed and decoded
barcode can be used as file name for the scanned tissue.
· The
entire barcode area is captured by one field of view of the preview camera.
· To see
the information of the barcode area, it is illuminated by the barcode
illumination.
· An
evenly illuminated barcode area and a well adjusted focus of the preview
objective increase the readability of the barcode.
· The
correct position and size of the barcode area will be defined in the step 16 of
the preview calibration procedure.
Important
Stickers and labels should be placed only beside the
cover slip; the opposite surface from beneath is glass only. If labels are
placed on the opposite side also, the slide will not be hold correctly in the
specimen holder and “slant fields of view” are produced during the scan
process; see: “Stage
skew check”.
“Barcode illumination”; “Adjust the barcode
illumination”, “The step 16 of the
preview calibration procedure” and
“Barcodes in practice”
When shall I check or define the scan area?
If the
X-Y-stage was exchanged- If the parallelogram was adjusted
- If any modifications on the specimen holder or its mounting was
taken
- If the scan area of the slide is too small or too much.
The scan area is defined during the system integration
procedure.
If the scan area was modified, the steps and checks,
described under “Adjust the
pixel position of the preview camera” must be
executed.
Physical
construction of the specimen holder
The scan area
values may vary from scanner to scanner (depending on the specimen holder and
parallelogram adjustment and mechanical tolerances); so the scan area
has to be defined for each scanner separately.
In all cases, the scan area values of X-min,
X-max, Y-min and Y-max are set just before the focus pin or the objective
touches the specimen holder; the accuracy of the limits is 100 steps.
The slide is hold by the specimen holder on the
longer, lower edge and with a slide clamp on its upper edge, on the barcode
area; see the red lines on the right. As you can see, the definition of the
limits X-min and Y-max are critical.
X-max and Y-min are given by the maximal usable slide
size and are not critical; they could be the slide edge of the largest, allowed
slide dimensions.
Because
there is a rounded corner on the specimen holder as shown on the right (X-min
and Y-max corner), there are two possibilities to define the scan area,
shown with the red and green lines.
The Y-max value
can be more decreased (the scan area will be shortened in
Y-direction) and the value of X-min can be decreased (the scan area
will be larger in X-direction). Technically, both solutions can be accepted,
but in practice, the users prefer the solution as shown with the green lines.
The difference between both solutions is more hundred steps in each direction.
Please take this into account if you are defining the scan area.
Check the found limit
position
To
decide, the focus pin is whether on glass or metal is touched, the following
actions can help.
· Move
the specimen holder with the finger nail carefully to the left
(SCAN, P250) by about 0.5 mm maximal so, that the mechanical preload
of the specimen holder is overridden; the slide is even disconnected from the
focus pin.
· Release
the specimen holder abruptly.
· The behavior
and the sound can be used to decide the focus pin is whether on glass or on
metal.
· To use
this method effectively, try this some times on different, non critical
positions on the slide; listening and memorize the sound for glass.
Define
the scan area
The scan area values to be used are set just
before the focus pin or the objective touches the specimen holder. The accuracy
of the limits is 100 steps.
1. Insert
a medium large slide or the Test slide #2.
2. Start
the SlideScannerService.exe program, Low level service part.
3. Go forward
to 26000 steps in +X direction.
4. Press
Home1 then Home2 for the Y-stepper motor.
5. Press
Home1 then Home2 for the X-stepper motor.
6. Go to
position X=4000 steps.
7. To
find the “rounded” corner of the specimen holder, go to the position Y-max.
This position is nearly 90000 step. The real value can be found in
the section [HardwareLimits]. If the specimen holder is touched by the
focus pin, move the Y-stepper backward by 100 steps until the specimen holder
is not touched and memorize this number of steps in Y-direction. If necessary,
go to Y-Home1,2 for the Y-stepper motor and forward again to the memorized
position (if steps where lost).
8. To
find the lower edge of the “rounded” corner of the specimen holder, decrease
the number of steps by 100 in X-direction until the specimen holder
is reached.
9. After each
movement of the specimen holder (either in X- or Y-direction) check the limit
as described in the paragraph above “Check the found limit
position”.
10. Decrease /
increase the number of steps in the X-direction and the Y-direction until the
correct corner point is found (see also “The physical
construction of the specimen holder”).
11. If the corner
point is found without touching the specimen holder and without lost steps,
update the parameter “ScannableAreaPositionXmin” with the found value of steps in
X-direction and the value of the parameter “ScannableAreaPositionYmax” with the
found value of steps in Y-direction in the file “MicroscopeConfiguration.ini”
section [HardwareLimits] and save the file.
12. With the 3200 steps
button go backward in -Y-direction (along the specimen holder side, defined by
the parameter X-min) until the inner edge of the slide is reached and check
after each click the correctness of the value for the parameter
“ScannableAreaPositionXmin” as described in the paragraph above “Check the found limit
position”. Collision with the specimen holder must not occur; otherwise
increase the value X-min of the scan area by 100 steps! Do the step 12
logically on each already defined edge.
13. To define the
values for the parameters “ScannableAreaPositionXmax” and
“ScannableAreaPositionYmin”, move the specimen holder into the opposite corner
and repeat the “flow chart” from step 3 logically. In step 6 start with the
position X = 26000 and in step 7 use the value of Y=34000 steps as a starting
point.
14. If the corner
point is found without touching the specimen holder and without lost steps
update the parameter “ScannableAreaPositionXmax” with the found value of steps
in X-direction and the value of the parameter “ScannableAreaPositionYmin” with
the found value of steps in Y-direction in the file
“MicroscopeConfiguration.ini” section [HardwareLimits] and save the
file.
15. Repeat step 12
logically with all four edges of the defined scan area. If anywhere
collision occurs, define the appropriate parameter value again and actualize it
in the file “MicroscopeConfiguration.ini”
section [HardwareLimits] and save the file; restart the program SlideScanner.exe.
Adjustments of the
preview unit
The
adjustment of the preview unit includes
- Adjust the aperture
iris size of the preview objective
- Adjust
the focus position of the preview objective
- Adjust
the preview camera rotation angle
- Adjust
the preview brightfield illumination position
- Execute the
preview calibration procedure of the brightfield preview
- Adjust the
preview pixel to the field of view of the scan camera
- Adjust
and calibrate the darkfield illumination
- Save the configuration file into the EEPROM.
- Adjust the
barcode illumination
- Check barcode reading
Prerequisites for
the BF preview calibration
The success of the preview calibration procedure is
bound to the following components and parameter values:
- The objective changer is in position 1 and this position is
equipped with a 20x objective.
- The flash light source is triggered by the CIS-camera.
- The CIS camera is mounted in the camera position 2 and the driver
is installed; power and trigger are connected.
- The VRmagic preview camera is connected and the driver is
installed.
- The hardware limits of the X-, Y- and focus unit are defined.
- The Home2 sensor position of the turret unit is already adjusted;
the first filter position is defined and the cover tube is inserted in the
10th filter position or the 10th filter position is
empty.
- The limits and positions of the camera changer unit are defined
correctly.
- The brightfield preview illumination was checked with the service
program and can be switched on / off.
When
shall I do a preview calibration?
- If the focus position of the preview objective was altered.
- If the preview camera was loosened, dismounted, changed.
- If adjustments are done on the parallelogram or the specimen holder
or the X-Y-stage was exchanged.
- If the scanned area of the tissue is different from the selected
area in the preview window.
Steps
of the preview calibration procedure
- Set the aperture
of the preview objective.
- Start the
preview calibration procedure
3.
Adjust the focus
of the preview objective.
4.
Adjust the rotation
angle of the preview camera.
5.
Adjust the
position of the preview brightfield illumination.
- Adjust the objective position
of the 20x scanner objective (if not adjusted previously).
- Define the barcode area
Set aperture of the
preview objective
The aperture becomes most important during
the darkfield preview.
- Loosen the aperture fixing bolt, set the aperture to a value of 3.0
and tighten the fixing bolt.
Remark To
adjust the aperture of the preview objective the horseshoe can be dismounted or
the focus unit with objective changer should be removed.
Start the service program and select the option
“Preview calibration”.
Load the preview slide in “Manual” or “Automatic” mode; select the
appropriate option.
- The manual mode should be selected if the magazine unit is removed.
- The automatic mode should be used if the magazine and slide loading
procedure is done by the magazine unit and the slide loader.
See also: “Insert or
remove a slide manually”
After inserting the preview calibration slide (Test slide #2)
the live view of the preview camera is shown in the camera window.
Use
the crosses of the calibration slide to adjust the focus of the preview camera
objective.
- Loosen the fixing bolt of the focus part of the preview objective,
adjust the focus position so, that the crosses in the center of the
circles are in focus and then tighten the fixing bolt.
Adjust the
preview camera rotation angle
Bring
the Y-stepper motor in the position where the edge of the slide is near to the
top edge of the preview window. Set the X-stepper value to 28800.
- Loosen the brightfield preview illumination adjustment
and fixing bolts a little bit and remove the shadow from the top edge of
the live view window, then tighten the bolts carefully; they are driven
into plastic!
Remark: The adjustment
of the background illumination position is here done only to see the top edge
of the slide as sharp as possible; so the camera rotation angle can be adjusted
properly.
To loosen the camera and objective
holder you need a 1.5 mm hex wrench.
- Hold the camera from beneath (the rear of the camera) and loosen
the camera
holder fixing bolt a little bit until the camera becomes barely
moveable.
By loosening one of the “Rotation angle”
bolts and tightening the opposite bolt, the rotation angle will be modified;
see also “Preview
objective and camera holder” above.
Important
Push the preview camera fully upward (until it stops)
before tightening the “Camera fixing” bolt; otherwise, the preview image will
be larger as required and many trouble might occur.
Adjust the
top slide edge parallel to the edge of the live view preview camera window
(in reality, the preview camera image will be rotated). The camera rotation
angle is acceptable if the top of the slide edge is parallel to the top
edge of the preview camera window; an acceptable deviation is often less
then 0.5 degrees.
Adjust
brightfield preview illumination’s position
Loosen
the position adjustment bolts of the brightfield preview illumination
and adjust its position so, that the shadow on the bottom edge of the preview
camera window is fully removed.
Remark: The shadow on the top edge of the preview will be cut
later, during the preview calibration procedure; “Step 2”.
Brightfield
preview calibration procedure
General
The preview calibration part of
the SlideScannerService program adjusts the pixel view of the preview
camera to the FOV (Field of view) of the scan camera, because both cameras are
physically in distance to each other.
The preview calibration includes:
1. Defining
the position of the preview camera’s pixel and the scan camera’s FOV.
2. Adjusts
the pixel position of the preview camera to the FOV position of the scan
camera.
3. The
preview camera takes three images from the slide. The parameters for assembling
the three parts are defined in the preview calibration process.
4. Defining
the barcode area.
The calibration
slide (Test slide #2) defines
positions inside the scan area with circles containing a cross. The
centers of the crosses are marker positions for the preview camera and the scan
camera also.
The calibration slide is used to calibrate the preview for scanners,
produced since summer 2016! For these scanners new calibration software is
used.
Attention: Do not mix the
versions of SlideScanner.exe and SlideScannerService.exe! Always use these programs with the same
version number. Otherwise the SlideScannerService.exe program could produce unwanted results and SlideScanner.exe does not work correctly or even freeze!
Calibration procedure for
scanners
This description
is true for scanners, produced until summer 2016.
Calibration program tools
X- Y- Stage area
X: steps Actual position
of the X-stepper motor
Y: steps Actual position
of the Y-stepper motor
X-Y step: 5000 Step range
for the blue arrow buttons
Scale 1, 10, 100
The step number of the numbered buttons can be varied
between at least 2 steps (Scale=1) and 2000 steps (Scale=100). This way, you
can find desired positions quicker.
· With a
right click on a numbered or arrow button the original direction will be
inverted. The stepper motor is moved with the number of steps written in the
button but the movement occurs in opposite direction.
Focus tools
Auto: Finds
the focus position via the auto focus function, realized in the program
SlideScanner.exe.
1600: Actual focus position field
Step rate: Step
rate for the blue arrows. By right clicking on an arrow button (or a numbered
button also), the original direction will be inverted by the actual number of
steps, given in the field “Step rate”.
Each
step of the preview calibration procedure is finished by pressing the NEXT
button.
With the Previous Step button you can go backward step
by step. At some steps this option is not available.
The command
and status window tells you the instructions, what to do and results of
calculations.
Steps of the preview calibration
This
description is true for scanners, produced until summer 2016.
Step 1: Automatic
slide handling: uses
the slide insert and remove options offered by the SlideScanner.exe
program.
Manual: moves
the specimen holder to a position where a slide can be easily inserted or
removed. The insert or remove procedure is done by the user, manually (e.g. if
the magazine unit is removed).
“Insert or
remove a slide manually”
Step 2: Cut
from top:
Go to the
position Y= 14000 with the Y-Stage tool, so that the top of the slide is
visible. If the preview camera is already adjusted and there is a shadow
visible on the top part, it has to be cut off with this option. Because the
entire preview is assembled from three preview camera images, this shadow have
to be fully cut. See also „Adjust the
preview illumination position”.
Step
3: Position the central circles between the red lines:
With
the tools in the “X- Y-Stage area” the desired result will be reached. An
accuracy of 200 steps is enough.
Step
4: Adjust the threshold until only the
circles are visible:
Remark: This step is
done only one time, if the three circles are in the middle of the preview
window.
Before
threshold adjustment
Adjust
threshold until the inside of the circles is empty, without dots or lines, but
the circles are fully visible.
The
desired result after the threshold adjustment
Step
5: Place the red squares over the circles:
The cross in the middle of the red
squares should be inside of the circle, but an exact match with the center of
the circle is not required. The squares are numbered; please place the squares
in the right order: 1, 2, and 3 from left to right.
- By dragging one of the red squares with the left mouse button all
three squares are moved at the same time, by dragging with the right
button, you can move each square separately.
· The
squares are numbered; please place the squares in the right order: 1, 2, 3 from
left to right.
Step
6: If the calculated circle centers are
correct...
The
correct result is shown on the right.
The blue crosses meeting the center
of the circles.
Step 7, 8 and 9:
Repeat the steps 3, 5 and 6 on the
top of the camera window.
Step 10, 11 and 12:
Repeat the steps 3, 5 and 6 on the
bottom of the camera window
Step 13: Move
the stage to the most homogenous background illumination
Some times on the left edge of the camera window
reflections are visible. By moving the stage to the right by several hundreds
of steps the reflections can be eliminated.
·
If the stage is moved too much in the direction X-min
of the scan area, the unevenly part of the illumination may be visible.
Without
uneven illumination
Step 14: Find the
centre of the spots
Fit the center of
the black cross with the centre of the red cross by using the X- Y- stage
control buttons.
Before you press the NEXT button for the first time,
you can correct the scan camera position and rotation angle and the scan 20x
objective focus position to see a sharp image.
After starting this step the auto focus option
adjusts the focus position for the scan camera. Now you can adjust the focus position
of the objective for the scan camera manually, if required.
After pressing the button “TEST Backlash” check
the position of the crosses to each other again and correct this if necessary.
After pressing the TEST button the X-Y-stage is moved to the next circle by
pressing the NEXT button (the same button with double task).
Repeat this procedure with all 9 circles.
· Since the software
version 1.15 only the 4 corner circles are used.
Step
15: Place the red squares onto the corner circles
The cross in the middle of the red
squares should be inside of the circle, but an exact match with the center of
the circle is not required.
By dragging one of the red squares
with the left mouse button all four squares are moved at the same time, by
dragging with the right button you can move each square separately.
· The
squares are numbered; please place the squares in the right order: 1, 2, 3 and
4 from top left to bottom right.
Step 16: Fit the barcode area into the red square
With the X-Y-stage control buttons bring the
barcode area of the slide into the red rectangle. The border, given by the red
rectangle should be a little bit away from the left slide edge. Because the
slide width tolerances of the allowed slide size are growing up or reduces the
right side of the slide, the right edge should be fully inside the red
rectangle if we are not using the widest possibke slide.
The lower edge of the slide should be equal with
the lower edge of the preview camera window.
Furthermore, we adjust now the barcode
illumination.
· Bend the LEDs
carefully so, that the barcode area is illuminated nearly homogenously.
· Modify the
exposure time of the preview camera to lower values, so the adjustment can be
checked easier.
After
finishing step 16 a dialog appears on the screen and you can check
the result of preview calibration visually, if you answering YES (Igen).
Step
17: Test the result
By selecting a higher resolution
with the “Scale factor” the possibility to find the centre of the circle is
higher. The goal of this option is that by clicking with the cursor in the
middle of a circle on the preview window (right window), to see the center of
the circle in the camera window.
This way, you can check the result
of the calibration before exiting the calibration program.
At the end of calibration, please do not forget to save
the resulting parameter values to the
file “MicroscopeConfiguration.ini” by pressing the ”Update
Parameters” button.
Exit the preview
calibration program by pressing the button DONE.
Next we scan the top left circle with the program
“SlideScanner.exe”.
Adjust
the pixel position of the preview camera
This description
is true for scanners, produced until summer 2016.
Adjust
the pixel position of the preview camera to the field of view of the scan
camera.
The goal of this
adjustment is, to see the same part of the tissue with the scan camera and the
preview camera also.
This task is done
by the preview calibration program, but the software is not able to find this
position correctly. Therefore, we must adjust the position of the pixel of the
preview camera more precise to the position of the field of view of the scan
camera “manually” by modifying pre-calculated parameter values.
As shown on the
right the scan program offers in the preview window the entire circle for
scanning (dilation=0; the red-brown area).
By checking the
scan result with the viewer, we can see, the circle is cut, so the offered area
is incorrect.
·
The size of the cut part is variable.
This chapter describes
which parameter values must be modified to fit both areas.
Important remark
·
The offered scan area, offered by the
preview camera’s view, will be named as “Spot”
·
The really scanned area is represented by
the “Circle”.
·
Of course, the entire area is scanned by
the scan camera, but our reference position will be the center of the circle in
relation to its surrounding.
·
In the picture above, the circle is cut,
this means, the offered area for scanning (offered by the preview camera) will
not met the real position of the circle on the slide.
·
Therefore, we have to shift the offered
preview area (the spot) to the left and a bit downward; so we will see the
circle in the center of the offered scan area.
·
All modifications, described below are
done with the offered scan area, offered by the preview camera’s view!
Pixel
position and corrections
and FOV and Preview
Scan the top left
corner circle
Start
the SlideScanner.exe, load a magazine
with the calibration slide, insert the calibration slide and set the
following options:
In the preview
section check the box “Use selection box”.
Select the upper left corner circle of the calibration
slide with the selection box as shown. Hereby it is important, that a small not
scanned area is included inside the selection box (white area).
Select the
options “Fill holes in scan area” and “Remove specks”.
Select the “Scan
area dilation” to zero.
With “Manual threshold” checked, adjust the threshold
of the preview so, that unwanted pixels are not disturbing the view.
Adjust the “Speck
size” as required.
Select
“Live view” in the section “Focus”.
Find any edge or a part of the cross with the
positioning tool (below the preview window) in the upper left corner circle in
the preview window and bring it into focus.
Check the option “Limited focus range” and click “Set
current”.
This way we are using the limited focus range for the
upper left corner circle during the scan process.
In the
dialog “Scan options” uncheck the checkbox “stitching”.
The focus frequency values are shown for the marlin
camera.
Define
a destination path for the slide to be scanned.
If the directory already exists, you can select it by
using “Browse”, otherwise you can type in a path in the field “Slide directory
path”.
Exit the dialogue “Scan options” with OK.
Type
in a file name and start the scan process.
With the viewer program we checking now the result, it
can be somewhat like this.
As
visible, the circle is situated in the bottom left corner of the spot, defined
by the preview camera. Therefore, the circle should be moved to the right and
upward in relation to the spot.
· In practice,
we are shifting the spot downward and to the left, the circle position stays
unchanged!
· The
spot represents the scan area, offered by the preview camera.
· The
circle represents the position of the scan camera in relation to the preview
camera (the spot).
· The
goal is now to move the circle into the center of the spot; in other words, the
position of the area, seen by the preview camera will be adjusted by modifying
parameter values so, that the circle will be in the center of the offered area,
seen by the preview camera.
Pixel
position and corrections and FOV
and Preview
Use
this calculation table to correct the position of the circle
This table
is available as an Excel file and by setting the desired input parameters (In
the green pull down menus) the configuration in front of you can be selected.
The result is shown in the yellow fields; this value should be added or
subtracted respectively from the named parameter value. The parameters are
found in the file MicroscopeConfiguration.ini section
[PreviewAndBarcodeScanning].
The “Status” line shows mistakes, if the selected
parameters can not be used in conjunction with the defined scan camera, but the
calculated result is always correct for actually set options!
· The status informs you, that the selected combination is incorrect, it
is a warning only!
“FOV
and Preview”; “Calculate offset
value” and “Sensor characteristics”
Preview corrections and pixel position
Scan
the 4 corner circles of test slide #2. To achieve a good scan quality it is
necessary to define a focus limit range in the option “Focus” of the scan program.
With the Pannoramic viewer program we are checking the relation
between tissue (the circle with cross) and the spot (visible surrounding of the
sample) of the scanned test slide #2. The goal is to fit the image, offered by
the preview camera (the spots) with the circles, the real position of the
tissue. Use the table above to calculate the values for fitting the tissue
nearly to the center of the spot.
The appropriate parameters are found in the file
“Microscope Configuration.ini” section [PreviewAndBarcodeScanning]. In
reality, we are moving the spot (the area, seen from the preview camera), not
the tissue (position of the scan camera). All corrections described here are
made with the preview image. The calculated values are added to or subtracted
from the actual values. For correction you can use integer values only,
fractions should be rounded.
With this
correction the entire tissue (all four corner circles) are moved seemingly up
or down respectively and only the circles connected with line “1” should
be observed after correction and scanning, in relation to the spots of the
previous scan result. In reality the spots are moved and not the sample. This
way, the pixels, seen by the preview camera are adjusted to the FOV of the scan
camera. In some cases, you are unable to fit the circles exactly; then you
should use an optimum value. The parameter value to be modified for this
adjustment is “PreviewImageTopLeftCornerPositionY” and is
situated in the file “MicroscopeConfiguration.ini” section
[PreviewAndBarcodeScanning].
To shift the spots downward, increase the value of the
parameter “PreviewImageTopLeftCornerPositionY”.
· In the
example, increase the value of the parameter “PreviewImageTopLeftCornerPositionY”.
Pixel
position and corrections and FOV
and Preview
With this
correction the entire tissue (all four corner circles) are seemingly moved left
or right respectively and only the circles, connected with line “
To shift the spots to the right, increase the value of
the parameter “PreviewCropLeft”.
· In the
example, increase the value of the parameter “PreviewCropLeft”.
Pixel
position and corrections and FOV
and Preview
The
pixel size of the preview will be corrected or modified in X-direction of the
entire sample. Because the influence of this parameter to the left hand part of
the sample (circles) is very small, you can really use it to correct the
position of the right hand circles, connected with line “
· In
the example, increase the value of the parameter “PreviewImageMicrometerPerPixelX”.
Pixel
position and corrections and FOV
and Preview
The pixel size of the preview will be corrected or
modified in Y-direction of the entire preview image. Because the influence of
this parameter to the upper circles is very small, you can really use it to
correct the position of the lower circles, connected with line “
· In the
example, decrease the value of the parameter “PreviewImageMicrometerPerPixelY”.
Pixel
position and corrections and FOV
and Preview
Prepare
the preview for scanning
Start
the Pannoramic SCAN software, load a magazine with the calibration
slide, insert the calibration slide and prepare the preview of the test slide
#2 as described above in the chapter “To scan the upper left corner
circle”.
In the preview tools select the tools:
· “Square
or Rectangle” (a)
· “Not
scanned” (b) and
· “Zoom
200%” (c).
By drawing rectangles, exclude the horizontal
and vertical center circles from the scan process as shown with 1 and 2 of
“Prepared preview”.
With
the limited focus the scan process of the test slide is done quicker and the
circles are scanned sharpen. To ensure, that all four circles are scanned
sharpen, the focus position must be checked for each of the circles, and
depending on the focus position of each circle the limited focus range must be
defined.
1. With
the positioning tool selected, find the center or any edge of the circle
(1).
2. Press
the button “Auto focus” (2).
3. Select
a step rate of 5 or 10 focus steps and check the found focus position of
sharpness (2a, 2b).
4. If the
sharp life view is found (3), check the checkbox “Limited focus range” (4) and press
the button “Set current” (5). The software defines the limited focus range by
the upper limit = actual focus position + 32 focus steps and the lower focus
limit = actual focus position -32 focus steps.
Check
or set the focus limit for the other 3 circles
5. Repeat
steps 1 to 3 with the next circle.
6. Check
the found focus position in relation to the existing focus range. If the found
focus position is inside the existing focus limits, check the next circle.
7. If the
found focus position is outside the existing focus limit, type a new value for
the upper or lower focus limit respectively as required (6). In the example on
the right a value of 1560 for the lower limit would be acceptable. If the
scanned circles are partly not sharp, might be the focus range must be defined
larger.
Correct the preview and adjust the pixel position
1. Insert
test slide #2 and prepare the preview so, that only the four corner circles will
be scanned. Check the focus position of all four corner circles as described
above and define a limited focus range. Use always a different file
name for each scanned slide.
2. Scan
test slide #2.
3. Switch
to SlideViewer.exe and load the just scanned test slide.
4. Check
the position of the upper two circles in relation to their spots. For better
visibility use the “color panel” “correction” option and shift the contrast
slider downward and the gamma slider fully up. So you get better contrast for
the spot and it will be more visible.
5. Calculate
the number of FOV for shifting the sample up or down respectively and modify
the appropriate parameters (PreviewImageTopLeftCornerPositionY and PreviewCropBottom) in
“MicroscopeConfiguration.ini” and save the file.
6. Exit
the entire SlideScanner.exe program and start it again to reload the
.ini files with the new parameter values. Repeat steps 1 to 6 until the tissue
fits their spots in Y-direction, see also above “Shift sample up / down. If
this adjustment is finished, go to step 7.
7. Repeat
Steps 1 to 6 logically, but now we adjust the spots by shifting them left
/right. See also above “Shift sample left / right. Repeat this
sequence until the position of the circles, connected with line “2” are in
the centre of their spots, so the X-position is defined. If this
adjustment is finished, go to step 8.
8. Repeat
Steps 1 to 6 logically, but now we are adjust the spots connected with line
“3”by shifting them left /right. This we achieve by modifying the parameter
value of Preview ImageMicrometerPerPixelX. See also above
“Shift the right hand spots”.
9. Repeat
Steps 1 to 6 logically, but now we are adjust the spots connected with line
“4” by shifting them up / down. This we achieve by modifying the parameter
value of PreviewImage MicrometerPerPixelY. See
also above “Shift the lower spots”.
Result of scanning the corner circles of the
calibration slide and finishing the preview calibration adjustment
· Please
do not forget to uncheck the option “Limited focus range” and to check the
option “Stitching”.
If the
preview calibration and the pixel size adjustment was done and the program
SlideScanner.exe is running, load a slide, increase the preview area size if
necessary and check the correctness of the scan area adjustment as
follows:
· After
the positioning tool is selected click on the edges and outside of the slide
(in the preview area window) and check after each click the correctness of the
position as described in the paragraph above “Check the found limit
position”. The limit of specimen holder movement is always
the scan area. If the focus pin is jamming or the check sounds
like metal, the scan area is too large and must be decreased at the
appropriate edge.
· Check
the corners also, especially the “rounded” corner.
Save
the file “MicroscopeConfiguration.ini”.
Endo of Preview calibration for scanners, before
summer 2016.
Theory
To define the required values (the rotation correction
angle and the position of the light beam line) a series of preview camera
images of the darkfield illuminated slide is required. For our purposes, we
using the grabbed images, made during a darkfield preview capturing process of
the program “SlideScanner.exe”.
If the darkfield preview capturing process is
finished, the files “GrabbedImage000 to
· DFP
means DarkField Preview
By opening the file “GrabbedImageXXX” with the program
“IrfanView.exe” two lines are visible near in the center of the image (see on
the right).
To create the
darkfield preview, two parameter values are important:
1.
The rotation correction angle of the light
beam line in relation to the top of the preview camera’s field of view
(DFPRotateAngle) and
2.
The distance of the light beam line from
the top of the preview camera’s field of view (DFPfirstLine).
- Both parameters can be found at the end of the section
[PreviewAndBarcodeScanning] in the file “MicroscopeConfiguration .ini”.
Because the light beam line position is always rotated
in relation to the top of the preview camera window, a correction angle is
required.
The correction
angle is calculated by the following formula:
- The correction angle α = arc tan (Y/X)
In the following, the values of X and Y will be
defined.
To define the angle with a high degree of precision,
use always the lower end of the beam thickness at the start and the end point.
·
Find also a file “GrabbedImageXXX” where
the horizontal distance between the points 1 and 2 is as large as possible (use
a tissue, large in width).
Draw
the rectangle, starting at the start point (1) (first click) to the end point
(2) (second click).
·
To increase the accuracy of the measurement,
the horizontal distance between start point and the end point should be as
large as possible.
If
the drawing of the rectangle is finished, the required values are found.
Y = the vertical difference of the left end
(the start point) and the right end (the end point) of the measured light beam
line.
X = the distance of the measured start and
end point in X-direction.
Calculate the
correction angle
· Correction
angle α = arc tan (Y/X)
Algebraic
sign of the rotation correction angle
The inclination direction of the light beam line or
the drawing direction of the rectangle can be used to define the algebraic sign
of the correction angle; see on the right.
· In our
example above the algebraic sign of the correction angle is positive (+).
Position of the light
beam line
- The position of the light beam line (DFPfirstLine) is defined in
relation to the top of the preview camera window (to the top of the
screen).
- Because the light beam will be rotated by the software in the
center of the image, the distance should be measured in the center (left /
right).
· If the
distance to the top is not measured in the center, the position of the light beam
line will be defined wrong and this may be a reason that the darkfield preview
can not be created by the program “SlideScanner.exe”.
Steps
of the darkfield preview calibration
1.
Create or prepare the file
“DebugSettings.ini”
2.
Create
the directory “DFPlog”
3.
Start the program “SlideScanner.exe”
4.
Create the darkfield preview images
5.
Open the program “IrfanView”
6.
Open the file “GrabbedImageXXX”
7.
Define the values “X”
and “Y”
8.
Define the value for
“DFPfirstLine”
9.
Calculate the correction
angle α
10. Update the values in
the file “MicroscopeConfiguration.ini”
11. Start
the program “SlideScanner.exe”
12. Create
the darkfield preview
13. Check
the darkfield preview quality
14. Remove created files and
directories
The
file “DebugSettings.ini” is used to check the software and software functions,
so this file does often not exist on the user’s computer. If the file does not
exist, we have to create it for our purposes.
To collect the made grabbed images, made during a
darkfield preview capturing process of the program “SlideScanner.exe”, the
software has to know that the made “GrabbedImagesxxx” has to be stored and
where the images should be stored. The section [DFPlog] of the file
“DebugSettings.ini” contains this information. Because this file and the
directory “dfplog” often do not exist on the user’s computer, we have to create
them or the section must be implemented into the existing file
“DebugSettings.ini”.
“What to do” in sequence
Check for the existence of the file
“DebugSettings.ini”; look in:
C:\ProgramData\3DHISTECH\SlideScanner\*.*
If existing: open it with
Notepad and check for the existence of the section [DFPlog]
If not exists: Create
the file “DebugSettings.ini” (see below)
If [DFPlog] exists: Memorize
the path and the correct name of the logging directory (upper and lower
case is important!!).
If [DFPlog] does not exist: Create it and memorize the path of the “LogDirectory” (see below)
Check for the existence of the folder “dfplog” or
“DFPlog”; the folder name must be the same as in the path definition for the “LogDirectory” in the file
“DebugSettings.ini”!
If the folder
exists: Memorize its name
and its path.
If the folder not
exists: Create it and memorize its
name and its path.
Important
If the file “DebugSettings.ini” already exists on the
computer (it may exist in some software versions or configurations), please
make a security backup and insert the following section, [DFPlog] into the
existing file.
Prepare
the file “DebugSettings.ini”
· Open
the text editor “Notepad” and copy the following 3 lines into the actual
opened, empty text file or the existing file “DebugSettings.ini”.
[DFPlog]
EnableDFPlog= true
LogDirectory= c:\dfplog; (example)
Save the actual text file (open with “NotePad”) as a “text
only” type with the file name “DebugSettings.ini” into the directory
C:\ProgramData\3DHISTECH\SlideScanner\*.*
Create the
directory “DFPlog” (example)
In the
specified path of the file DebugSettings.ini parameter “LogDirectory=” the path
of the log directory is defined; in our example the directory should be created
in the folder “C:\”.
Press the buttons: “Start”,
“Computer”, “Hard disk drive C:”, “New Folder” and name it as “dfplog”.
· In
this folder we will find the images “GrabbedImagesxxx” after the darkfield
preview creation procedure of “SlideScanner.exe” is finished.
Prerequisites for the darkfield preview calibration
The success of the darkfield preview calibration procedure
is bound to the following components and parameter values:
- In the section [Microscope] of the file
“MicroscopeConfiguration.ini” the value of the parameter
“ObjectGuideXYZType” has to be ”OGXYZ_FLASH3”; see also: “Configure the preview
unit” and “Configure the
X-Y-stage unit”.
- The brightfield
preview calibration procedure is finished successful
- The pixel
size adjustment was done
- PreviewLightType=PreviewLightUnitType_Type2; if this value is not
set, the darkfield preview procedure can not be selected; the DF software
part is disabled.
- The darkfield illumination
focus position is adjusted
- The length of the Y-direction has to be at least 89600 motor steps
(hardware limit)
- The hardware
limits in Y-direction have to be defined correctly.
- The darkfield illumination was checked with the service program and
can be switched on / off.
Remark:
The darkfield illumination is not implemented if the
P250 is configured for brightfield
scan only and so a darkfield preview can not be created!
The slide
- Use a slide with a large tissue in width and high contrast.
- It is a good idea, to make fingerprints onto the bottom of this
slide in the widest part of the tissue, so the slide bottom becomes also
visible. To see the bottom of the slide is required, if the light beam
line position will be defined.
Prerequisites for the manually calculation
of the values
Calculator
An electronically scientist calculator supported by
windows or any other kind of calculator can be used which is able to calculate
the arcos tangent.
Program “Irfan
View”
If IrfanView does not exist on the user’s
computer you can download it from the internet or you may use the stored
version.
Download
from the web: http://download.cnet.com/IrfanView/?part=dl-IrfanView&subj=dl&tag=button
If there is no internet connection
available, start: “iview435_setup.exe”
Handling “IrfanView”
- If the image “GrabbedImage000” is
loaded, the next or previous image can be reached by using the mouse wheel
for scrolling or with the keyboard keys “Cursor right” and “Cursor left”.
By
clicking the start point in the image and moving the cursor to the right, a
rectangle can be drawn. The coordinates are shown in the top line of the
program “IrfanView” if the click on the end point was done.
In
the file
C:\ProgramData\3DHISTECH\SlideScanner\DebugSettings.ini
Check the following entries:
[DFPlog]
EnableDFPlog=true
LogDirectory= <drive>:\DFPlog; memorize the path and the correct name of
the logging directory (upper and lower case is important!!).
- Find and open the folder “<drive>:\dfpog” and delete the entire
content.
Calibrate the darkfield preview
manually
This chapter describes the calibration procedure “on
foot” without using any darkfield calibration software; see also “Execute the
darkfield calibration software”
To
create the darkfield preview, two parameter values are important:
1.
The rotation angle of the light beam line
in relation to the top of the preview camera’s field of view (DFPRotateAngle)
and
2.
The distance of the light beam line from
the top of the preview camera’s field of view (DFPfirstLine).
- Both parameters can be found at the end of the section
[PreviewAndBarcodeScanning] of the file “MicroscopeConfiguration .ini”.
The rotation
angle is calculated by the following formula:
- Correction angle α = arc tan (Y/X)
Y = the vertical difference of the left
end (the start point) and the right end (the end point) of the measured light
beam line.
X = the distance of the measured start and
end point in X-direction.
Position of the
light beam line
- The position of the light beam line (DFPfirstLine) is defined in
relation to the center (left and right) and the top of the screen of the
program “IrfanView”.
Start the scanner program “SlideScanner.exe”.
Load a slide with a tissue, large in width and with
much contrast; the slide bottom should
not be clean!
Cover the scanner against ambient light if the cover
is opened or removed!
After the brightfield preview is done, select the
button “Darkfield preview” in the tab “Preview” and execute the darkfield
preview capturing procedure of the program “SlideScanner.exe”.
If the
darkfield preview images can not be assembled to a preview, an error
message occurs at the end of the process.
- If this error message occurs or the quality of the darkfield
preview is bad, the calibration of the darkfield preview unit is
necessary.
Procedure to define the
wanted rotation correction angle value
1.
Start the program “Irfan View”.
2.
Open the folder “dfplog” and open the file
with the file name: “GrabbedImage000”.
3.
With the keyboard keys “Cursor right” and
“Cursor left” the next or the previous grabbed image can be reached or scroll
with the mouse.
4.
Find an image, where the shown light beam
line is large in width.
5.
Click
on the start point of the tissue and the bottom of the line thickness (see
“first click position).
6.
Now move the cursor to the right and click
onto the same point of the line (see “Second click position).
· Observe
the drawing direction of the rectangle
7.
After the second click is done, the size
of the drawn rectangle is shown in the top line of “IrfanView”.
- If the rotation angle of the light beam line is not zero, a
rectangle will be drawn during moving the cursor.
The algebraic sign of the light
beam line rotation angle is defined by the drawing direction of the
rectangle.
· For
best results, the resulting rectangle can be drawn two or three times, if
required.
- Because we have drawn the rectangle in our example from the bottom
left corner (first click (1)) to the top right corner (second click (2))
(B), the rotation correction angle is positive.
- Notify the values X, Y and the drawing direction of the rectangle
for the algebraic sign.
- In our example: X =
1450; Y= 5; the algebraic sign of the correction
angle value = “+”.
8.
Open
the calculator (All programs\Accessories\Calculator) and calculate the light
beam line rotation angle as follows:
- Rotation angle α = arc tan (Y/X)
Example
Press the calculator buttons in the following
sequence:
1.
5
2.
/
3.
1450
4.
=
5.
Inv
6.
tan
Rotation angle
calculation result value is: 0.19
- The algebraic sign is defined by the drawing direction of the
rectangle; it is positive.
The result
DFPRotateAngle=
0.19
Procedure
to define the line position
Now it
is important to have a slide with specks on the bottom also, because the line
position is defined in relation to the top and the bottom light beam line.
With “IrfanView” you can scroll between the images, if
necessary.
Procedure to define the wanted line
position value
·
Position the cursor in the center (left
and right) of the image and about 1/3 of the distance (between the top and the
bottom line) below the top line and click.
·
Now
move the cursor to the top of the image, hereby a rectangle may be drawn, but
we are interested only in the Y-value.
·
If the
top of the image is reached, the value, used for the parameter “DFPfirstLine”
is shown in the top line of the program “IrfanView”.
DFPfirstLine = 731
Update the file
“MicroscopeConfiguration.ini”
· Update
the parameter values in the section [PreviewAndBarcodeScanning] of the file
“MicroscopeConfiguration.ini” and save the file.
· Leave / quit the program
“SlideScanner.exe” and start the program again (only this way, the new values
of “DFPfirstLine” and “DFPRotateAngle” will be used), create a darkfield
preview and check the darkfield preview quality visual; see also above “When shall I
use the brightfield or
the darkfield preview?” For this, please use a tissue with cleaned cover slip and cleaned slide
bottom surface.
Finishing
If
the darkfield preview calibration procedure is finished, please remove all
created parts:
· Remove
the section [DFPlog] from the file “DebugSettings.ini” or delete the file
“DebugSettings.ini” (if it was created) from the folder
“C:\ProgramData\3DHISTECH\SlideScanner\”or the security backup of the file
“DebugSettings.ini” may be copied return; and
· Delete
the folder “<drive>:\DFPlog”.
Principle
of the darkfield calibration program
The darkfield calibration
is used to define and correct the rotation angle and the position of the light beam line.
Two
components are important to create a good darkfield preview:
- The rotation angle of the light beam line and
- The position of the light beam line
The rotation angle “α” is
defined by two red lines; these lines can be moved with the handle (the red
squares) (by dragging the handles with the mouse).
If the rotation angle is found, the line position will
be defined; it is in the middle of the beam line.
The middle of the beam line is also the center of the
rotation if the angle will be corrected.
1) Move
the first red line by dragging the left side handle to the lower edge of the
beam line on the left side, see (1).
2) Move
the second red line by dragging the right side handle to the lower edge of the
beam line on the right side, see (2).
3) Move
the beam position line (blue) below the center of the light beam line (3); now
all required parameters are found.
Execute
the darkfield calibration software
1) Start
the program SlideScannerService.exe and the option “Darkfield calibration”.
2) Load a
magazine with a slide; if the slide is reach in contrast, the adjustment would
be done easier, because the light beam line will be thicker.
3) Insert
the slide.
Cover
the scanner unit or the entire scanner from stray light if the cover is
removed!
4) Press
“Take Image”; the entire slide will be moved over the position of the light
beam line and all found particles (tissue and specks) are used to make the
light beam line visible for the camera, because the exposure time is very high.
The option “Take Image” can be used more times also, until a well visible light
beam line is found; if necessary, the exposure time can be increased. Please
make sure, that the scanner unit (or the entire scanner) is in a dark space
during this process (covered from stray-light (ambient light)), otherwise the
quality of the image may be unusable.
5) After
the image had been taken, move the scroll bar on the right side to the middle
of the window until the beam lines are found.
6) You
can see two beam lines; the upper line contains the tissue, situated on the top
of the slide, the line below shows the found particles of the bottom of the
slide; this will never be used.
7) Bring
the lower scroll bar to the left side and you can find the line handle for the
first red line. Move the red line to the lower edge of the upper beam line by
dragging the handle; as shown on the right.
8) Bring
the lower scroll bar to the right side and you can find the line handle for the
second red line. Move the red line to the lower edge of the upper beam line by
dragging the handle; as shown on the right.
9) Now,
bring the lower scroll bar to the middle and you can find the handle to define
the center of the beam line. Move the violet line to the center of the beam
line by dragging the handle; as shown on the right.
10) The result will
be shown in the parameter window; by pressing the button “Ini update” the
result will be written into the section “PreviewAndBarcodeScanning” of the file “MicroscopeConfiguration.ini”.
Exit the darkfield calibration program and
start the program “SlideScanner.exe”.
After
the slide was inserted and the brightfield preview was created,
select the radio button “Darkfield preview” in the tab Preview of the
program “SlideScanner.exe”; the darkfield preview will be created in about
30 seconds.
If the calibration was successful,
the darkfield preview remembers to the brightfield preview.
Keep in mind, that the cover slip surface and the slide
bottom surface needs more cleanness for the darkfield preview as for
the brightfield preview.
If the creation of the darkfield preview in
the program “SlideScanner.exe” is finished with the fault report, shown on the
right, the darkfield calibration procedure should be repeated.
The reason for this fault may be the incorrect
rotation angle or the position of the beam line is incorrect.
Also might be, that the stray light was too much
during the darkfield calibration procedure and so the real positions
for the angle and / or the line position was found incorrect.
Barcode
reading and adjustments
For
technicians and sales managers!
These instructions
describe the procedure to define the barcode area and the adjustments necessary
for reading barcodes with the Pannoramic scanner P250, equipped with
- The preview camera VRmc-8+ PRO and
- The preview objective, TAMRON
23FM16SP
If the barcode reading is not allowed in the dongle for
the SlideScanner software, barcodes can not
be read; nevertheless the barcode dongle is connected.
Check the barcode reader unit for necessary
adjustments:
· If the barcode print quality on
the barcode area is high or acceptable and barcodes can be read, but some
barcode types are unreadable (with also a high print quality), then adjustments
may be needed.
Requirements
- Service program for Pannoramic scanners
(SlideScannerSrevice.exe) with actual license file
- Pannoramic SCAN and Pannoramic Viewer software
(SlideScanner.exe, SlideScannerViewer.exe) with actual license file or
dongle
- Dongle for barcode
reading
- Installed Matrox library
for barcode reading (MIL 9.0; received with the installation CD)
- 1.5, 2.5, 3 and 5 mm hex key wrenches,
Before
you start adjusting the components of the barcode reader, try to read barcodes of different
kind and codes and do the following checks; see also barcodes in practice:
1. If there is no
barcode readable or available:
· Check
the Matrox
driver installation
· Check
the presence of the dongle
for barcode reading
· Check
the validity of the license file / dongle
· Check
the focus position
of the preview camera
· Check the
size of the preview objective
aperture
· Check the selected barcode type in the dialogue “Barcode settings”
2. If several barcodes are
unreadable:
· Check the barcode print quality on
the slide’s barcode area
· Check
the uniformity of
illumination on the barcode area
· Check the size of the barcode area
· Check
the focus position
of the preview camera
· Check
the size of the preview objective
aperture
· Check
the selected barcode type in the dialogue “Barcode settings”
Define the barcode area
We assume that the preview calibration procedure is
already done and the barcode area is defined properly. For details to define
the barcode area please refer to the “Step
16: Fit the barcode area into the red square”.
Driver
installation
Attention:
· Do not copy the MIL Runtime folder to your desktop!
Instead copy the folder to C:\. Because there are a lot of long file- and
folder names inside the MIL Runtime folder, the maximal path length will exceed
the limit, and the install procedure can not be executed successfully. Never
install the MIL Runtime via network!
· For further information please refer to “Setup the Matrox Imaging Library”.
Start the
Pannoramic scan software and check the barcode readability.
Adjust the preview camera parameters and barcode
illumination for barcode
reading and preview capturing
For a successful barcode reading the adjustment
of the barcode
illumination, the correct setting of the focus position of
the preview camera is essential. For this adjustment a slide with a white label
area and a barcode test set is required.
Attention
If you adjusted
the focus position of the preview camera objective you have to do the preview
calibration process again. For details to execute the preview calibration
please refer to the “Preview calibration above”.
A fully white barcode area can be created easily by
sticking a blank adhesive label onto the slide and the edges are cut around
with a utility knife. The white barcode area is required to check the
uniformity of illumination.
Adjust the barcode
illumination
Start the program “SlideScanner.exe” with the service password.
Insert a slide with white label area and select the tab “Service” and
“Microscope control”. In the pull down menu “Video source” select the “Preview
camera”. After click on “live view” check the checkbox “Barcode area
illumination” and set the “Backlight illumination intensity” to zero.
To reach a uniform barcode illumination, please bend
the LED’s carefully and observe the live view. By modifying the PCB position,
the uniform illumination of the barcode area can be also reached.
The following dialog is implemented since the
version 1.11 in the program “SlideScanner.exe” and can be found in
“Options”, “Barcode settings”.
1D Barcode Types, 2D Barcode Types
Check the appropriate radio button for the barcode
type you are expected to read. In the file “Barcode print check.pdf”
you will find enclosed the barcode set and you can compare the view of barcode
to the samples.
1D
Barcodes with check digit
This barcodes including a check of correct reading the
barcode and this is added as a digit of the barcode (always the last character
position). With checked checkbox “Exclude check digit” this check digit is not
displayed and is also not a part of the read barcode. For test possibilities
you can uncheck this checkbox, the check digit will be displayed and is a part
of the read barcode.
Fore ground color
The foreground color is black. If the barcode is
reverse printed (white barcode on black background) select the foreground color
as white.
Preprocess Barcode Image
Sharpen
The sharpness is calculated meanwhile the barcode
image is taken.
Gamma modification
A slider will be opened and you can adjust a value for
gamma in the range of
Contrast modification
A slider will be opened and you can adjust a value for
contrast in the range of
Special Barcode Settings
Barcode string length
You can define a string length, coded in the barcode.
This way, the barcode detection will be quicker. If the number of
coded characters in the barcode and the number of characters to be read defined
here is not identical, an error message occurs, “There is no barcode”. If zero
is selected (default) any string length can be read.
Maxi code dot spacing
The dot spacing for the dots in the maxi code can be
defined. Maxi code barcode can be created with dot spacing different from the
default value (0). For successful barcode decoding this dot spacing value must
be defined here.
Other
Use barcode compensation Image
This is one of two possibilities to increase the
readability of barcodes. The compensation image, taken with the “Grab New”
option is used to compensate the barcode image for best barcode decoding
results. The image is stored and will be used until this checkbox is checked.
Grab New
A new compensation image is taken of the barcode area
and will be stored; the previously took compensation image will be overwritten.
It is important, that an exact white (not light gray) area is inserted and the
surface should be very smooth. An only white paper surface is too rough.
Barcode exposure time increase
This is another possibility to increase the
readability of barcodes. Because the automatically calculated exposure time
often produces a too dark view of the barcode area, you can increase the
brightness of the view; and so you increase the readability of barcodes. The
calculated exposure time reflects to the value of 100 percent. To find a well
usable value for the exposure time, the start value of 100 percent should be
increased by a step rate of 10 percent until the optimum was found.
Check barcode reading with different barcodes and types
Now we are checking the readability of defined
barcodes. For this purpose a set of barcodes is required that fulfill the
requirements in pixel size and mechanical dimensions for the smallest, readable
barcodes. In the appendix you find a set of barcodes for testing. If this page
is printed onto an adhesive label sheet and each label is stuck on a slide, you
created your own barcode test set. For further details see the appendix. After
all these barcodes are recognized successfully by the Matrox Imaging
Library, check some samples of the user’s unreadable barcodes. See also chapter
“7 Barcodes in practice”.
1. Start
the program “SlideScanner.exe” and in the tab “options” of the first selector
screen select the option “Barcode Settings”.
2. Insert
a slide with barcode.
3. In the
selector guide “Barcode settings” select the barcode type to be decoded. The
type, shown on the slide label and selected in the “Barcode settings” must be
equal.
4. Press
the button “Read barcode”.
5. The
software starts the barcode reading and decoding, the result is written in the
field below the button “Read barcode”. If the barcode can not be decoded in 5
attempts the error message “There is no barcode” is displayed.
Attention:
Some 1D barcodes can be read with different types and the contents of
the barcode is different also. To achieve the correct barcode content select
always the appropriate barcode type in “barcode settings”!
1. Repeat
steps 2 to 4 with all slides of the barcode check set.
2. Read
barcodes from the users “Hard to read” collection.
The following examples show barcodes, coded
with Data-matrix. The left hand column shows the original barcode area of
the slides and on the right hand column you find the same slide as it is seen
by the preview camera for barcode analyzing and decoding. In the following we
analyze the barcode print. Slides with visible faults in the barcode should be
excluded from the tissue creation process. All the following barcode prints was
read with the same illumination setup. Unreadable barcode prints could also not
be read after illumination modifying. To ensure a high print quality and to
increase the readability of the barcode the slide’s barcode area should be
clean and dry, free of moisture, before printing. The printers, even ink jet
printers should be maintained in the defined time interval and the ink
cartridge should be changed if the “ink low” message occurs. Ink jet printers
have often an integrated service part with a “Clean” option in their drivers.
Run the “clean” and “Test print” options if the print quality is reduced.
Figure 1: The
slide has a LASER printed barcode paper label. A good solution, the barcode print
is rich in contrast and therefore easily and safely readable.
Figure 2: The
barcode was printed with an ink jet printer directly onto the surface of the slide.
May be there was too much ink, the slide was moved before the ink was dry or
moisture is on the barcode area meanwhile printing. Contrast poor, blurry or
diffuse barcode print is unreadable.
Figure 3: The
barcode was printed with an ink jet printer direct onto the surface of the
slide. A nozzle does not work during printing. A missed jet in the print may be
readable or not, it depends on the position, where the jet is lost. Maintain
the printer.
Figure 4: The
barcode was printed with an ink jet printer direct onto the surface of the
slide. Some jets have not enough ink, so the contrast is partially reduced. A
contrast reduced print may be readable or not. Maintain the printer and / or
change the ink patron earlier.
Figure 5: The
barcode was printed with an ink jet printer direct onto the surface of the slide.
Some jets have not enough ink, so the contrast is partially reduced. A contrast
reduced print may be readable or not. Maintain the printer and / or change the
ink patron earlier.
Figure 6: The
barcode is engraved by a LASER beam. The contrast is reduced because a white
surface (=background) is “printed” (as a result of engraving) with glass, so
the contrast cannot be much. This barcode needs very uniform adjusted
illumination, exact adjusted white balance and shutter values.
Appendix
Create
your own barcode check set.
In the file “Barcode print check.pdf” you will
find enclosed the barcode set. The content of this page generates a full,
specified barcode test set.
Please print the file “Barcode print check.pdf”
perhaps with a LASER printer onto an adhesive label sheet with 27 labels
organized as 3 columns with 9 lines of labels, the upper border is 5
mm and the lower border is 3 mm, with no border on the left and no
border on the right.
The size of one label is 70 mm x 32
mm without space to each other.
After printing, stick each label onto a slide and cut
it around with a cutter.
Take into account that the slide clamp lower part
in Pannoramic SCAN cuts out a bit of the barcode area. The shadow
should not disturb the barcode print.
To prevent the barcode print from dust and finger
prints during use, stick a strip “tesa Film crystal clear” 19
mm in with or equivalent translucent strip over the barcode
print. So the barcode print becomes cleanable. If the adjustments
are correct and no mistake occurred meanwhile creation, all this barcodes are
readable; even if the label is stacked on slanted.