Preview unit; SCS

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

Configure the preview unit

Preview components

Preview illumination unit

Areas of the slide

Mechanical adjustments

BF preview calibration

DF preview calibration

Barcode reading

Requirements

• 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!

Configure the preview unit

· 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]

SerialNumber=PCON_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

ShutterMotorType=Shutter_Motor

PowerSwitchBoardType=PowerSwitchBoard_Type1

ConfocalUnitType=ConfocalUnitType_Aurox

WaterFeederType=WaterFeeder_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]”

Components

Preview unit’s mounting

The entire preview unit is situated on the edge of the scanner plate, parallel to the magazine unit.

The position is fixed with two fixing pins and hold by the mounting bolt.

See also: “Scanner plate” and “Horseshoe mounting”

Remove the preview unit

· Disconnect the cables for the preview illumination, the barcode illumination and the preview camera’s USB cable

· Remove the mounting bolt and then the entire preview unit can be removed frontward.

Mount the preview unit

· Fit the fixing pins of the scanner unit into the position fixing holes of the horseshoe and push the entire preview unit onto the edge of the scanner unit.

· Drive in the mounting bolt.

· Connect the cables for the preview illumination, the barcode illumination and the preview camera’s FireWire cable

Preview unit

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”.

Preview camera and objective

In the scanner PCON 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.
• Set the value of the aperture to 3.0.

Barcode illumination

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.

See also “Barcode adjustments”.

Preview illumination unit

• The brightfield preview illumination is used to make the preview of the tissue visible for the brightfield scan procedure during the darkfield preview 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.

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 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 brightfield illumination is connected with the wire BGJ-1.
• The darkfield illumination is connected with the wires DLJ-1 and DLJ-2, these connections are interchangeable to each other.

Watch video: “Assembling”

Preview illumination unit’s mounting

The preview illumination unit is mounted with 2 bolts onto the edge of the scanner plate.

Principles 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; the barcode area can be viewed in a separated window.

 See also: “Scan area” and “Barcode area”

“Creation of preview” and “Preview and field of view”

Create a darkfield view

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).

Darkfield illuminated preview

• 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 the cover slip requires higher cleanness than 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!

Areas of the slide

Scan area

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.

See also: “Define the scan area”.

Barcode area

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”.

See also: “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, the 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.

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 and in the PCON

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!

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 steps. 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 “To check the found limit position”.

10. Decrease or 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 “To 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.
• 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 or off.

• The scan area is already defined.

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

1. Set the aperture of the preview objective.
2. 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.

6. Adjust the objective position of the 20x scanner objective (if not adjusted previously).
7. Define the barcode area

Set the 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.

Adjust the focus position of the preview objective

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 centers 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 by adjusting the illumination position, then tighten the bolts carefully; they are driven into plastic!

Remark: This adjustment is 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 fixing 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 and rotate able.

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 (in direction of the preview mirror) 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 the 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”.

• The shadow on the bottom of the preview camera window has to be fully removed!

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.

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 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

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).  

See also: “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 20x objective focus position.

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

 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.

See also: “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 needed.

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.

 See also: “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!

See also: “FOV and Preview”; “Calculate offset value” and “Sensor characteristics”

Load the EXCEL table

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.

Shift the spots up or down

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”.

See also: “FOV and Preview”

Load the EXCEL table

Shift the spots left or right

With this correction the entire tissue (all four corner circles) are seemingly moved left or right respectively and only the circles, connected with line “2”, should be observed after correction and scanning in relation to 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 “PreviewCropLeft” and is situated in the file “MicroscopeConfiguration.ini” section [PreviewAndBarcodeScanning].

To shift the spots to the right, increase the value of the parameter “PreviewCropLeft”.

· In the example, increase the value of the parameter “PreviewCropLeft”.

See also: “FOV and Preview”

Load the EXCEL table

Shift the right hand spots

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 “3”.  To correct the position shown on the right, you should increase the value of the parameter “PreviewImageMicrometerPerPixelX” by 0.1 to move the spot to the right (the tissue seemingly moves to the left in relation to the spot). If you adding to or subtracting from the parameter value 0.1 the spots are moving nearly by one FOV.  In some cases, you are unable to fit the circles exactly, then you should use an optimum value.

·  In the example, increase the value of the parameter “PreviewImageMicrometerPerPixelX”.

See also: “FOV and Preview”

Shift the bottom spots

_1The 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 “4”.  To correct the position shown in the right hand picture, you should decrease the value of the parameter “PreviewImageMicrometerPer PixelY” by 0.1 to move the spot upward (the tissue seemingly moves downward). If you are adding to or subtracting from the parameter value 0.1 the spots are moving nearly by one FOV. In some cases, you are unable to fit the circles exactly; then you should use an optimum value.

· In the example, decrease the value of the parameter “PreviewImageMicrometerPerPixelY”.

See also: “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 “The prepared preview”.

Set limited focus

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 or 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 orright.  See also above “Shift sample left or 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 orright. 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 or 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”.

Finally

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 “To 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” to the EEPROM of your scanner.

Darkfield preview calibration

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 199” can be found in the folder “<drive>:\DFPlog”.

· 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 or 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

File “DebugSettings.ini”

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 or off.

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 = “+”.

Calculation of the angle

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 or 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 to reading barcodes for Pannoramic scanners, 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 and 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 adjustments

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 or dongle

· Check the focus position of the preview camera

· Check the size of the preview objective aperture

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

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 Barcode Settings dialog

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.

Barcodes in practice

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 direct 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.