S_M_D_optics

Optics, illumination; S_M_D

For technicians and partly for sales managers!

This chapter handles the components of the brightfield illumination and the optical path for Pannoramic scanners; previously released descriptions about the brightfield illumination are no longer valid. Because our products are developed continuously, some items in the shown menus may differ to the actual software version you are using.

To help resolve problems with the illumination and optics, a hardware description of the implemented components and adjustment procedures are added.

Contents

Overview

Components and construction

Optics adjustments

Brightfield illumination

Illumination adjustment

Adjust uniform illumination with LUT

Image path adjustment

Check the optical path adjustments

Manual camera changer

Overview

The emitted light of the light source is collected by the aspheric lens in the illumination tube and the produced parallel light rays are sent to the illumination mirror (DESK, MIDI only). The mirror reflects the light in an angle of 90 degrees to the condenser.

The condenser concentrates the light to that area of the tissue that is just observed by the objective’s pupil and the scan camera; the condenser illuminates the field of view (FOV) evenly.

The light, emitted by the tissue is collected by the objective; the objective performs the image, together with the tube lens.

Into the space between objective and tube lens optical components can be inserted, like the fluorescent filter block (SCAN, MIDI) or in the DESK, the image mirror.

The image, created by the objective together with the tube lens can be modified in its size by using camera adapters with different magnifications.

The reached magnification, seen by the CCD of the main (scan) camera is the result of the product of objective magnification and camera adapter magnification.

Example: If the objective magnification is 20x and a camera adapter with a magnification of 0.63x is implemented, the resulting magnification is 12.6x.

Remark: The magnification of the camera adapter can not be varied as desired, the construction of the image path and the size of the CCD of the used camera limits the usable camera adapter magnification.

The CCD of the camera transforms the incoming light into electrical charge, this is read by the camera electronics and the composed data stream (the image) is transferred to the software.

“Optical path and Field Of View”

“Influence of the camera adapter” and “Useable resolutions of scan (main) cameras”

Components and construction

The used components are nearly identical in all the three scanner types (S_M_D)); but the mechanical construction requires some detailed modifications. Differences are named as they occur in the description.

Lamp housing

The lamp housing is designed to reflect light as much as possible to the aspheric lens. Essentially, the distance of the filament to the aspheric lens is important; for best illumination results, the filament should be in the focus of the aspheric lens; this distance is ~13mm. To reach this, the position of the lamp socked (and so the filament position) can be adjusted.

“Adjustment procedures”.

To exchange the lamp, the two knurled bolts should be removed.

Lamp

A halogen lamp with a power of 5W is used as light source to illuminate the tissue.

“Adjustment procedures”.

Attention

Never touch the lamp glass with fingers! The lamp will burn out in a half of an hour. If you have done so, clean the lamp glass with alcohol before switching on the lamp.

Important

· Please do not use a lamp with a power less than 5W! If the power of the halogen lamp is reduced, the delivered amount of light will be also less and may be not enough to illuminate the FOV; even if the tissue is a little bit thicker or the 40x objective is used; the software will interrupt the scan process or will reporting “Illumination error” during start up the software.

· Please do not use a lamp with a power more than 5W! If the power of the halogen lamp is increased, the control output of the brightfield scan illumination may be destroyed!

Lamp tube

In scanners the illumination of the tissue is very important. The lamp (illumination) tube contains the optics to produce light with a high density and coherent rays; so, the field of view can be illuminated evenly.

1. Ring nut

2. Aspheric lens. The focus position is ~13mm.

3. Distance ring “a”; SCAN: the aperture is near to the diffuser

4. Diffuser

5. Distance ring “b”

· The convex side of the aspheric lens shows to the lamp.

· The rough surface of the diffuser shows to the lamp.

· The distance ring “a” (3) has no aperture in MIDI and DESK.

· In the SCAN, the position of the lamp tube is fixed; during in MIDI and DESK the position of the lamp tube can and should be adjusted.

“Adjustment procedures”.

Illumination mirror; MIDI, DESK

Because in MIDI and DESK the illumination housing is mounted onto the scanner plate from beneath, the illumination mirror reflects the incoming light, in an angle of 90 degrees, to the condenser.

· For best illumination results the mirror position must be adjusted; see also “Adjustment procedures”.

Condenser

The condenser concentrates the incoming light to the field of view (FOV).

Because the size of the illuminated part of the tissue is critical, the condenser position can be adjusted; the focus position is 10.9mm nominal.

· Maintenance is not required

Remark

The best illumination results would be reached if we would use an objective also to illuminate the field of view; but because objectives are very expensive, a condenser is used.

· In optical aspects we can say, the condenser is a simplified objective.

· See also “Focus unit”.

· See also “Adjustment procedures”.

Condenser ; Wikipedia

Condenser; © microscopy-uk.org

Objective

In microscopes, the objective gathers the light, emitted from the tissue to be observed and focuses the rays to produce an image. The character of the objective is given by the magnification and the numerical aperture.

The position of the objective and the distance to the tissue is very important to produce a sharp image. Because in Pannoramic scanners this distance can be modified by moving the tissue position (focusing) both positions are important, the objective position and the nominal focus position.

Remark

In the standard version of SCAN, MIDI and DESK the 20x magnification is implemented. In exceptions, the user ordered the option 40x magnification also and so an exchange from 20x to 40x magnification or vice versa may be requested.

“the focus unit”.

“Adjustment procedures”.

To exchange the objective, please refer to the appropriate chapter “Exchange the objective” in the chapter “How to exchange spare parts and units”

“Optical path and Field Of View”

Objective; © Objectives_for_Microscopes_from_Carl_Zeiss.pdf; stored

Slide, tissue and cover slip

Important

If the scan program takes the compensation images after the BF part of SlideScanner.exe was started and the program stops with the error message

· “The parameter is incorrect”,

please check the components of the optical path; the camera exposure time is outside the allowed range!

· The illumination unit illuminates the tissue; the position of the illumination mirror is correct

· Condenser inserted and condenser position is correct

· No filter block inserted in the optical path (10^th filter wheel position) and the filter wheel hardware limits are set correctly

MIDI and SCAN with Objective changer unit

Since March, 2014; also named as “MIDI modular” and “SCAN modular”

· The focus unit got an objective changer unit.

The scanner, equipped with the objective changer unit are named as “MIDI with objective changer” or “MIDI OC” (3Dmic10) and “SCAN with objective changer” or “SCAN OC” (3Dmic11) and requiring the software version 1.18 or higher; see also: “What is new… in the software version 1.18”

The objective changer unit of the P250 is mounted onto the focus block of the SCAN and the MIDI.

The focusing principle and mechanics are not modified, these parts working as known from the S_M_D; see “Focus unit”.

The objective changer part is the same as described in the P250; see: “Objective changer”

Cabling: The turret unit cable is connected to the bus connector of the objective changer drive unit and the bus cable of the drive unit is connected to the stepper motor electronics of the turret unit.

Camera tube

On the side, near to the objective, the tube lens is situated; this performs the image (together with the objective). Into the space between objective and tube lens further optical components can be inserted, like the filter block for the fluorescent scan or a image mirror like in the DESK. For best image quality, the tube lens should be mounted into the camera tube until it stops!

The camera adapter 60 C1” can be also connected to the 60N interface.

· The camera tube of the DESK is equipped with an adapter 60 C1” so a 60N interfaced camera adapter can not be connected.

· Since the year 2014, the DESK is also equipped with a 60N photo port.

“Optical path and Field Of View”

Camera tube mounting; SCAN, MIDI

The tube is mounted so, that the correct position can be adjusted; this way the chromatic aberration is corrected and minimized.

· In MIDI the tube mounting is rotated by 90° CW.

· For adjustments, loosen the four clamp mounting bolts to make the tube mounting barely moveable.

See also “Adjustment procedures”.

Camera tube mounting; DESK

In the DESK, the camera tube is mounted into the mirror tube; and this is mounted directly onto the focus unit with a dovetail mounting.

By adjusting the mirror tube position, the chromatic aberration is minimized.

Camera adapter

The camera adapter is situated between the camera tube and the scan camera and offers the possibility to insert lenses or other optical means like filters into the image path.

If lenses are inserted, the camera adapter modifies the image size and the magnification.

The usable magnification of the camera adapter depends on the scan camera’s CCD size, its resolution and the construction of the optical path.

“Influence of the camera adapter” and “Useable resolutions of scan (main) cameras”

Camera adapter CARL ZEISS; microshop

Scan (main) camera

The charge coupled device (CCD) of the scan camera transforms the incoming light (the image) into electrical charge; and this is read out by the electronics of the camera.

· See also “System technique” for usable scan cameras.

· See also “Adjustment procedures” to “Adjust the camera rotation angle”.

Optics adjustments

General

Even illumination is important in microscopes and in all of our scanners as well. A well adjusted illumination ensures that any approved camera can be used properly with our scanners without further adjustments.

The entire adjustment procedure can be divided into two main parts,

1. The FOV illumination adjustment and

2. The image path adjustment.

The adjustment parts can be done nearly separately from each other, but always the illumination path is adjusted first and only then will be adjusted the image path. If the adjustments are done, the entire result should be checked again!

The adjustment is always done from the light source to the tissue and from the tissue to the CCD of the camera. Because distances are not measureable, the actual adjustment result is used to adjust the next component. This procedure requires adjusting or checking the position of previously adjusted components again!

Brightfield illumination

The goal of the brightfield illumination adjustment is, to illuminate the FOV, seen by the objective pupil (and the scan camera) evenly and with a density of light as much as possible.

· If the FOV is not fully and evenly illuminated, the quality of the virtual tissue becomes poor (“Stripping” or “Color shading” occurs), and

· If the illuminated field is too large, the exposure time of the camera will increase and the scan procedure slows down, because the light density is reduced.

Because the image, delivered by the scan camera is used for the adjustments of the illumination path, some adjustments (Objective- and focus position) for the image path have to be done before the illumination can be adjusted.

Furthermore, because we using colors to adjust the illumination path, the final correctness of the illumination path must be checked with the LUT-adjustment again, after the chromatic aberration and the camera rotation angle is adjusted.

Brightfield Microscopy

Illumination adjustment

Because the physical solutions in MIDI and DESK are modified in relation to the SCAN (in the SCAN the illumination mirror does not exist and the lamp tube position is not adjustable) the adjustment sequence is shown for the MIDI and DESK, the appropriate steps are left out during the SCAN adjustment.

Adjust the objective position and the focus position; then hold the distance between objective and tissue constant during the entire adjustment procedure by using always the found number of focus steps!

Adjust the condenser position

Find the correct lamp tube position

Find the correct mirror position

Adjust the lamp socket (filament) position.

Do the adjustment of previously named components again as required; steps 2 - 5.

Adjustment possibilities 6-9

should be used only, if the adjustments 2-5 does not deliver the desired results; with this manipulations, the tolerances of the lamp can be affected or eliminated.

Often it helps and is easier to exchange the lamp instead!

Adjust the lamp position in relation to the socket (pull the lamp out of about 1mm),

Adjust the mirror position in relation to the scanner plate (pull the mirror out of about 1mm) and

Bend the lamp toward the lamp tube (Scan only, because the tube distance can not be adjusted).

Loosen the mounting bolt for the Lamp tube (if SCAN: loosen the mounting bolt for the lamp housing also) and rotate the lamp tube. Some times there can be reached more proper results in the SCAN.

Adjust the objective and focus position

This Adjustment assumes that the focus unit is adjusted, except the objective position.

· For further information and adjustments see “Focus unit”.

1. Start the slide scanner program, insert a slide with a tissue and create a live view; the known focus position of the tissue should be nearly to 1600 steps, the nominal focus position.

2. Produce a live view and set the focus motor position to 1600 steps (or the known focus position).

3. Loosen the objective mounting by loosening the objective fixing bolt.

4. Drive the knurled objective nut so, that the objective moves toward the tissue or away from it until the focus is found.

5. During tightening the fixing bolt of the objective observe the live view and correct the objective position as necessary.

6. With the option autofocus check the adjustment.

7. Repeat from step 2 if necessary.

“Mount the objective” and “Adjust the objective and focus position”

Adjust condenser position

The adjustment of the condenser is important for the bright, uniform and optimal illumination of the FOV. This reduces so the exposure time of the camera and increases the quality of the scanned tissue. If the objective position was modified, the correctness of the condenser position has to be checked again!

“Condenser mounting”

The position of the condenser affects the following:

· The size of the visible FOV (color shading)

· The image contrast

· The image resolution (the numerical aperture) and

· The exposure time.

1. Create a live view with the scan camera in the focus tab and set the focus motor position to 1600 steps.

2. With the preview positioning tool find a “clean” FOV outside the tissue and inside the cover slip, without dust.

3. Loosen the condenser’s fixing bolt.

4. Rotate the condenser toward to the objective, to find the start position for the adjustment; the brightness will increase.

5. Rotate the condenser in opposite direction (away from the objective) and look at the live view. Beware of the condenser cover (shutter), don’t close it and don’t bend it. You will see two surfaces (from the diffuser) coming into focus (see “Condenser 1 and 2”). While rotating the condenser you can also observe that the brightness decreases.

6. After the second surface just disappeared (Condenser 2) and the live image is smooth and bright, stop moving the condenser and tighten its fixing bolt, see “Condenser position” (the pictures was done with previously adjusted illumination. If you are starting the adjustment, the figure “Condenser position” might be is not so smooth).

7. If the brightness decreased too much, repeat the steps 10 to 13.

8. Check the correct condenser position in the focus positions 1200, 1600 and 2000 steps. There must not be significant differences.

Adjust the lamp tube position

Loosen the fixing of the lamp tube (MIDI, DESK) and bring the lamp tube into the proper position as shown.

Tighten the lamp tube fixing bolt.

If DESK: To reach the lamp housing and the lamp tube fixing bolt, the scanner plate must be dismounted from the base plate.

2. Disconnect the preview illumination cable and remove the three mounting bolts of the scanner plate; then the scanner plate can be rotated by 90 degrees to the left.

Adjust the illumination mirror position

1. Illumination mirror mounting bolt

2. Illumination mirror

3. Illumination tube

4. Illumination tube mounting bolt

· By rotating the mirror to the left or to the right respectively and by observing the live view on the screen, the right mirror position can be found.

· The mirror position should be checked and/or adjusted more times during the adjustment sequence.

Adjust the lamp socket (filament) position

Drive the adjustment bolts “a”, “b” and “c” in or out, until the optimal position of the filament in relation to the aspheric lens is found.

· Remove the knurled bolts of the lamp socket mounting and modify the lamp position in relation to the socket if required or to exchange the lamp.

Adjust the uniformity of the illumination with the look up table

General

To adjust the uniformity of the bright field illumination we recommend using the Marlin F-146C camera or equivalent. This adjustment assumes, that the focus - and condenser position is already adjusted!

The Marlin F-146C: The hardware control for unequally illuminated FOV can be switched off for this camera and our software use this possibility.

The Stingray F-146C: This camera can be used also, because the camera driver is the same.

Other cameras: Like Hitachi have another driver implementation or other possibilities and can therefore not be used with the LUT-adjustment.

· In the following, the term “Look Up Table” will be used as “LUT”

Requirements:

· Scan software with valid dongle or license file

· Service program with valid license file

· LightCalibration_marlin.csv control file for the marlin camera, Path: … website\Descriptions\Files_\LUT-calibration\

· LightCalibration_stingray.csv control file for the Stingray camera, Path: … website\Descriptions\Files_\ LUT-calibration\

· The installed Marlin F-146C or Stingray F-146C camera

· AVT Smart View 1.6.1. or higher the actual driver for the Marlin and Stingray camera

· Hex key wrenches M1.5, 2.5, 3, 5

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 it may freeze!

Preparations before the LUT adjustment starts

1. Start SlideScanner.exe, insert a slide with cover slip.

2. After the preview is done, select the option Focus and click the button “Live view”, select the positioning tool, click inside the preview window, inside the cover slip and find a well usable FOV without tissue or dust. The focus position should be 1600 steps.

3. Kill the program SlideScanner.exe with the task manager.

4. Start the AVT Smart View 1.6.1. or higher and select the option “Always scale image to window” in the “VIEW” menu.

5. Start the “Settings dialog” in the menu ”CAMERA” and “Settings”.

6. In the window “Format” check the options “F7 mode 0”, “RAW 8”, ISO speed 400 and in “Debayering” select 2x2 or 3x3 (see “Format”).

7. Select the tab “Ctrl 1”, set “Gain” and “Brightness” to zero and leave “Gamma” unchecked (see Ctrl1).

8. Select a “Shutter” value in the range between about 50 to 100 and push the buttons play and then at the white balance.

9. In the menu “VIEW” and “additional information” select the option “Histogram” (see the menu “View”).

10. Increase or decrease the shutter value until a medium brightness live view is visible (see Ctrl1).

MIDI and DESK: Turn also the illumination mirror carefully left or right and move it up and down until the max. brightness is reached.

11. The goal is now to bring the functions of red, green and blue nearly into cover in the middle of the range between 127 and 255 (modify the shutter value for the red function and the white balance UB and VR values for green and blue) (E). Possible peaks in the functions can be eliminated (minimized) later, with the LUT-adjustment.

To reach the desired results, please adjust the following parts:

1. As you can see, the only fix distances are given by the position of the objective in relation to the position of the tissue (the focus position) and the condenser position. Therefore, these distances are defined first.

· See also above “Adjust the objective and focus position” and “Adjust the condenser position”.

2. The next fixed distance is the distance of the illumination tube (aspheric lens) to the lamp (filament), therefore the lamp tube position should be found next (MIDI, DESK).

3. The mirror position is important to reflect the entire emitted light to the condenser; if the position is wrong it affects both parameters, the evenly illumination and the light intensity.

4. Because the filament position is critical, the distance and the position can be fine adjusted with the lamp socket position adjustment.

Because there are several adjustment possibilities, the adjustment steps should be done more times until the best result is found.

Adjustment

An essential tool for the bright field illumination adjustment is the “Look Up Table” (LUT); it is implemented in the AVT Marlin camera driver. With this tool small deviations from the actual or optimal adjustment (even if we adjust the mechanics) becomes visible as color alteration on the screen. Expected results can be found on the end of this chapter.

Driver settings for the LUT adjustment

1. Click on the tab LUT and stop the live view by pressing the Stop button.

2. Select the LUT file by click on and select the file “LightCalibration.csv” (for the camera “Marlin” or “Stingray” as required) from its source folder, we suggest to use the folder 3DHISTECH\ Pannoramic SCAN (the file is found on the install CD or use the files LightCalibration_marlin.csv or LightCalibration_stingray.csv respectively).

3. Import data from column: select “COL B” and Starting at: “Row 1”.

4. In the pull down menu next to the button “Upload” select the option “Data from the file below”,

5. Press the button “upload” and

6. Check the box “LUT operation on” and uncheck “Shading correction on”.

7. After pressing the button “Play” you will see often a rainbow colored live view or a nearly full black screen, if the adjustment was done before.

Steps of the LUT adjustment

The goal is to reach a nearly one color screen. The centre and the border of the screen may be in different colors; this depends often on the shutter value. See also at the end of this chapter. The mechanical shutter (condenser cover) should be always fully open and the turret position has to be correct (without filter!); otherwise the LUT adjustment can never be performed correctly!

Attention:

Never touch the lamp glass with fingers! The lamp will burn out in a half of an hour. If you have done so, clean the lamp glass with alcohol before switching on the lamp.

8. Increase the shutter value (tab “Ctrl1”) until a fully white screen is visible (the live view is burned out) (white color can occur twice, use the last one where the whole screen is white!).

9. Decrease the shutter until the first colors occur, then decrease the shutter carefully until the dominant color is green. This is our default color. If adjustments are executed, this color should be always used by actualizing the shutter value in the tab “Ctrl1”. If you are unable to reach the green color, leave the LUT adjustment and adjust the white balance again. Some times it helps also to reset the camera by unplugging the FireWire cable and plugging again after about 10s.

10. Adjust the filament position by driving the adjustment bolts “a”, “b” and “c” of the lamp socked and check the live view. Find the position with maximum brightness and decrease the shutter value until the dominant color is green again. Repeat these steps several times.

11. MIDI and DESK: Turn also the illumination mirror left or right and move it up or down until the max. brightness is reached.

12. MIDI and DESK: Adjust the position of the lamp tube again.

13. SCAN: Rotate the illumination tube.

14. Repeat the steps 8 to 13 several times until the optimal brightfield illumination is found, it is a puzzle game! Keep in mind, that the position of the turret is also important. If the turret position is not adjusted right or a filter is inserted, the correct LUT-result can never be reached!

15. The entire optical path is affected by the mounting of the scanner plate and the mounting of the turret plate also. If the adjustment results are poor, loosen the scanner plate mounting and the turret plate mounting. By moving the scanner plate and / or the turret plate, the LUT-picture is also changed. Find the correct position for the turret plate and the scanner plate and tighten them in the right sequence; see also “The scanner and turret unit mounting”. There must not be trapped any cable, especially the cable for the DC-controller, between the lower part of the scanner plate and the truss.

16. If you see full color rings on the screen the condenser should be adjusted to correct the view. And check the mechanical shutter (condenser cover) position! It must be fully open! MIDI or DESK: The lamp tube should be more inserted into the lamp housing; find the optimal position.

17. If there can not be found a sufficient result, try with another halogen lamp also.

18. If the AVT camera driver should be leaved, please do not forget to uncheck the box “LUT operation on”!

19. If the desired result is reached scan several tissues with the Marlin camera and check the LUT adjustment. If there are blue or pink stripped areas on the preview in the slide viewer program (stripping or color shading occurred), the LUT adjustment must be corrected, perhaps scan the same tissue with a different camera.

LUT-Adjustment result

The following images show an acceptable, finished LUT adjustment. The shutter was increased in steps by one from 83 to 90 (Pannoramic SCAN), Marlin camera and 1.0x camera adapter.

To check or adjust the white balance leave the LUT by uncheck the checkbox “LUT operation on”.

The same scanner with adjusted illumination and a shutter value of 63. Peaks in the functions should be eliminated (or as little as possible).

To eliminate peaks in the functions you can also adjust the condenser position carefully again.

Remark

The reachable result depends highly on the used marlin camera (variance of the product) and the quality of the selected field of view (clean FOV).

Image path adjustment

The entire image path adjustment includes the adjustment of the following parts:

1. The objective position

This adjustment ensures that tissues with different thicknesses can be scanned in focus; of course, it was adjusted previously for the brightfield illumination, but the objective position should be checked and adjusted again. If the objective position is incorrect, the tissue or parts of it can not be scanned in focus; see also “Check the optical path adjustments”.

2. Camera tube position

The position of the camera tube (lens) affects the color trueness of the scanned tissue; the chromatic aberration becomes visible in more blue, and more red or yellow colored cell borders on the opposite sides; see also “Chromatic aberration” and “Adjustments”.

3. Camera rotation angle

If the camera rotation angle is out of the limits, the stitching is not correct and the borders of the FOV’s becoming visible in the virtual tissue with the viewer program, the sample does not fit on the border of the FOV; see also “Stitching’.

Chromatic aberration

The appearance of chromatic aberration can be divided into two main reasons:

1. The used materials (the composition of the glass) in the lens system; different wavelengths of light will be focused to different positions; and

2. The arrangement of the lenses to each other (centermost), with other words, the straightness of the optical path (lens system).

· For any kind of optical aberration see “Optical aberrations”

Chromatic aberration of a FOV is seen as unevenly colored cell borders. Because the first item is given by the used optics (the construction of the objective and lenses) and can not be affected by the technician, we minimize the chromatic aberration by making the optical path straight and centered.

For this purpose, in the SCAN and the MIDI the position of the tube in relation to the turret plate is modified (with loosened tube mounting bolts); in the DESK the chromatic aberration is affected by the position of the mirror tube and the plane of the image mirror.

After the chromatic aberration adjustment was finished, the camera rotation angle has to be adjusted (again).

The adjustment of the chromatic aberration is done in the real focus position and in the center of the FOV to be observed. To check the result of the adjustment, the focus position can be modified by some steps in positive or negative direction. In this way, the correctness of the adjustment becomes more visible. If the yellow color occurs evenly on the inner and outer part of the circle in the center, the adjustment is acceptable; see “Focus position +4 steps”.

The images was done in the focus position of the live view, except otherwise specified and with a zoom factor of 2,73

Reduce the chromatic aberration

Chromatic aberration becomes visible if the optical light path is not exactly perpendicular (mirrors) or centered (lenses); it is corrected by different positioning of the tube. For this purposes use a well visible tissue. This adjustment assumes that the LUT adjustment is already finished! To adjust the chromatic aberration use and observe always the center of the FOV, never the border, because the border has always more chromatic aberration as the center!

Example: If the otherwise dark spots in the tissue have blue boundaries on the top, and red or yellow on the bottom (see also above “Chromatic aberration”), move the tube to the red (yellow) direction.

For Pannoramic SCAN: Keep in mind, that the camera is mounted 30° from the plumb-vertical, therefore the directions up, down, left and right are also turned 30° with respect to the room’s coordinates; see “The camera angle”.

Reduce the chromatic aberration

1. Start the program “SlideScanner.exe” and load a slide with tissue.

· Important: Check the proper position of the slide in the specimen holder.

2. After the preview is done, select the option Focus and click on the button “Live view”, positioning tool and click inside the tissue and find a well usable FOV with a lot of cells. Use the “Auto focus” button.

3. Fit the camera view to window size with the button 1:1 and zoom in by using the zoom tool until a zoom value of 2,73 is reached. By moving the horizontal and the vertical scroll bar to the middle of their acting range, the center of the FOV is in the center of the screen.

4. If the zoom value is large enough (between 2.6 and 3), you can see something like this “Aberration”. If yellow, red or brown colors are visible at the boundaries of spots, the optical system has chromatic aberration; check this behavior on different positions of the tissue also.

Procedure for SCAN and MIDI

5. Loosen the tube fixing bolts until the tube becomes just barely moveable.

6. Move the tube in the direction, where the red or yellow color of the spot or cell occurs. (With the Pannoramic SCAN: Take into account, that the camera is mounted parallel to the magazine loader edge (30 degrees), so that the directions up, down, left and right are also turned 30 degrees; see also “Camera rotation angle”. Remember, the chromatic aberration will be adjusted always in the center of the field of view!

To check the result

7. After pressing the button “auto focus”, use a focus step size of 2 steps and go from the auto focus position in plus direction. If the cell gets a brown or yellow ring in nearly constant thickness the aberration seems to be adjusted.

8. Repeat step 7 and check this result on different positions of the same slide (tissue) with live view.

9. Scan a tissue or a part of it and check the result with the SlideViewer. When you can find more positions where the aberration is visible always on the same side of the cells, repeat from step 6 (if DESK then from step “b”).

10. When you can find parts of the tissue where the chromatic aberration is visible on different sides of the spots, the chromatic aberration seems to be adjusted.

11. Scan two further tissues with different samples and check the results (repeat the steps 9, 10).

12. If the boundaries of the spots (see “corrected”) are colored evenly the optical path is correct.

13. Tighten the tube mounting bolts and check the result, by repeating the steps 7 to 10. If necessary, repeat the steps from step 5.

14. Before scanning tissues the scan program “SlideScanner.exe” has to be restarted, otherwise stitching errors may occur.

After the chromatic aberration adjustment was finished, the camera rotation angle has to be adjusted (again).

Procedure for DESK

With this adjustment, the chromatic aberration can be reduced in the vertical (Y) direction only. If the aberration in the X-direction (left- right) is significant, the image mirror position must be adjusted; see below “Image mirror position adjustment”.

a. Do the steps 1 – 4 as described above.

b. Loosen the mirror tube mounting bolt.

c. Loosen the aberration adjustment bolt fixing nut and drive the adjustment bolt backward (CCW) some turns.

d. Pull the mirror tube in direction to the objective until it is stopped by the adjustment bolt.

e. Observe the live view and drive the adjustment bolt forward (CW).

f. If the chromatic aberration in vertical direction disappeared stop the adjustment.

g. Tighten the mirror tube mounting.

h. Hold the position of the adjustment bolt constant with the hex key wrench and tighten the position fixing nut.

i. To check the result, continue the adjustment for SCAN and MIDI at step 7.

Image mirror position adjustment (DESK)

· Attention: If the mirror position bolts are fixed with red glue, the correct position is adjusted previously and the reason for the horizontal aberration may be elsewhere. Check the tightness of all optical components first and the quality of the tissue! Check also tissues with different thicknesses!

· Always check the proper position of the slide in the specimen holder also.

With this adjustment, the image mirror can be positioned in the image path into the angle of exact 45 degrees in relation to the objective and the tube lens; and so the chromatic aberration will be minimized.

· The bolt pairs “a”, “b” and “c” defines the plane of the image mirror; “a” and “b” define the angle of inclination mainly left or right and the bolt pair “c” defines the angle of inclination mainly up or down.

· By loosening the mounting bolt of the appropriate bolt pair and driving in the plane position bolt; or driving outward the plane position bolt and tightening the appropriate mounting bolt, the mirror plane is modified.

· Before checking the result, all the mounting bolts should be tightened!

Adjust the image mirror plane

1. Produce a well usable live view as described above “Adjust the chromatic aberration”.

2. Drive the chromatic aberration adjustment bolt (see the image above, “Mirror tube”) nearly into the middle of its adjustment range and pull the mirror tube toward the objective until the adjustment bolt is reached.

3. Modify the position of the mirror plane by driving the bolt pairs “a”, “b” and “c” as required and minimize so the chromatic aberration.

4. Does the chromatic aberration adjustment procedure as described above, see “Procedure for DESK”.

Stitching

Stitching errors have two main reasons:

1. Improper adjusted camera rotation angle and

2. The hysteresis in Y-direction is too much.

The camera angle becomes important during stitching. If the angle of the scan camera is out of the limit, the stitching does not working well, so the FOV’s, seen with the viewer does not fit to each other. An acceptable camera angle has less then +-0.5 degrees deviation from zero.

If the camera angle is correct and stitching errors occur, check the hysteresis in Y-direction.

next chapter “Y- and X-hysteresis” and also “X-Y-stage unit”

Remark

The shown stitching errors existing always parallel inside of the same scanned tissue, it means, if one occurrence is found, all others can also be found on different areas of the same scanned tissue (if the scanned area is large enough).

Stitching

Adjust the camera rotation angle

In the selector menu and ‘Options” start the item “Microscope settings”.

In the tab “Base settings” set the values for the parameters numbered with (1)-(5) as these are true for the scanner to be set up; then change to the tab “Camera rotation” (6).

Load a magazine (7), select the desired slide position (8) and insert the slide (9).

In the preview window find a FOV with tissue, Press the button “Live view” (10) and “Auto focus” (11). If the focus position is found, click outside the tissue and inside the cover slip on a “white” position.

Set the “Auto exposure time” and the “White balance” by clicking on the appropriate icon on the lower screen border.

Click inside the tissue and find a well usable FOV with cells.

Find the focus position (11).

Select a “Step size” of 10 or 20 µm (12) and move the object guide to the left or to the right as desired (13) and observe the movement of a cell near to or on the horizontal red line.

· If the cell deviates from the red (horizontal) line in the center upward or downward respectively, correct the camera angle continuously (by moving the camera adapter on its mounting) until the cell moves on the red line (14) or exact parallel to it.

If the cell moves from the left border to the right border of the screen (or reverse) nearly on the red line, the camera angle is correct (14).

Press the button “Measure camera rotation” (15).

Now the program arranges two FOVs to each other and shows so graphically the fitting of the FOVs in the centre of the live view; the numerical value of deviation is shown in the lower part of the left sided adjustment window.

· If the value of the rotation angle is shown in red, the position must be adjusted more precise (16). Correct the camera position and press the button “Measure camera rotation” (15) again, until an acceptable angle is found.

· An acceptable camera rotation angle has less than 0.5degrees deviation from zero.

If the rotation angle can be accepted, the angle value is shown in black (17).

Save the calculated rotation angle to the appropriate file by pressing “Save” (18); and in the next following dialog answer with “YES” to save the file.

Leave the menu “Options” by clicking on “Exit”.

Check the optical path adjustments

Objective and focus position

As discussed previously, the correct objective and focus position is important to be able to scan tissues of different thicknesses in focus.

This fact we are using to determine the correct objective position.

1. Find at least three, better are 5 slides with tissue of different thickness and of different kind.

2. Insert the (next) slide; check the correct position of the slide in the specimen holder!

3. Produce a live view of the tissue, press “Autofocus” and notify the focus position.

4. Repeat step 3 on 5 different positions of this tissue; the distance of the positions should be as much as possible.

5. Calculate the average focus position of this slide and notify it.

6. Repeat from step 2 until the average focus position of all the selected tissues is determined.

7. Calculate the average focus position of all the tissues.

8. If the average focus position deviates more then 50 steps from the nominal focus position, calculated with the used slide thickness, the objective position should be corrected.

9. If the objective position was modified, please check the correctness of the condenser position again.

Condenser position

Check the correct condenser position in the focus positions 1200, 1600 and 2000 steps. There must not be significant differences.

· For best scan results, the clean FOV should be evenly illuminated over the entire focus range.

· If the condenser is misaligned, the roughly surface of the diffuser becomes visible!

Remark

“Clean FOV” means a Field of View, seen by the scan camera without tissue, dust or dirt, between slide and cover slip.

above “Adjust the condenser position” and “Focus unit”

Y- and X-hysteresis

The software divides the sample to be scanned, seen by the preview camera into fields of views; the size of the FOV depends on the resolution and the size of the scan camera’s CCD and the magnification of the camera adapter. Each field of view contains a small part of the neighbor FOV. In this way, stitching becomes possible. Because the capturing of the FOV’s is done on a meandering course, the Y-direction is often changed. If the hysteresis in Y-direction is too much, stitching will not work correctly; therefore, we have to check the hysteresis in Y-direction. The maximal allowed hysteresis is 4 mm (=4 motor steps). We comment that this hysteresis decreases itself by some motor steps after some sample scan procedures, even if the X-Y-stage is brand new.

Because the X-direction is never changed during a sample scan process, the X-hysteresis is not critical and can be some steps more (max: 8 steps).

· To reduce the Y-hysteresis, see also “X-Y-stage unit” and “X- and Y-carriage drive unit”.

Watch video: “Tissue scan process” (P250)

Check the maximal hysteresis in Y-direction

Start the program “SlideScanner.exe” with the service password. In the tab “Focus” produce a sharp life view.

In the tab “Service” select “Microscope control”. In the part of the X-Y-control select a step size of two steps and go upward, until the tissue moves.

Now go in opposite direction and count the clicks until the tissue moves again. If more then 3 clicks are required, the hysteresis is too much.

The correction of the hysteresis can not be done in the field.

Chromatic aberration

Scan a tissue and check the chromatic aberration with the Slide Viewer program.

above “chromatic aberration”.

Stitching

Scan a tissue and check the stitching with the Slide Viewer program for stitching errors. See also “Typical stitching errors” in the chapter above.

Stage skew check

The stage skew check is used to determine the inclination of the specimen holder and so the inclination of the slide. If the inclination is too much, parts of the tissue are in focus during other parts of the same FOV are not in focus.

The Stage skew check should be done:

If the parallelogram was removed.
If the parallelogram or the specimen holder was exchanged.
If the entire X-Y-stage unit was changed.
If the Focus unit was exchanged.
If any spare part was changed and this spare part is in connection with the perpendicularity of the optical axis to the slide.
If the mounting bolt positions or the adjustment bolts position of the parallelogram was altered.
See also “Parallelogram adjustment”.

To check the inclination angle of the specimen holder, a series of screen shoots is done of a cell (circle) in the center of the FOV and in the upper and lower and left and right corners respectively.

There are 7 screenshots taken in each position; 3 before the found auto focus position and 3 screenshots after the auto focus position. Then find the screenshot of each position where the cell (circle) is most in focus. If there is a difference, more then 2 focus steps to the found focus positions, the specimen holder is slanted and has to be adjusted; this adjustment can not be done in the field; probably the specimen holder or the parallelogram is deformed.

Important: Always check the proper position of the slide in the specimen holder first.