P250_Optics_Illumination.htm

Optics, illumination; P250

For technicians and partly for sales managers!

This chapter handles the components of the brightfield illumination and the brightfield optical path for the scanner Pannoramic 250. Because our products are developed continuously, some items in the shown menus may differ to the actual software version you are using; the description is based on the software version 1.15.

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

Contents

Optics and illumination

Overview

Components and construction

Illumination path adjustments

Image path adjustment

Chromatic aberration

Reduce the chromatic aberration

Stitching

To adjust the camera rotation angle

Check the optical path adjustments

The Y- and X-hysteresis

Stage skew check

Overview

The optical path includes the following components:

Brightfield illuminated optical path

The emitted light of the pulsed xenon flash light source, triggered by the software, crosses the “ND filter” in the “ND filter housing”, the “Diffuser” and the “Yellow filter” in the “Collimator lens holder”. All these filters are used to prepare the emitted light of the light arc to illuminate the field of view (FOV) with the homogeneity and intensity of the needed light wave lengths. The collimator lens in the collimator lens holder produces parallel light rays and these are arriving to the condenser.

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

The light, passed thru the tissue is collected by the objective.

Into the space between objective and tube lens optical components can be inserted, like the fluorescent filter block during the fluorescent scan process; hereby the turret unit inserts the light from the fluorescent light source to illuminate the tissue.

The image, created by the objective together with the tube lens, arrives to the mirror of the camera changer unit. Depending on the position of the mirror, the image is reflected to the camera position 2 for bright field scan operation or to the camera position 1 if the fluorescent scan mode is selected.

The image can be modified in its size by using camera adapters with different magnifications.

The reached magnification, seen by the CCD of the camera in the position 1 or 2 respectively 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 limit the usable camera adapter magnification.

The CCD of the camera transforms the incoming light into electrical charge, this is read by the electronics of the used camera; 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

Flash light source

The flash light source creates the light arc, used to illuminate the Field Of View (FOV) in the brightfield scan mode.

The pulse frequency can be more then 1kHz; it means, the scan camera might make more than 1000 images /second.

To create the arc during the camera is ready; the flash light source, as well as the shutter of the camera is triggered (synchronized) by the firmware of the control electronics; the flash light pulse is started if the shutter of the brightfield camera is already open.

Configure the flash light source

Since the software version 1.15 the units of the scanner are configured in the file “MicroscopeConfiguration.ini”, section [Microscope].

The actual version of the flash light source in the scanner Pannoramic 250 is “BrightfieldLightSourceType=FlashLight2010”.

[Microscope]

BrightfieldLightSourceType=FlashLight2010; brightfield scan procedure with 40x magnification is impossible; see also the section [Microscope]

BrightfieldLightSourceType=FlashLight2012; brightfield scan procedure with 40x magnification is possible; see also: “ND filter unit” and “Upgrade to software version 1.16”

Remark

If the Upgrade of the hardware to the software version 1.16 is done the value has to be modified to “FlashLight2012”

Important

If the switch “TRG” or “Vref” is not set to “External” the camera installation may be not finished correctly in the dialog “Microscope settings” or the scan program fails the camera installation with the error message.

The flash light source does not need adjustments.
Maintenance is not required.

Neutral density (ND) filter unit

The ND filter unit is used to adjust the brightness of the light, emitted by the pulsed light arc.
The Home position of the stepper motor is defined by the transition from black to white of the home sensor.
The stepper motor rotates the filter disc and so, the desired intensity of the brightfield illumination can be found.

Mounting

· The flange of the ND filter unit is mounted with four mounting bolts to the scanner plate.

Remove the mounting bolts and pull the entire ND filter unit to the right.

· No adjustments are needed

· Maintenance is not required

Configure the ND filter unit

Since the software version 1.15 the units of the scanner are configured in the file “MicroscopeConfiguration.ini”, section [Microscope].

The actual version of the ND filter unit in the scanner Pannoramic 250 is “NDFilterType=NDType2”.

[Microscope]

NDFilterType=NDType2; see also the section [Microscope]

ND filter disc

The ND filter disc consists of sectors with different intensity of gray filter zones from white (fully translucent) to black.
The appropriate gray level intensity of the ND filter disc is selected by the software during the calibration of the exposure time for the brightfield camera; by rotating the disc with the ND motor, the intensity of the illumination can be selected / adjusted.
Usually, the fifth sector after white is used, but due to the aging process of the light arc (after some years) the used sector may be closer to white; this way, the aging of the light source can be handled also.

Collector lens

The collector lens concentrates the light, emitted from the light arc, and sends it to the ND filter.

· No adjustments are needed

· Maintenance is not required

Collimator lens holder

In microscopes the illumination of the tissue is very important. The collimator lens holder contains the optics to produce light with a high density and coherent rays; so, the field of view can be illuminated evenly.

Components of collimator lens holder

A. Housing; insert the diffuser foil first

B. Insert the yellow filter next.

C. Insert the distance ring; it keeps the convex surface of the lens away from the yellow filter.

D. Collimator lens; the surface of the thinner lens part shows to the light arc.

E. Ring nut

Because the blue and violet part of the emitted light is very much; the yellow filter helps to create a “more white” light.
The diffuser foil insures the homogeneity of the light rays.
The collimator lens creates nearly parallel light rays.

· No adjustments are needed.

· Maintenance is not required.

Upgrade to software version 1.16

· Since the software version 1.16 the user is able to scan tissues in the brightfield scan mode with the 40x objective also.

· Because the 40x objective needs “more light” in relation to the 20x objective, and the actual construction of the illumination path can not deliver the required amount of light, modifications in the illumination path are required; these modifications are done with this upgrade.

Remark

If the Upgrade to the software version 1.16 is not executed, the software version 1.16 can be started and used, but a brightfield scan procedure with the 40x objective will be done in a very poor quality.

Upgrade is required, if:

· The serial number of the scanner is ending with 02 (P250-00xx02) (or less) and in the file “MicroscopeConfiguration.ini” the value of the parameter “BrightfieldLightSourceType=” is “FlashLight2010”!

Upgrade is not required, if:

· BrightfieldLightSourceType=FlashLight2012; the upgrade is already included!

Requirements

· The diffuser foil and the yellow filter foil of the collimator lens holder have to be exchanged into a version that absorbs less light.

Upgrade kit of 40xBF scan for P250; order number: HP-P250-PAX-0200

· A: In earlier developed scanners P250 the “Yellow filter” was placed in this position! Please remove this filter if the upgrade is done; see the image above.

With this solution, the 40x objective get “more light” and the brightness of the virtual tissue, scanned with the 40x objective will increase.

· Please exchange the named foils and make the modifications as described in the chapter “P250_upgrade the illumination”

Finishing

If the Upgrade of the hardware to the software version 1.16 is done the value of “BrightfieldLightSourceType=” in the file “MicroscopeConfiguration.ini” has to be modified to “FlashLight2012”

BrightfieldLightSourceType=FlashLight2012; see also the section [Microscope]

Remark

Scanners with the serial number P250-00xx03 and higher already including this modification and for these scanners the named upgrade is not required or if the value of “BrightfieldLightSourceType=” in the file “MicroscopeConfiguration.ini” is already modified to the value FlashLight2012!

“Possible scan modes with different cameras and magnifications in the software version 1.16; 1.17beta and 1.17

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 “the focus unit with objective changer” for the condenser position adjustment.

· Maintenance is not required

Condenser ; Wikipedia

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, the objective position and the nominal focus position are important.

Remark

Since the software version 1.16 the BF scan session may be executed with the 20x or the 40x objective likewise.

Upgrade to the software version 1.16

“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 Flash illumination unit illuminates the tissue

· The ND filter unit supports enough light

· 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

· Camera changer unit’s mirror stays in the correct working position

If the scan software SlideScanner.exe shows the error message

· “Error occurred” and stops working, please read the temperature values with the service program!

· See also: “Temperature sensor, fan and fan control”

Camera changer unit

The camera changer unit allows the mounting of 2 scan cameras at the same time and to deflect the image to the appropriate scan camera, depending on the scanning mode.

The camera changer separates the common fluorescent (FL) and brightfield (BF) light path, arriving from the tube lens to the appropriate camera, depending on the mirror position.

In the brightfield scan mode, the image is reflected to the camera position 2 and the camera position 1 is selected if the fluorescent scan mode is activated.

The brightfield scan camera is always situated in the camera position 2 and the fluorescent scan camera in the camera position 1.

In the standard version of the P250 the brightfield camera is the CIS-camera, while in the fluorescent scan mode the PCO-edge camera is used.

On the side near to the objective (tube mounting), 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. For best image quality, the tube lens should be mounted into the camera tube until it stops!

The functionality of the camera changer unit is discussed separately.

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

Detailed information can be found in the chapter “Camera changer unit”

Camera changer unit mounting

The camera changer unit is mounted so, that the correct position can be adjusted. With this adjustment the chromatic aberration is corrected and minimized.

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

“Chromatic aberration” and “Reduce chromatic aberration”.

Attention

The camera changer mounting of the P250 differs in length from the camera tube mounting in the SCAN and the MIDI!

Camera adapter

The camera adapter is situated between the camera changer 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 highly on the scan camera’s CCD size, its pixel resolution and the construction of the image path.

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

Camera adapter Carl Zeiss; Product selection

Influence of the camera adapter

The useable magnification of the camera adapter depends on the size of the sensor (useable geometry x and y in pixels), the used objective magnification and the construction of the image path (Length of the camera tube).

· The resulting magnification of the image path is defined by the product of Objective Magnification multiplied by the Camera Adapter Magnification.

Example

If the Objective Magnification is 20x and the camera adapter magnification is 0.63x the resulting magnification of the image path will be 12.6x.

Image magnification = 20 x 0.63 = 12.6

Advantage

By reducing the image magnification, the dimension of the FOV will be increased; the scan speed increases because the number of FOVs to be scanned is reduced.

Disadvantage

The resolution of the virtual tissue is reduced.

Conclusion

· The camera adapter fits the image, seen by the objective into the focus of the camera sensor and influences the resulting magnification of the image path and the size of the FOV.

· If the camera adapter magnification is 1x, then no lenses are inserted, and the sensor is in the focus of the tube lens; the optical magnification is defined by the objective magnification.

· If the camera adapter magnification is 0,63x, then the lens of the camera adapter enlarges the FOV; the resolution of the scanned tissue is decreased.

· If the camera adapter magnification is 1,6x, then the optics of the camera adapter makes the FOV smaller, and the resolution of the scanned tissue is increased!

Brightfield 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.

If the camera uses a CMOS image sensor instead of the CCD device, the necessary modifications are handled by the software.

· See also usable scan cameras and the camera “CIS-VCC-F52U25CL”

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

· Detailed introduction of CCD’s: “http://www.microscopyu.com/articles/digitalimaging/ccdintro.html”

· Digital Camera Resolution Requirements for Optical Microscopy; interactive

“Possible scan modes with different cameras and magnifications in the software version 1.16; 1.17beta and 1.17

Optical path and Field Of View

The pupil of the scan objective is very close to the tissue, so, the small area on the tissue will be enlarged by the objective and the camera adapter.

The seen area on the slide is always defined by the size of the camera’s CCD; more precise, the effective number of pixels horizontal and vertical and the optical means in the image path.

The objective type “Plan-Apochromat” requires a tube lens to create the image. In opposite to other objective types, an infinite space exists between the objective and the tube lens, in which the light rays are parallel.

So, optical means, like the filter block in fluorescent scan sessions can be inserted (by the help of the turret unit)

· The filter block’s components do not affect the magnification of the image path!

Fluorescent scan camera

· See also “CCD versus CMOS image sensor”.

· What is the difference between CCD and CMOS image sensors in a digital camera?

usable scan cameras and camera “PCO.edge”

Camera “AxioCam MRm REV.3”

“Adjustment procedures” to “Adjust the camera rotation angle”

Illumination path 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 of the optical path 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 measurable, the actual adjustment result is used to adjust the next component. This procedure requires adjusting / checking the position of previously adjusted components again!

Illumination adjustment

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 required.

The adjustment of the illumination path is reduced to the adjustment of the objective position and the condenser position.

The successful adjustment of the condenser requires the nominal focus position; so the focus position of the objective must be adjusted correctly before we can adjust the condenser position.

If the FOV is not fully and evenly illuminated, the quality of the virtual tissue becomes poor, 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.

· In the P250, the adjustment of the illumination path is reduced to the adjustment of the objective position and the adjustment of the condenser position.

Adjustment procedure

Measure the thickness without cover slip of the slide to be used for the objective position adjustment and calculate the number of focus steps to be set in the focus unit; calculate the focus position; see also: Check or adjust the objective position

Adjust the objective and focus position

1. Start the scan program “SlideScanner.exe”,

2. Insert a slide with the known focus position for P250.

3. In the tab “Focus” create a live view and set the focus unit to the known focus position of the slide.

4. Now adjust the objective position (with the delivered wrenches) until the tissue becomes in focus.

5. Tighten the counter nut of the objective nut.

6. Execute the auto focus command.

7. The found focus position should not have more then 50 steps in distance to the known or calculated focus position.

8. If the deviation is too much, adjust the objective position more precise.

Remark

In scanners, delivered after spring 2014 this adjustment is simplified; see

“Focus unit with objective changer”; “Dismount or mount the objective”; “Objective position”; "Solution since spring 2014", “Check or adjust the objective position”.

Adjust the condenser position

Create a live view with the BF scan camera in the tab “Focus” and adjust the condenser position.

“Adjust the condenser position”; “Condenser”.

Image path adjustment

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

1. 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 / 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 changer unit position

The position of the 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 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 P250 the position of the tube in relation to the turret plate is modified (with loosened tube clqamp mounting bolts).

· 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.

A zoom factor of 2,73 is very helpful for this adjustment.

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 of the FOV, the adjustment is acceptable; see “Focus position +4 steps”.

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

Reduce 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. To adjust the chromatic aberration use / 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 red or yellow boundaries on the top, and blue boundaries on the bottom (see also above “Chromatic aberration”), move the tube to the red, yellow direction.

1. Start the program “SlideScanner.exe”, type in the service password 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. Switch to “Service’ and “Microscope control”; check the checkbox “Cross line on image”

4. Fit the camera view to the size 1:1 with the button 1:1 and zoom in by using the zoom tool until a zoom value of 2,73 is reached.

5. 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 on only 1 side and the opposite side is blue, the optical system has chromatic aberration; check this behavior on different positions of the tissue also.

Move the scroll bars so, that the cross is visible on the screen. Observe always the center of the field of view; in the near of the cross. To find the desired positions, set a step size in the tool “Object guide” and move the stage by using the movement buttons.

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

7. Move the camera changer on its mounting in the direction, where the red or yellow color of the spot / cell occurs; see also “Position of camera changer unit”.

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

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

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

11. 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.

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

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

14. Tighten the tube mounting bolts and check the result, by repeating the steps 8 to 11. If necessary, repeat the steps from step 6.

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

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

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 FOVs, seen with the viewer does not fit to each other. An acceptable camera angle has less than +-0.5 degrees deviation from zero.

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

“Y- and X-hysteresis” and “X-Y-stage unit”

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.

If the rotation angle can be accepted, the angle value is shown in black (17); an acceptable value has less than 0.5degrees in deviation.

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 -300, 500 and 1300 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.

“Adjust the condenser position” and “Focus unit”

Y- and X-hysteresis

General

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 μm (=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.

“X-Y-stage unit” and “X- and Y-carriage drive unit”.

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 description 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 while 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 / lower and left / 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.