Optics, illumination; D_II

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and the optical path for Pannoramic scanners; previously released descriptions about the brightfield illumination are no longer valid.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.

 

 

 

       Enhancements_2016

 

 

Contents

 

Overview

Components and construction

Optics adjustments

            Brightfield illumination

            Image path adjustment

            Check the optical path adjustments

 

 

·        For safety regulations regarding human health and scanner functionality please refer to:   Precautions

 

 

 

 

 

 

 

                         Overview

 

 

The BF optical path can be devided principially into an illumination part and an image part.

The border between both is the specimen.

The bottom of the specimen is illuminated, while the top of the specimen emits the image.

 

Illumination

In the DESK_II the specimen's illumination is done by an RGB illumination unit, an illumination mirror and the condenser.

The RGB illumination unit creates monochrome light in the wavelength of Red, Green and Blue, sequentially, so, the specimen's Field of View is three times illuminated.

The control of the illumination is mainly done by the shutter time of the camera (triggering), the timing may be done hardware or software controlled.

The created monochrome, parallel  wavelengths are reflected by the illumination mirror in an angle of 90° to the condenser and this focuses the lightrays to the field of view, observed by the Objective.

 

Image

The Quality of the image is mainly influenced by its size, resolution, brightness and contrast.

Size and resolution is influenced by the camera adapter and the sensor parameters of the scan camera, while other image parameters are mainly influenced by the construction of the image path, the image illumination and image magnification.

The objective gathers the light rays, passed through the image and arranges these, together with the tube lens to an image.

The size and resolution of the image may be varied in limits by the magnification of the camera adapter.

 

Watch video:            Optical path

 

 

 

 

 

 

 

 

 

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.

 

 

 

 

 

 

                         RGB BF illumination unit

 

The construction of the BF optical path uses only a monochrome camera, so only monochrome images can be produced.

 

To create color information of the tissue with a monochrome camera, we illuminate the tissue with monochrome light.

 

If the tissue is illuminated by blue light, and we are making an image of the Field of view, the gray scaled camera image contains the intensity of the blue parts in the tissue.

 

Because the pixel resolution of the camera is very high and the resolution of the image's gray scale is 12bit per pixel, very detailed information of the blue part in the FOV related to the appropriate pixel can be reached.

 

If we repeating the procedure with the colors Green and Red, 3 images of the same FOV are produced and so, the software knows detailed color information about each pixel of the Field Of View.

 

By using the software coloring method the true color information of each pixel is found.

 

By using cameras with a large image sensor low shutter time and high pixel resolution (small pixel size), the scan time of the tissue can be held in acceptable boundaries and the result is an image with high resolution and high color fidelity.

 

 

 

 

   RGB BF illumination

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                            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

 

Watch video:            Condenser

 

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.

 

·    See also “the focus unit”.

·    See also “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

 

 

Exchange of the objective

 

As mentioned before, the focus unit may be equipped with a 20x or a 40x objective. Because the focus position of the 40x objective is very close to the tissue, the scan area, defined for the 20x objective is no longer valid, to avoid collision of the objective with the specimen holder.

If the objective will be changed from a 20x to a 40x magnification, the scan area must be defined again and therefore the preview calibration must be executed again also. During defining the scan area for the 40x objective, the position of the focus pin and the position of the objective pupil should be checked to avoid touching of the specimen holder! Adjust the limits in the nominal focus position!

If the objective is changed from the 40x to a 20x magnification it is not bound to define the scan area again, if the smaller area can be accepted. In all cases, if the scan area was modified, a preview calibration is bound to do.

 

 

       Working distances” and “How to exchange the objective in DESK

                        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

 

 

 

 

 

 

 

 

 

                                            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 photo port.

 

 

       Optical path and Field Of View

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                            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, the magnification and the resolution of the image.

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

 

 

Watch video:            Camera adapters

 

                  Camera adapter          CARL ZEISS; microshop

           

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                   Influence of the camera adapter

 

The useable magnification of the camera adapter depends on the size of the camera's 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 in the focus of the camera sensor (with its length) 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!

 

 

 

 

 

 

 

 

 

 

 

 

                                            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.

 

In the DESK_II the default camera is the monochrome "Grasshopper 3", manufactured by POINTGREY.

 

 

             Prerequisites” for usable scan cameras, and Grasshopper3.

 

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                   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 sensor; 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 image mirror in the DESK type scanners or 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!

 

 

 

 

 

 

 

 

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 required.

 

·    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 again, after the chromatic aberration and the camera rotation angle is adjusted.

 

 

 

       Brightfield Microscopy

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                            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 1050, 1850 and 2650 steps, there must not be significant differences.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 position 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 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 Condenser and objective position is correct! 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.

 

 

       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 (minimal) 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.

 

·    See the 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 bottom 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. 

 

       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  is a bit more complicated and should not be done in the field.

 

 

       X-Y-Stage unit and

                        How to exchange the Y-drive unit

 

 

 

 

 

Chromatic aberration

 

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

 

·   See also the chapter 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 and mounted again.
  • 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 misaligned or deformed.

 

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

 

See also the “X-Y-stage unit”.

In the example on the right the most difference is 2 steps and therefore the inclination of the specimen holder is acceptable.

 

1.        Start the program SlideScanner.exe with the service password, insert the slide with circle, produce a live view and press auto focus.

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

 

2.     Find the circle and bring it nearly into the center of the live view, press auto focus.

 

3.     Select the tab “Service” and “Microscope control”.

 

 

 

 

 

 

 

 

4.     Select a step rate about 5 or 10 steps for the object guide.

5.     Check the checkbox “Cross line on image” and with the object guide movement buttons bring the center of the circle to the center of the cross; the circle is now in the center of the FOV.

 

6.     Uncheck the checkbox “Cross line on image”

 

7.     Zoom in until a value of 2,73 is reached.

 

8.     Grab the center of the circle (FOV) into the middle of the screen.

 

9.     Memorize the auto focus position and go backward with the focus position about 20 steps; and then go forward to the auto focus position -3 steps with a step size by 1. This way, the probably hysteresis of the focus unit and other mechanics is eliminated.

10. Make a screenshot and create a directory named “Focus stack”, name the file as C (for center) and the number of the actual focus steps, e.g.  “C 1659” if the memorized focus position was 1662 steps and save the file into the directory “Focus stack”.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11.   Increment the focus position by 1, make the next screenshot and save the file.

 

12. Repeat step 11 until all the 7 screenshots are done.

 

13. Now move the circle with the object guide positioning buttons to a corner position, e.g. to the top left corner. The corner is found correctly if the circle can not be grabbed in direction to the center (see also the green arrows in the image above “Field of view”).

 

14. Repeat the steps from step 9 logically until the screenshots are done in all four corners. The file names should be TL xxxx, BL xxxx, TR xxxx and BR xxxx (for Top Left and so on).

 

Find the screenshot with the circle most in focus for each series and notify the file names.

 

Decide the specimen holder has either to be adjusted or not as shown in the image above “The field of view”).