Pcon_Optics_Illumination.htm

Optics, illumination; PCON

For technicians and partly for sales managers!

This chapter handles the components of the brightfield illumination and the brightfield image path for the scanner Pannoramic Confocal. 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 1.19.

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

Contents

Overview

Brightfield illuminated optical path

Configure light sources and units

Components and construction

Brightfield RGB illumination unit

Condenser

Slide Tissue Cover slip

Immersion Liquid Feeder

Illumination path adjustments

Image path adjustment

Overview

The optical path includes the following components:

RGB illumination unit

· Mirror and diffuser

Brightfield illuminated optical path

RGB illumination

The construction of the BF optical path uses only a monochrome camera, the camera PCO.edge 5.5MP, 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 gray scale of the image is made by 14bit 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 resolution, 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.

Brightfield image path

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

The image, created by the objective together with the tube lens, arrives to the imaging system Aurox CC88.

The spinning aperture disk is moved sideward and will not be used to produce the brightfield illuminated image.

The image is prepared by lenses and mirrors and will arrive to the camera.

· In the brightfield image path a filter cube must not be inserted!

The image, seen by the objective, illuminates only one half of the camera’s image sensor.

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.

Configure light sources and units

· The path of the file MicroscopeConfiguration.ini, in the software version with the operating system Windows® 7 is:

C:\ProgramData\3DHISTECH\SlideScanner\MicroscopeConfiguration.ini

[Microscope]

SerialNumber=PCON_xxx

MicroscopeType=3DMic10

MicroscopeSubtype=Confocal

ScanCameraType=

PreviewCameraType=CVrmc_m8_pPro

BarcodeReaderType=PreviewCamera

LoaderType=SL_1Mag_12Slide_Sensor_Horizontal2

CameraChangerType=CC_none

ReflectorTurretType=RT_None

BrightfieldLightSourceType=RGBLedLight

ObjectiveChangerType=OC_2Pos

ObjectGuideXYZType=OGXYZ_FLASH4

FlashUnitType=NoFlashUnit

NDFilterType=NDType_None

PreviewLightType=PreviewLightUnitType_Type2

ShutterMotorType=Shutter_Motor

PowerSwitchBoardType=PowerSwitchBoard_Type1

ConfocalUnitType=ConfocalUnitType_Aurox

WaterFeederType=WaterFeeder_Type1

Components and construction

Brightfield RGB illumination unit

Condenser

Objective

Slide Tissue Cover slip

Brightfield RGB illumination

The illumination unit consists of:

Housing

Housing with:

· two dichroic beamsplitters to route the light rays of Red, Green and Blue to the BF illumination mirror

· Dichroic beamsplitters are mounted in an angle of 45º in relation to the light sources

· the mounting of illumination modules

· Illumination mirror with diffuser

· Mountings to the scanner plate of the PCON; see image above

· Electronics (power supply and control of the LEDs; not shown here)

· The illumination components are mounted to the Illumination unit housing by bolts!

· Adjustments are not required.

· Maintenance is not required.

Part Number: FF580-FDi01-25x36

Dichroic beamsplitter 2; 488nm

· All light wavelengths above 488nm (the red, yellow and green light) passing thru the dichroic beamsplitter all wavelengths below 488nm, the blue and violet light, will be reflected.

In other words:

· The lower wavelengths, below 488nm are always reflected while the higher wavelengths, above 488nm pass through the beamsplitter!

Part Number: Di02-R488-25x36

Working principle

By using the dichroic beamsplitters the required wavelengths for the colors Red, Green and Blue can be filtered from the white light, emitted by the LED.

· Also important in this construction is the emitted wavelength spectrum of the white power LED.

· The violet light, in the range from 390 ~ 420nm does practically not exist.

· The visible white light is defined in the range of approximately 400 ~ 700nm.

Significant colors can be assumed in the following wavelength ranges

Color	Range	Typical
Color	[nm]	[nm]
Violet	390 ~ 430	410
Indigo	430 ~ 450	440
Blue	450 ~ 495	460
Green	500 ~ 560	535
Yellow	560 ~ 590	575
Orange	590 ~ 620	610
Red	620 ~ 690	660

Illumination path

The illumination module creates always white light in the wavelength range of ~400 to 700nm.

· The shown color of the illumination tube is only used to show the arrangement of the light sources in relation to the beamsplitters.

· The illumination modules are switched on separately, so only 1 wavelength range will be created at a time.

· Detailed information about the working principle will be shown in the following.

The Red light source emits light in the range of 400 ~ 700nm

· The unwanted wavelength range from 400 ~ 580nm (yellow, green and blue) will be filtered out (reflected) by the dichroic beamsplitter with a nominal wavelength edge of 580nm.

The Green light source emits light in the range of 400 ~ 700nm

· The unwanted wavelength range from 590 – 700nm (yellow, orange and red) will be filtered out (passes thru) by the dichroic beamsplitter with a wavelength edge of 580nm.

· The blue part will be filtered out (reflected) by the dichroic beamsplitter with a wavelength edge of 488nm.

The Blue light source emits light in the range of 400 ~ 700nm

· The dichroic beamsplitter would also reflecting violet light, but because the power LED emits only blue light (from about 420nm) in practice, the violet part does not exist.

Power LED module

The power led module creates white light and is used to illuminate the Field Of View (FOV) in the brightfield scan mode.

· Because the brightfield image is created from the colors RGB the module exists 3 times in the brightfield illumination unit; the wire color is used to find the appropriate connector easily.

The pulse frequency may be more than hundred Hz; it means, the scan camera can make more than 100 images /second.

To switch on the LED during the camera is ready; the led module is triggered (synchronized) by the PCO.edge camera.

· The LED module is inserted into the Illumination tube until it stops!

· Adjustments are not required.

· Maintenance is not required.

See also: “RGB BF scan illumination”

Illumination tube

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

Because the brightfield image is created from the colors RGB the illumination tube exists 3 times in the brightfield illumination unit; there are no differences in the construction.

· The illumination tube is mounted to the Illumination unit by 2 bolts!

· Adjustments are not required.

· Maintenance is not required.

· The white light, emitted by the LED will be collected by the aspheric lens and will be arranged to parallel light rays.

· The light rays crossing the diffuser and are send to the dichroic beamsplitter.

Illumination module

An illumination module consists of the LED module and the illumination tube.

· The Illumination module does not contain wavelength range filtering components!

· Adjustments are not required.

· Maintenance is not required.

· To ensure, that the distance of the illumination module to the condenser is equal for all three colors, the light path of blue got a light path length equalization tube!

· The construction does not contain wavelength range filtering components!

· Adjustments are not required.

· Maintenance is not required.

Mirror and diffuser

· Adjustments are not required.

· Maintenance is not required.

Connections

· Connect the appropriate cable to the specified connector

See also: “Power and control” and “RGB BF scan 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.

· See also the “condenser unit” for the condenser position adjustment.

· Maintenance is not required

See also: “Confocal unit”

Camera adapter ring

The C-mount camera adapter ring is situated between the camera and the Aurox CC88 spinning disk unit.

· The usable magnification of the C-mount Camera Adapter Ring is always 1:1!

· Drive the C-mount Camera Adapter Ring manually first onto the camera until it stops

· Loosen the fixing bolt

· Move the latch as shown to open the clamp and insert the C-mount Camera Adapter Ring with camera

· Release the latch and adjust the camera rotation angle

· Tighten the fixing bolt

Mount the Aurox CC88 spinning disk unit

· Drive the fitting ring onto the 60N Photo port manually, until it stops

· Loosen the mounting bolt on the Aurox unit a bit.

· Put the Aurox unit onto the fitting ring and so, that the fluorescent light input shows to the Lumencor SPECTRA light engine.

· Tighten the mounting bolt

Scan camera

· In all scan modes, the camera PCO.edge with a resolution of 5.5Mp is used until otherwise specified.

· The camera produces monochrome, gray scaled images.

· See: Camera “PCO.edge”

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

· “CCD versus CMOS image sensor”.

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

· CMOS Image Sensor Technology

· Random Access CMOS - Sensoren in der Bildverarbeitungspraxis

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 or 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 possible.

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 PCON, 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 PCON.

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 until the tissue becomes in focus.

5. Fix the objective position by tightening the fixing bolt 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.

Adjust the condenser position

Create a live view with the BF scan mode in the tab “Focus” and adjust the condenser position; use the green light source.

See also: “Adjust 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 or 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. Photo port and tube lens position

The position of the tube lens as well as the position of the 60N photo port affects the color trueness of the scanned tissue; the chromatic aberration becomes visible in blue, and red or yellow colored cell borders on the opposite sides. Because both components are mounted separately, both components have to be adjusted well; see also “Chromatic aberration” and “Straightness of the image path”.

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’.

Straightness of the image path

The straightness of the image path is affected by all components, included between the condenser’s mounting until the sensor of the camera.

The entire adjustment includes:

1. Straightness of the optical axis between condenser and objective (objective disc mounting);

see “Align the objective into the optical axis”

2. Straightness of the tube lens mounting position is in relation to the objective mounting;

see: “Align the tube lens”.

3. Straightness of the 60N photo port position is in relation to the tube lens mounting;

see: “Align the 60N photo port”.

4. Inclination of the Aurox unit is in relation to the momentarily defined optical axis (requires software aided adjustment).

Remark

· Not finished yet; procedures will be described in future updates

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 the 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 PCON the position of the tube lens and the position of the photo port in relation to the objective (changer) will be modified (with loosened tube lens and photo port 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, the adjustment is acceptable; see “Focus position +4 steps”.

The images are 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. 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 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 or 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 or 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 component’s mounting bolts and check the result, by repeating the steps 8 to 11. If necessary, repeat the steps from step 6.

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

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

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 than +-0.5 degrees deviation from zero.

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

See the next chapter “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 then 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.

See also: “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”.

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 difficult to do and should not be performed in the field.

· See also “X-Y-stage unit” and “X- and Y-carriage drive unit”

· Further information: “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 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 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 or lower and left or 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.