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PureFocus850 Installation Guide
Author
Date
Version
Status
Aims
Simon Bush
12
March 2021
th
2.2
Released
This document contains an in depth
guide to the PureFocus850, including a
full installation walkthrough for both
automatic and manual setup. The
troubleshooting section has been
greatly expanded and representative
signal morphologies are shown
throughout.
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Table of Contents
Related Manuals for Prior Scientific PureFocus850
Summary of Contents for Prior Scientific PureFocus850
- Page 1 March 2021 Version Status Released Aims This document contains an in depth guide to the PureFocus850, including a full installation walkthrough for both automatic and manual setup. The troubleshooting section has been greatly expanded and representative signal morphologies are shown...
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Page 2: Table Of Contents
Thank you for purchasing this product from Prior Scientific – we are confident it will be a reliable and useful addition to your microscope system. Please take the time to read and understand this manual before using this product – it contains not only important operating instructions but also vital safety information. - Page 3 7.6 Software Limits via Range Flag Section 8: OEM features 8.1 Interface selection 8.2 Focus search Section 9: ASCII commands 9.1 Signal settings commands 9.2 Focus signal commands 9.3 Servo settings commands 9.4 Flag settings commands 9.5 Objective parameters commands 9.6 Digipot settings commands 9.7 Focus commands 9.8 System commands...
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Page 4: Section 1: Important Safety Information
PureFocus850. If you have any questions do not hesitate to contact Prior Scientific. If you intend to use this unit in a manner not specified by Prior in this manual, contact Prior Scientific beforehand. -
Page 5: Section 2: Product Description
Section 2: Product Description 2.1 What is the PureFocus850? The Prior Scientific PureFocus850 is an advanced, integrated, unit comprising of an IR laser diode, precision optical components, detector and signal processing electronics with on-board micro controller. The system allows optimum visual focus to be found and maintained on a microscope system for a range of different sample types, microscope objectives and imaging methods. - Page 6 Due to the nature of the line sensor there is freedom to choose what range of pixels either side of the centre point are used in calculating a focus error signal. This allows the rejection of reflections from spurious reflective surfaces and offers great flexibility when dealing with various different samples. The addition of adjustable laser collimation also allows the reflective surface, used by PureFocus for holding focus, to be located at a different plane to the microscope’s imaging plane, where the specimen resides.
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Page 7: Types Of Microscope System
2.3 Typical sample types The PureFocus850 is designed with these common sample types in mind: • Permanent fixed biological slides with a 1.5 thickness coverslip • Cell culture dishes/wellplates •... -
Page 8: Section 3: Hardware Installation
Section 3: Hardware Installation 3.1 Types of microscope system PureFocus850 works on a range of different microscopes which use infinity corrected optics. The PureFocus head sits in the infinity space of the optical path between the objective and tube lens. - Page 9 Each system will contain a target for optical alignment of the PureFocus850 infrared laser beam with the microscope. Each target has a range of thread sizes in order to be compatible with a variety of microscope nosepieces. PF200 Alignment target for RMS, M26 and M32 threads...
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Page 10: Installing Your Pf850 Mounting Kit
Once installed, connect the head to the controller via the PF400 cable. Connect the controller to the mains using the H407 power supply. Connect the focus system to the 15-pin D-connector on the PureFocus850 controller. Use the PF404 cable to link BNC outputs from piezo nano-positioning systems to this connector. -
Page 11: Pf850 Controller Guide
Turn the blade screw clockwise until it reaches the limit. It is set close to the limit during factory processing. Do not force the screw. Switch on the controller and start the PureFocus850 GUI. Reset the PureFocus to factor defaults by clicking Parameters > Factory Reset. This will close the GUI. - Page 12 (see advanced features). If no sample is detected by the PureFocus850 (in any mode), this is indicated by the appearance of the letter NS next to the objective number indicator. This overwrites the ‘F’ focus indicator even if the sample is in focus.
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Page 13: Section 4: Optical Alignment
(Fig. 4). Open the ‘camera’ application, select USB Camera 2.0, and then click start. Start the PureFocus850 GUI. Go to the offset submenu and select ‘Go to Limit’. The offset lens will move in the PureFocus850 head. - Page 14 Open the laser submenu in the PureFocus850 GUI and adjust the laser power to ensure the signal is clearly visible but not reflecting off the target. Typical values for this are 400-1000, however this varies between systems and values outside of this range are acceptable if required.
- Page 15 Using a flathead screwdriver, rotate the setup screw to reduce the spread to the laser signal to a minimum (Fig. 6). Using the 45 screw, adjust the lateral position of the laser line so that it aligns over the centre of the target (Fig.
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Page 16: Laser Setup Considerations
4.3.1 Sensor centre setup for slice mode systems In the PureFocus850 GUI, go to the offset submenu and click ‘Go to Factory Home’. The offset lens will move. Go to the laser submenu and increase the value to 100. Click set. - Page 17 4.3.2 Sensor centre setup for line mode systems In the PureFocus850 GUI, go to the offset submenu and click ‘Go to Factory Home’. The offset lens will move. Go to the laser submenu and increase the value to 100. Click set.
- Page 18 Use the digipot, in MAN FOC mode, to achieve focus. Click Setup, which will open a graph that indicates the laser intensity over the PureFocus850 sensor. Adjust focus until a peak appears in the signal. At this stage, the peak may be very small or may not be visible at all, even at high laser power.
- Page 19 See section 4.3.3 for full details. Click the Auto Set Centre button, and click ‘Yes’ in the following pop-up box. This will set the centre of the PureFocus850 sensor, which is used to positively verify whether the sample is in focus.
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Page 20: Creating Signal Imbalance
4.3.3 Laser Setup Troubleshooting The appearance of the peak does not necessarily correlate with sample focus. It is normal if the sample is out of focus whilst the peak is present. If the signal is a flat line with no features and no features appear following refocusing, increase the laser power in 500 unit steps and see if any features appear. - Page 21 Rotating anti-clockwise, adjust the blade screw on the side of the PureFocus Head until the blade blocks the left half of the peak, up to the sensor centre that was set up in the previous step (Fig. 12). Note that the blade will not remove any background signal. The blade screw has considerable tolerance built in so expect to undertake a large number of screw rotations during this step.
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Page 22: Removing Background Signal
4.5 Removing background signal Move the dustcap into the light path, leaving any illumination and the PureFocus850 switched on. The signal above the background will disappear. Click Measure Average Background. Click ‘Yes’ in the following pop-up to apply this background setting to all objectives. -
Page 23: Section 5: Focusing System Setup
4.5. WARNING: this step is essential for operation of the PureFocus850. If the focus direction is set incorrectly this can create a positive feedback loop. This can cause a system crash which may... -
Page 24: Stepper Motor Focusing System
5.1 Stepper motor focusing system Prior Scientific focus motor systems should be installed on the left hand focus knob when possible. Move the lowest magnification, longest working distance objective into the optical path. Go to the mode submenu and select the ‘Stepper’ option. -
Page 25: Section 6: Autofocus Parameter Setup
Complete the following autofocus parameter setup first using the PF209 setup slide in order to ensure user familiarity with the various controls. Once the offset value has been saved and PureFocus850 servo has been successfully activated using the setup slide, review and adjust the parameters in line with your samples by repeating the procedure. -
Page 26: Objective Selection
6.2.1 Focusing on the sample Prior Scientific recommends using a 5x, 10x or 20x objective when setting up an objective for the first time, although any objective can be used. Once steps 6.3-6.7 have been understood, those steps will... -
Page 27: Setting The Offset
6.3 Setting the offset The offset between the reflective plane that the PureFocus850 detects and the focal plane of interest needs to defined for each objective. Materials samples typically have an offset close to zero but the optimal performance is achieved by following the below procedure. - Page 28 Proceed to step 6.3.4. Note however that higher magnification objectives may not allow enough laser signal to operate the PureFocus850. If low peak maximums are already apparent at 5-20x magnification, or no peak can be identified, refer to Section 10: Troubleshooting.
- Page 29 In rare cases, a sample may require a negative offset (Fig. 15). This is where the focal plane of interest sits above the main reflective interface in the sample. The PureFocus850 has a limited negative offset range that can be used if required.
- Page 30 Section 10: Troubleshooting. 6.3.4 Checking error swing Error swing is essential for operation of the PureFocus850. The speed and magnitude of swing contribute to the focus recovery range and focus stability. Error swing is indicated by the green bars in the centre of the main interface of the PureFocus850 GUI.
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Page 31: Checking For Back Reflections
6.4 Checking for back reflections Reflections from other reflective surfaces, such as lenses inside the objective, may produce unwanted signals on the PureFocus850 sensor. It is important to minimise the impact of these signals on the error swing. Remove the sample and turn off all illumination sources. The error signal is now derived solely from the reflection of the PureFocus850 laser from components of the optical path. - Page 32 If the signal is localised to a small number of pixels, often in the form of a large peak located away from the pinhole centre, reduce the pinhole half width in the pinhole submenu such that the signal is removed from the pinhole boundaries. This will exclude this signal from the error value calculation (Fig.
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Page 33: Setting Focus Recovery Speed
The objective pre-set values loaded during section 6.2 contain PID values. PID is a common control loop mechanism that allows the PureFocus850 to maintain focus on the sample. The magnitude of the proportional (P), integral (I) and derivative (D) gains define the rate at which the PureFocus850 recovers sample focus and focus stability. - Page 34 6.5.1 Stepper motor control Stepper motor systems can be used effectively using suitable P-gain values. Bring the sample into focus. The error value should be close to zero. Put the controller into SRV mode by pressing the Servo on/off button on the controller. If the image is unstable, reduce the P gain by a factor of 10.
- Page 35 Objective pre-set values for piezo control also have a defined output limit. This will limit the distance the piezo can travel. If set to low the piezo system will have a limited travel range, potentially preventing focus recovery. If set too high the piezo system will repeatedly attempt to drive over its limits.
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Page 36: Fine Offset Adjustments
6.5.3 D-gain Derivative (D) gain is not strictly required for any focus systems to achieve near-optimal performance. Prior Scientific strongly advise that only users with experience of PID control mechanisms attempt to use this value. 6.6 Fine offset adjustments Offsets values calculated using the auto-find function or by a manual setup are likely to be close to the optimal. -
Page 37: Saving Your Settings
Focus correction is dependent on the error value, which is generated by an imbalance of reflected signal on each side of the PureFocus850 sensor. The error value must be close to zero when the sample is in focus and increase linearly to +1 or -1 either side of focus (see section 6.3-6.4 for more details). - Page 38 If you have not set up an offset before, refer to section 6.3.2. Click on Setup in the main PureFocus850 GUI. A signal should be present at the pinhole centre. Click on Laser in the main PureFocus850 GUI.
- Page 39 Repeat for each objective in the objective set. Individual objectives, especially if they use a different imaging method or light source, may also exhibit excess background when compared to others in the objective set. Mouse over the background signal to determine an approximate value, which will be displayed as the Y-coordinate underneath the setup graph.
- Page 40 (Fig. 19). 7.2.4 Signal to noise considerations Laser power and pinhole width have opposite effects on PureFocus850 performance. Raising the laser power increases focus recovery speed, but increases background signal. Decreasing the width of the pinhole helps to remove unwanted signals from the error value calculation, but can also limit the focus recapture range if set too narrow.
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Page 41: Enhanced Focus Control Via Focus Flag
Slowly adjust the focus on the sample using the controller digipot. Check the error value displayed on the main PureFocus850 GUI. Determine the maximum error value which can be allowed whilst also maintaining focus on the sample. Increase the value into the error parameter field in the Focus Flag submenu to this value. - Page 42 Bring the sample into focus, ideally by switching the controller into SRV mode. Open the Pinhole submenu in the main PureFocus850 GUI. Click on setup to open the setup graph.
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Page 43: Sample Detection Via Sample Flag
Rotate the digipot on the controller. The focal plane will change as the autofocus corrects for the movement of the offset lens. Once a new focal plane has been selected, click Offset in the main PureFocus850 GUI. Click Set, and select ‘1’. Repeat for any further focal planes, using the next available number in the list. -
Page 44: Software Limits Via Range Flag
Similarly, immersion objectives may produce complex signals with multiple interfaces. Any of these interfaces can be used by the PureFocus850 assuming the error value is zero at a given offset value. The signal reflected at each of these interfaces can be used, via the interface flag, by software developers to trigger unique scanning behaviour in the PureFocus850. - Page 45 Use the digipot to adjust the focus on the sample. Observe each time a peak is visible across the pinhole centre. Each of these peaks is a distinct interface in the sample. It is advantageous to use from the strongest peak in the sample as focus capture range and focus stability will be enhanced, however this is not mandatory.
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Page 46: Focus Search
8.2.1 Search and Lock This can be used when the sample flag is false and PureFocus850 is unable to capture focus. The routine will carry out a ramp of the Z-axis across a specified peak to peak (pk to pk) range and at a specified speed. - Page 47 Set the pk to pk range for the scan. The value refers to the maximum Z-axis distance will move during the scan. Refer to the Z position field in the main PureFocus850 GUI. The default setting, 10, gives a peak to peak range of 1 micron.
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Page 48: Section 9: Ascii Commands
Section 9: ASCII command sets The PureFocus850 communicates with a baud rate 460800, 8 data bit, no parity, 1 stop bit, no flow control. All commands/responses are terminated by <CR> character. 9.1 Signal settings commands Command Response Description PINHOLE Centre, width... -
Page 49: Servo Settings Commands
TEST, t Begins test mode: t=0, disabled t=1 continuously prints TARGET,INPUT,ERROR,OUT PUT to the terminal window t=2 continuously prints A,B,C,D to the terminal window 9.3 Servo settings commands SERVO Queries current servo state, 0 = off, 1 = on SERVO, N Turns servo on or off respectively,0=off, 1=on Query proportional gain for... -
Page 50: Flag Settings Commands
9.4 Flag settings commands SAMPLE Returns current sample state: s=0, sample not detected s=1, sample detected s=1 when A+B>SAMPLEL SAMPLEL Returns the low threshold l for determining the sample state for the current objective SAMPLEL,l Sets the low threshold l for determining the sample state for the current objective FOCUS... -
Page 51: Objective Parameters Commands
INTERFACEH,h sets the high threshold h for determining the interface state for the current objective INTERFACEL returns the low threshold l for determining the interface state for the current objective INTERFACEL,l sets the low threshold l for determining the interface state for the current objective INHIBIT Returns the servo inhibit status:... -
Page 52: Digipot Settings Commands
PINHOLE,750,750 REGION,D,0,0 BACK- GROUND,0,0,0,0 LENSSO,1,0 LENSSO,2,0 LENSSO,3,0 LENSSO,4,0 LENSSO,5,0 9.6 Digipot settings commands LENS Returns current function of the digipot control: l=0, focus motor/piezo l=1, offset lens Return speed setting of digipot for focus control, n= 1-100% OF,n Set speed setting of digipot for focus control, n= 1-100% Return speed setting of digipot... -
Page 53: Focus Commands
LENSSO,n Save current offset lens position to stored offset lens position n=1 to 5 LENSSO,n,p Save position p as stored offset lens Position p in steps n=1 to 5 9.7 Focus commands Reports the microns/revolution u of the focus drive in stepper mode (default is 100) or the full travel range (microns) in piezo... - Page 54 SMZ,n Set maximum focus speed n in 1-100% range, n=1 to 100 Return maximum focus acceleration n in 1-100% range, n=1 to 100 SAZ,n Set maximum focus acceleration n in 1-100% range, n=1 to 100 SMZ,U Return maximum focus speed n in microns/s SMZ,n,U Set maximum focus speed n...
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Page 55: System Commands
9.8 System commands DATE Prior Scientific Instru- ments Returns date of software OptiScan LF Version (n) compilation and build number, complied (date & time) SERIAL decimal number Returns serial number of control box RESET Resets unit and all parameters RESTART... -
Page 56: Section 10: Troubleshooting
10.1 No laser line emitted visible on the target Open the main PureFocus850 GUI. The laser power is set to zero by default during installation. Increase the laser power to 500 and the laser line will become visible. Increase the laser power further if necessary. -
Page 57: Laser Signal Is Too High Or Too Low In Setup Mode
WARNING: The following procedure will cause the laser to be emitted up towards the user. Do not look into the exit aperture of the PureFocus850 head or stare at the laser line visible on the tissue paper. Do not undertake this procedure if you are not able to work safely. -
Page 58: Additional Peaks Are Visible In Setup Mode
10.3 Additional peaks are visible in setup mode Additional peaks are a common feature in certain sample types e.g. cell culture dishes. They can also be features of certain objectives or other components in the light path. Use the pinhole half width function and laser power to eliminate these peaks from the error value recalculation. - Page 59 objective. The laser power can then be increased such that only the peak is visible on the graph. Note this will impact focus stability; see to section 7.3 for methods to improve focus stability. Illumination can also create significant amounts of background signal if it has a high infrared component, which produces a signal morphology similar to the below.
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Page 60: No Or Imbalanced Error Value Swing
Attempt to deliberately misalign the dichroic in one axis by making very small adjustments to control. When making this adjustment, observe the sensor signal in Setup Mode to confirm that the signal size is not compromised. 10.5 No or imbalanced error value swing 10.5.1 No error value swing... -
Page 61: Focus Recovery Is Too Fast Or Focus Unstable
If there is no error swing after calculating an offset, it is likely that one of the above situations is true. The peak identified during offset calculation is not derived from the sample, and therefore cannot be used by the PureFocus850. 10.5.2 Imbalanced error swing... -
Page 62: Performance Is Good But The Flags Are Inactive
10.9 Performance is good but the flags are inactive For many users, flag setup is not required and it will not impact performance of the PureFocus850. See sections 7.3, 7.4, 7.6 and 8.1 to set up the focus, sample, range and interface flags respectively. -
Page 63: A Suitable Offset Cannot Be Calculated For An Objective
Refer to section 6.7. If your signal morphology is complex, the PureFocus850 may be using the incorrect reflection from the sample to offset from. There are a number of options for dealing with this scenario. - Page 64 Thicker coverslips may therefore prevent the use of these objectives with the PureFocus850. The PureFocus850 has a limited negative offset range (typical maximum = -13,000). Certain materials objectives, or samples which have their focal plane of interest above their first reflective layer, use this range.
- Page 65 ‘set pinhole centre’. The vertical blue pinhole centre line will now be aligned with the peak. Typical values for the pinhole centre are between 300 and 500, but others are acceptable. Lower values can be used but this restricts the size of the PureFocus850 sensor significantly (Fig. 25).
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Page 66: Z Scan
If the unit is not working, first contact either your distributor or Prior Scientific directly. If you wish, or are advised to, return your unit, a RMA number must first be obtained by filling in a returns form located on the prior.com website. We will then send you an RMA number which should be added to a decontamination form again located on the Prior Scientific website. -
Page 67: Section 12: Controller Z Connector Pinout
Phone: +86 (0)512 6617 5866 Section 12: Controller Z connector Pinout The 15 way D connector on the PureFocus850 controller allows connection to either a stepper motor or a piezo actuator system which can accept an analogue 0-10V input. Pin connections are as follows:... -
Page 68: Appendix 1: Windows Usb Driver Update
Appendix 1: Windows USB Driver Update Recently the PureFocus850 controller has had an upgrade/change to the USB connection hardware. The company FTDI Ltd. manufactures the hardware and constantly updates the associated Windows USB Driver. The new USB hardware and interface driver is Windows Hardware Quality Labs (WHQL) tested and will automatically download the latest drivers when connected to a PC with internet access. - Page 69 Right clicking on the PF850 USB port in Device Manager will allow you to click on the Properties menu. In the Properties window, click on the Driver tab and view the Driver Date: If it is older than 2016 or if the Driver Provider is not listed as FTDI, see the image below, a new driver needs to be installed.
- Page 70 It is recommended to uninstall the existing FTDI drivers. There is an uninstaller tool that can be accessed from http://www.ftdichip.com/Support/Utilities.htm#CDMUninstaller From the website download the CDM Uninstaller as shown below...
- Page 71 Extract all the files and run the CDMuninstallaerGUI. Click the Add button on the right and the Vendor ID and Product ID will be populated inside the blank area. Download the latest USB Driver version from http://www.ftdichip.com/Drivers/VCP.htm Download the proper 32- or 64-bit driver as shown below. Extract all of the files to the desktop or other download folder.
- Page 72 Choose Browse my computer for driver software Browse to the location of the saved Driver and click Next Windows will then update the Driver using the downloaded files. A successful installation will show a new date in the USB properties as shown below.
- Page 73 If at this point a COM port has not been assigned to your USB connection, retry the “Update Driver More to PRIOR than meets the eye Version issue No. - 2.0 For more information visit www.prior.com40 41 Software”, as in some instances Windows will not assign a proper COM port the first time the driver is updated.
- Page 74 Click OK twice to save the Latency Timer change.
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