Prior Scientific PureFocus850 User manual

PureFocus850 Installation Guide
Author
Simon Bush
Date
12th March 2021
Version
2.2
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
throughout.

2
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. Use this product only as specified in this
manual. If you wish to use it differently, contact Prior Scientific beforehand.
Please do not hesitate to contact us with any comments or questions regarding this product.
Contents
Section 1: Important Safety Information
4
1.1 Important Safety Information
4
Section 2: Product Description
5
2.1 What is the PureFocus850?
5
2.2 Working Principle
5
Section 3: Hardware Installation
7
3.1 Types of microscope system
7
3.2 Types of Illumination
7
3.3 Unpacking your system
7
3.4 Installing your PF850 mounting kit
10
3.5 Installing the PF850
10
3.6 PF850 Controller Guide
11
Section 4: Optical Alignment
13
4.1 Aligning the PF850 with the optical path
13
4.2 Laser setup considerations
16
4.3 Setting the sensor (pinhole) centre
16
4.4 Creating signal imbalance
20
4.5 Removing background signal
22
Section 5: Focusing System Setup
23
5.1 Stepper motor focusing system
24
5.2 Piezo nanopositioning focusing system
24
Section 6: Autofocus Parameter Setup
25
6.1 Installation Review
25
6.2 Objective selection
26
6.3 Setting the offset
27
6.4 Checking for back reflections
31
6.5 Setting focus recovery speed
33
6.6 Fine offset adjustments
36
6.7 Saving offset values
36
6.8 Parameter setup for remaining objectives
36
6.9 Saving your settings
37
6.10 Flags
37
Section 7: Advanced features
37
7.1 Non-typical sample types
37
7.2 Signal to noise
37
7.3 Enhanced focus control via Focus Flag
41
7.4 Sample detection via Sample Flag
43
7.5 Using multiple offsets
43

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7.6 Software Limits via Range Flag
43
Section 8: OEM features
44
8.1 Interface selection
44
8.2 Focus search
46
Section 9: ASCII commands
48
9.1 Signal settings commands
48
9.2 Focus signal commands
48
9.3 Servo settings commands
49
9.4 Flag settings commands
50
9.5 Objective parameters commands
51
9.6 Digipot settings commands
52
9.7 Focus commands
53
9.8 System commands
55
9.9 Advanced commands
55
Section 10: Troubleshooting
56
10.1 No laser line emitted visible on the target
56
10.2 Laser signal is too high or too low in setup mode
57
10.3 Additional peaks are visible in setup mode
58
10.4 Background signal in setup mode is high or uneven
59
10.5 No or imbalanced error value swing
60
10.6 Focus recovery is too fast or focus unstable
61
10.7 Focus recovery is too slow
61
10.8 Focus recovery does not occur despite a good error value swing
61
10.9 Performance is good but the flags are inactive
62
10.10 Focus locks when my sample is not in focus
62
10.11 A suitable offset cannot be calculated for an objective
63
10.12 Z Scan
66
Section 11: Spare parts, repairs and returns
66
Section 12: Controller Z connector Pinout
67
Appendix 1: Windows USB Driver Update
68

4
Section 1: Important Safety Information
1.1 Important Safety Information
Class 1 laser product, laser wavelength 850 nm, laser output < 0.77 mW
CLASSIFIED TO BS EN 60825-1:2014
It is important to follow these safety warnings to avoid potential injury or damage. Please read and
understand these warnings, operating instructions and specifications before using the 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.
SAVE THIS MANUAL AS IT CONTAINS IMPORTANT INFORMATION AND INSTRUCTIONS.
Before using the system, please follow and adhere to all warnings, safety and operating instructions
located either on the product or in this User’s Manual.
•Do not expose the product to water or moisture.
•Do not expose the product to extreme hot or cold temperatures.
•Do not expose the product to open flames.
•Do not allow objects to fall on or liquids to spill on the product.
•Do not touch the glass plate fitted between the circular dovetail and the top plate. Any dust,
dirt, fingerprints will cause degradation of image quality.
•Do not poke inside the open aperture in the base plate of the unit. There are delicate optical
components which are easily damaged if touched.
WARNING. This unit emits non-visible laser radiation at 850 nm from the dichroic aperture as
indicated by a warning label on the unit. The total output power is below the class 1 emission limit of
770uW and is therefore eye-safe. However, staring into the aperture should still be avoided.
The supplied mains adaptor must always be used with an earthed mains socket. The equipment
should be positioned in such a way that the mains switch, power supply and system power switch are
easily accessible.
If the equipment is used in a manner not specified by the manufacturer, the protection provided by
the equipment may be impaired.
Only the exterior of this product should be cleaned using a damp lint-free cloth. If internal
contamination is suspected, please contact your supplier for advice.
DANGER. Under no circumstances unscrew the lid of the unit. Disassembly of the unit will void the
warranty. This product does not contain consumer serviceable components. Service and Repair
should be performed by authorised service centres only.
Use only the proper type of power supply cord set (provided with the system) for this unit. Failure to
do so could instantly destroy the electronics and laser diode. The unit requires 24 VDC at 2 Amperes.

5
Always switch off the unit using the on/off switch SW1 or unplug the PSU (CON3) when plugging/
unplugging the stepper motor (CON4) or DIGIPOT (CON2). It is safe to plug/unplug the USB
connector (CON1) with the unit powered.
Keep this manual in a safe place as it contains important safety information and operating instructions.
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.
The PureFocus system allows powerful automated autofocus functionality to be added to existing
microscope systems by installing the unit into the infinity space (between objective and tube lens).
The system has been designed to fit on many popular microscopes using infinity corrected optics,
both upright and inverted types, using the relevant mounting kit. The PureFocus controller outputs
signals suitable for controlling piezo or motor focus drives and is compatible with Prior piezo actuators
and Prior stepper motors, by simply attaching to the fine focus knob of the microscope.
With the laser autofocus system the user has the ability to work with a range of sample types with a
reflective surface, including permanently mounted glass slides, live specimens in aqueous solution,
metallurgical, semiconductors and other samples with multiple reflective layers. The system can also
work with plastic vessels such as well plates.
PureFocus works with both epi and transmitted illumination, and can be used for fluorescence
applications with the 850 nm source being outside of most fluorescence bands.
A fully standalone system gives the end user the option of using the PureFocus controller with digipot,
display and buttons allowing all basic functionality options without the need for a host PC. The inbuilt
signal processing electronics generates focus correction information internally every 1 ms allowing for
fast focus capture and tight closed loop action. For more advanced functionality PureFocus can be
fully remote controlled via USB communication, using our ASCII commands set.
2.2 Working Principle
The PureFocus operates using a sensor with multiple pixels. Half the aperture of a collimated laser
beam is blocked via a knife edge and directed into the back of the microscope objective. The laser
light is focussed to a line on a reflective surface at the sample and then reflected back through the
objective and directed towards the sensor forming a corresponding line on the sensor at the centre
point. Due to half the aperture of the laser being blocked, motion of the reflective sample up or down
causes this line to move either left or right on the sensor, giving information to automatically control
the focus of the microscope and keep the sample in focus (Fig. 1).

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. This allows for continuously variable offsets to be added. This is especially useful
when dealing with biological samples where the specimen will reside under a coverslip, which has a
reflective top surface (Fig. 2).
To form a focus error signal firstly the system sums the pixel values to the left and right of a defined
centre pixel on the sensor, these summations are named A and B respectively. The number of pixels
that are summed either side of the centre to generate A and B can be chosen. The command
PINHOLE allows the centre point and width to be set. The sensor has 1500 pixels so the maximum
possible width is 750 pixels, for a centre point in the exact centre of the sensor at pixel 750.

7
The position signal is then computed as
POS = (A-B)/(A+B)
which is a signal that nominally swings between -1 and +1. A target value is subtracted from the
position signal to form an error signal
ERROR = TARGET - POS = TARGET - (A-B)/(A+B)
This signal is fed into a PID controller and the output of this controller is sent either to a Prior stepper
motor or to an analogue voltage for driving an external piezo controller.
PureFocus also computes two further values from the line sensor, C and D, which aid in operation. C
is simply the value of the centre pixel. D is the summation of pixels across an arbitrary section of the
sensor, which is settable to any range of pixels. This D value is useful for detecting if the system is
focussed to the correct interface when working with samples that contain more than one reflective
surface.
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
•Wafer samples e.g. semiconductor
•Flat metallic samples
Other sample types are discussed in section 7.1.

8
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.
On inverted microscopes where the objective moves and the sample is fixed the PureFocus head sits
directly behind the objective nosepiece moving with the objective nosepiece as it changes focus
height, this is achieved using a mounting kit for the particular microscope being used.
On upright microscopes where the objective is fixed and the sample moves the PureFocus head can
sit at any position in the infinity space of the optical path, remaining fixed to the body of the microscope
using a dovetail kit for the particular microscope being used.
3.2 Types of Illumination
3.2.1 Transmitted light/brightfield illumination
When working with transmitted light illumination, PureFocus could be susceptible to illumination
light reaching its sensor. In this case it is better to set the illumination light source to the lowest
brightness that still gives an acceptable image at the camera/eye pieces.
3.2.2 Reflected light/epi illumination and fluorescence
For reflected light illumination and fluorescent applications it is beneficial to position the
PureFocus head below beam splitters and fluorescence filter cubes which could attenuate or
prevent the 850 nm PureFocus light reaching the sample.
3.2.3 Phase contrast and DIC
PureFocus can work with a range of phase contrast objectives. The polarisation optics in DIC
microscopes must be positioned after the PureFocus head to be compatible.
3.3 Unpacking your system
All standard PureFocus850 systems contains the following equipment:
Part Number
Description
PF185(M)
PureFocus850 Head (Line Mode)
PF100
PureFocus850 Controller
PF300
PureFocus850 Setup Camera
PF209
PureFocus850 Setup Slide
PF400
Head-controller connector cable
H407
Controller power supply
W3045
USB cable

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
PF201
Alignment target for M25, M27 and M32 threads
If purchased alongside a piezo nano-positioning system, a specialist cable will be provided in order
to connect the system to the PureFocus850 controller.
PF404
BNC to 15-pin D-connector cable
Different microscopes require different mounting kits in order to install the PureFocus850.
Part Number
Description
LF335
Flange set for Olympus upright microscopes
LF320
Flange set for Nikon upright microscopes
LF310
Flange set for Leica upright microscopes (contact Prior)
LF312
Flange set for Leica upright microscopes (contact Prior)
LF341
Flange set for Zeiss upright microscopes (contact Prior)
PF202
Mounting kit for Olympus IX73 (2-deck)
PF208
Mounting kit for Olympus IX73 (2-deck)
PF214
Mounting kit for Olympus IX73 (1-deck)
PF203
Mounting kit for Olympus IX71
PF213
Mounting kit for Olympus IX81
PF211
Mounting kit for Nikon Ti
PF212
Mounting kit for Nikon Ti2
PF210
Mounting kit for Olympus BX41/43/51/53/63
If the PureFocus850 mounting kits above are not suitable for your microscope, please contact Prior
Scientific.
3.4 Installing your PF850 mounting kit

10
Please follow the dedicated guides for each mounting kit for full instructions, available at
https://www.prior.com/download-category/help-sheets-installation-instructions.
3.5 Installing the PF850
3.5.1 Installing the PF850 head
The mounting kit installation guides cover the specifics of how and where to mount the various
components directly onto the PF850 head and onto the microscope.
Special considerations should be made depending on the imaging technique and if you have
purchased a line mode system.
Factor
Action
Fluorescence imaging
Mount the PF850 head between the nosepiece and the
beam splitter/filter cubes. Additional mounting brackets may
be required.
Phase contrast/DIC imaging
Mount the PF850 head between the nosepiece and any DIC
optics. Phase objectives are compatible with the PF850.
Line mode
For upright microscopes, mount the PF850 at 30-60
degrees with respect to the arm it is mounted on. This
maximises the effectiveness of the line mode focus-recovery
algorithm. For inverted microscopes, the orientation of the
PF850 is fixed, meaning the sample should be oriented so
that any linear features are viewed at 30-60 degrees with
respect to the orientation of the PF850 head
3.5.2 Connecting the PF850 controller
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.

11
Connect the controller to the PC via the USB cable provided (Fig. 3).
3.5.3 Pre-alignment checklist
Download the PureFocus850 GUI application and help sheet from www.prior.com/downloads
Insert the alignment target into the nosepiece.
Insert a dustcap into the nosepiece if available. If a suitable dustcap is unavailable, leave an empty
position on the nosepiece.
Insert your required objectives into the remaining positions on nosepiece.
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.
3.6 PF850 Controller Guide
The controller complements the GUI by allowing manual input of a number of instructions. This
allows the PF850 to be used independently from computer control during day-to-day operation, once
setup is complete.
3.6.1 Controller Modes
The controller can be toggled into various modes using the Servo On/Off and Offset/Focus buttons.
There are four mode combinations, which are indicated on the right hand side of the controller
display.
The Servo On/Off button activates or deactivates the servo, which is responsible for holding the
sample in focus. This changes the controller display; SRV indicates servo activation and MAN

12
indicates servo inactivity. Whilst in SRV mode, the digipot cannot be used to control the focusing
mechanism as the PureFocus850 repositioning algorithm is actively controlling it.
Offset/Focus button allows control of the focusing mechanism or the PureFocus offset lens
mechanics using the digipot. Which piece of hardware is being controlled is indicated by the
controller display; FOC indicates control of the focusing mechanism and OFF indicates control of the
offset lens mechanics.
See below for the functionality of these mode combinations.
Mode combination
Functionality
MAN FOC
The servo is inactive and the digipot controls the focus mechanism.
SRV FOC
The servo is active and the PF850 is automatically focusing on the
sample. The digipot will not move the focus mechanism until servo
deactivation.
MAN OFF
The servo is inactive and the digipot controls the offset lens
mechanics. Focus position cannot be changed in this mode. This
mode is primarily used during manual setup.
SRV OFF
The servo is active and the digipot controls the offset lens mechanics.
Moving the offset lens will cause a change in focus position in real
time, as the servo causes the focus motor to move a distance
corresponding to the increase or decrease in the magnitude of the
offset.
The function button currently is not used. It may be utilised for OEM applications.
3.6.2 Display Indicators
The Obj Select button changes the parameters used to control the servo to match the objective that
is currently in use. Repeatedly pressing this button loads the parameters for objectives 1 to 6 with
reference to the GUI, and will display OBJ1-OBJ6 corresponding to which settings are currently in
use.
When the PureFocus850 is holding focus in SRV mode, this is indicated by the appearance of the
letter F, which appears next to the objective number indicator. This indicator is dependent on the
focus flag discussed later in this document (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. This is dependent on the sample flag discussed later in this document (see
advanced features).

13
Section 4: Optical Alignment
The PF850 is mounted in the infinity space of the microscope. In order perform correctly it needs to
be aligned with the optical path. First, the refractive and reflective optical elements of the PF850 head
must be aligned with the microscope objectives. The focal point of the laser on the sensor must then
be determined, half of the laser blocked in order to generate signal imbalance either side of focus,
and the average background illumination reaching the PureFocus850 sensor calculated. Optical
alignment of the system is only required once.
4.1 Aligning the PF850 with the optical path
Rotate or move the nosepiece to place the alignment target into the optical path.
Place the PF300 alignment camera onto the microscope stage using an appropriate sample holder.
Connect the camera to the PC via the USB, using the 1.8-metre extension lead if required. Move
microscope stage to manoeuvre the camera close to the centre of the alignment target (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.
A pink laser signal should be visible on the camera. Ensure the centre of the target is in the centre of
the field of view. Set the PureFocus850 controller into MAN OFF mode and rotate the digipot to extend
the laser signal to the approximate diameter of the first visible ring of the target.

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.
To set the alignment correctly, the setup lens, 45 degree and 0 degree screws are required (Fig. 5).

15
Using a flathead screwdriver, rotate the setup screw to reduce the spread to the laser signal to a
minimum (Fig. 6).
Using the 45oscrew, adjust the lateral position of the laser line so that it aligns over the centre of the
target (Fig. 7).
Using the 0oscrew adjust the position of the laser line so that equal halves of the line are on either
side of the centre of the target. Adjust the length of the line using the digipot if necessary.

16
Make any final adjustments to both the 45oand 0o screws to ensure optimal alignment of the laser line
with the target. Once completed, remove the camera from the microscope stage and the alignment
target from the nosepiece.
4.2 Laser setup considerations
Before setting up the laser to allow autofocus function, consider the nature of your microscope
system. The PF209 setup slide used for this procedure has a number of different options. Please
note that Line mode systems (PF850M) have a slightly different procedure than spot mode systems.
4.2.1 Biological samples
Use the coverslip sample, which most closely mimics permanent slides or petri dish samples.
Transmitted light only (diascopic) microscope systems can only be setup using the coverslip
sample. Alternatively, the mirror sample can be used if sufficient ambient light is available to
illuminate the sample. If using a reflected light technique (e.g. fluorescence) for the majority
of imaging, the mirror sample can be used. The sample provided on the PF209 also may not
fluoresce at the required wavelength.
4.2.2 Samples requiring reflected light
Use the mirror sample, which is the simplest reflective sample. This sample closely mimics
wafers. When using biological samples on microscopes without diascopic illumination, Prior
Scientific recommends this sample for ease of use. Please note that large increases in laser
power may be required when setting up the samples you intend to image.
4.3 Setting the sensor (pinhole) centre
Please refer to section 4.3.1 for slice mode systems (PF850) and section 4.3.2 for line mode
systems (PF850M).
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.
Go to the mode submenu and ensure slice mode is selected.
Set the PureFocus controller to MAN FOC mode.
Move an objective into the light path. Prior Scientific recommends a 10x objective for this
procedure due to the working distance relative to higher magnifications, but any objective of
higher or lower magnification can be used.
Place the PF209 onto the stage and focus on either the coverslip sample or the mirror sample.
Use the digipot, in MAN FOC mode, to achieve focus.
Go to the setup submenu, which will open a graph that indicates the laser intensity over the
PureFocus850 sensor. Adjust focus until a peak appears in the signal. Make this peak as sharp
as possible. It is optimal for the peak to fill almost the entire y-axis of the graph, which can

17
usually be achieved by increasing or decreasing the laser power. 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 (Fig. 8).
The blue line, which indicates the pinhole centre, will move to the middle of the peak. Half of
the total signal will be on the left side of the line, and other half will be on the right.
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.
Go to the mode submenu and ensure line mode is selected.
Set the PureFocus controller to MAN FOC mode.

18
Move an objective into the light path. Prior Scientific recommends the use of the highest
magnification objective that provides a clear peak maximum close to the top of the setup graph.
If a low magnification objective is used, further adjustments may be necessary when setting up
individual objectives. It is strongly advised to maximise the size of the peak using the 45 degree
adjustment, then reduce the laser power.
Place the PF209 onto the stage and focus on either the coverslip sample or the mirror sample.
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.
If the peak is visible, slightly adjust the position of the 45oscrew to increase the size of
the peak (Fig. 9).
If the peak is not visible at all, go to the mode menu and temporarily put the system into
slice mode. This will amplify the signal by a factor of 10, which may bring the peak above
the noise. If the peak is still not visible, adjust the focus to identify the peak. Adjust the
45oscrew to increase the size of the peak. Remember to switch back into line mode

19
before continuing. If necessary, adjust the 45ofurther to maximise the size of the peak.
After maximising the size of the peak, if the peak crosses the top of the graph reduce
the laser power until the top of the peak is visible (Fig. 10).
Make this peak as sharp as possible by rotating the digipot. It is optimal for the peak to fill
almost the entire y-axis of the graph. 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.
Check that a peak is visible with each of the other objectives, before returning to the highest
magnification objective.

20
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.
If the signal is very noisy and no peak is visible, decrease the laser power in 500 unit steps and
see if any features are retained.
If the peak is short, increase the laser power in 100 unit steps until it reaches the optimal height.
If the peak goes over the top of the y-axis of the graph, reduce the laser power in 100 unit steps
until it reaches the optimal height.
If there are multiple features on the graph, which have intensities above the baseline noise,
this can affect the Auto Set Centre function. If the calculated centre of the sensor does not
correlate with the peak, go to the pinhole submenu and reduce the pinhole width in order to
exclude these features. Please refer to the section 7.2.1 for further guidance on this topic.
(Line Mode only) If you are having trouble with the 45oadjustment step with your highest
magnification objective, switch to the next highest magnification objective. Note the extra care
will be required when configuring the highest magnification objective with your sample later in
the procedure.
4.4 Creating signal imbalance
Ensure the controller is in MAN FOC mode.
Ensure the setup submenu graph is open.
Rotate the digipot in such a way that the single peak splits into a broad signal with peaks on either
side. The direction of rotation is system dependent. If the signal does not split, and just becomes
broader and less intense, rotate the digipot in the opposite direction. Increase the laser power such
that the split peak fills as much of the vertical axis as possible