Prior PureFocus850 User manual

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Version issue No. - 1.7 For more information visit www.prior.com
PureFocus850
User manual

Version issue No. - 1.7
Thank you for purchasing this product from Prior Scientic – we’re condent it will be a
reliable and useful addition to your microscopy 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. This product must be used only as specied in
this manual. If you wish to use it di󰀨erently, contact Prior Scientic beforehand.
Please do not hesitated to contact us with any comments or
questions regarding this product.
Visit Prior Scientic online at www.prior.com

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Contents
Section 1
Safety Information 2
Section 2
Product description 3
Section 3
Unpacking the system 4
Section 4
Introduction
4.1 Working principle 6
4.2 Sample types 8
4.3 Types of microscope system 9
Section 5
Installation
5.1 Installing the head 10
5.2 Initalizing the head 11
5.3 Controller Motor Connections 14
5.4 Setting up for stepper focus control 15
5.5 Setting up for piezo focus control 15
Section 6
Operation
6.1 Controller keypad and digipot 16
6.2 Controller display 16
6.3 Inital setup and operation 17
6.4 Routine operation with the GUI 20
Section 7
ASCII command set 25
Section 8
Replacement, Repairs, Returns and Spare Parts 34
Section 9
Troubleshooting 35
Appendices
Appendix 1: PF850 FTDI USB Field Issues and Solution 36

Version issue No. - 1.7
2
Section 1
Safety information
Class 1 laser product, laser wavelength 850nm, laser output < 0.77mW
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 specications before using the PureFocus850. If you
have any questions do not hesitate to contact Prior Scientic. If you intend to use this unit in a manner not
specied by Prior in this manual, contact Prior 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 ames.
• Do not allow objects to fall on or liquids to spill on the product.
• Do not touch the glass plate tted between the circular dovetail and the top plate. Any dust, dirt,
ngerprints 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 visible laser light from the aperture in the base plate of the unit. The total
continuous power does not exceed 1mW thus it falls into a CLASS 1 Laser Product. As such the
user should not stare directly into the laser beam although the normal eye reex response will o󰀨er
protection. The laser power is less than most commercially available laser pointers sold in novelty
shops.
• 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 24VDC at 2 Amperes.
• Always switch o󰀨 the unit using the on/o󰀨 rocker 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.

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Section 2
Product description
The Prior Scientic 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 di󰀨erent 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 innity space (between objective and tube lens).
The system has been designed to t on many popular microscopes using innity 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 ne focus knob of the microscope.
With the laser autofocus system the user has the ablitiy to work with a range of sample types with a
reective surface, including permanently mounted glass slides, live specimens in aqueous solution,
metallurgical, semiconductors and other samples with multiple reective 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 uorescence
applications with the 850nm source being outside of most uorescence 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 option without the need for a host PC. The inbuilt
signal processing electronics generates focus correction information internally every 1ms 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 may be sent.

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Section 3
Unpacking the system
This guide is an introduction to installing a Prior PureFocus850. Please note that instructions for specic
microscopes and parts may vary. Consult Prior Scientic if you are unsure.
PF850 PureFocus Controller, Head, Cables & PSU LF523 Lower Dovetail Flange Olympus BX60
PF100 PureFocus controller LF320 Flange Set (Nikon 150)
PF301 PureFocus controller OEM LF526 Upper Dovetail Flange Nikon Optiphot 150
PF185 PureFocus Head LF527 Lower Dovetail Flange Nikon Optiphot 150
PF209 Sample Set Up Slide LF312 Flange Set (Leica LB)
PF300 PureFocus Setup Camera Jig LF532 Upper Dovetail Flange Leica DM4000
PF200 PureFocus Alignment Target Type 1 LF533 Lower Dovetail Flange Leica DM4000
PF201 PureFocus Alignment Target Type 2 LF341 Autofocus Flanges (Zeiss Axioskop 2/40)
PF202 IX73 PureFocus Mounting Kit LF534 Upper Dovetail Flange Zeiss AxiosKop 2 / 40
PF203 IX71 PureFocus Mounting Kit LF535 Lower Dovetail Flange Zeiss AxiosKop 2 / 40
LF335 Flange Set (Olympus BX) PF208 IX73 Basic PureFocus Mounting Kit
LF522 Upper Dovetail Flange Olympus BX60 PF400 Connecting Cable
W3045 USB Cable
If for whatever reason you fail to recieve all the items you require please contact [e-mail],
where a member of sta󰀨 will happily help with your issue.
Please note you will have to download the GUI from the prior.com website.
Downloads are free and additional infomation is available on our website on how to use the GUI.

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1. PF system
In your shipment you should recieve:
PF head
PF controller
Connecting cable
Power supply
USB cable
Stepper motor (optional)
Piezo BNC cable (optional)
Dovetail kit (optional)
Inverted microscope kit (optional)
2. Alignment kit
Alignment target
IR alignment camera
3. Setup slide
Set up mirror
Sample for life science
Sample for material
science
The sample for life science consists of a thin specimen permanently mounted under a standard #1.5
coverslip.
The sample for material science consists of a a bare piece of stainless steel.
PF head
PF controller
Connecting
cable
Power
Supply
USB cable

Version issue No. - 1.7
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Section 4
Introduction
4.1 Working principle
The Pure Focus operates using a sensor with mutiple 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 reective surface at the sample and then reected 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 reective 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.
Knife Blade
Beamsplitter
Laser Diode Source
Adjustable
Collimating Lens
Line Sensor
Dichroic Mirror
Objective Lens
Sample
Incident Beam
Reflected Beam
Out of focus Out of focus
Tube
Lens
Camera / eye piece
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 reections from
spurious reective surfaces and o󰀨ers great exibility when dealing with various di󰀨erent samples.

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The addition of adjustable laser collimation also allows the reective surface, used by PureFocus
for holding focus, to be located at a di󰀨erent plane to the microscopes imaging plane, where the
specimen resides. This allows for continuously variable o󰀨sets to be added. This is especially useful
when dealing with biological samples where the specimen will reside under a coverslip, which has a
reective top surface.
Offset
Reflective plane
Imaging plane
20x/0.75
To form a focus error signal rstly the system sums the pixel values to the left and right of a dened
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.
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 to either the 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 reective
surface.

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8
4.2 Sample types
PureFocus works with a variety of di󰀨erent samples:
Permanently mounted slides
These are specimens that are xed in a mounting media usually with refractive index matched to the
glass coverslip (n≈1.5). When working with dry objectives with such samples a reective signal is
only available from the outside of the coverslip. It is recommended to work with #1.5 coverslips with
PureFocus.
If the specimen is high contrast (such as a stained section) as you scan over the slide, the level
of scattered/reected light from the inside of the coverslip where the specimen resides can vary
signicantly. In such cases it is benecial to position the knife edge `past-centre’ so more than half of
the laser line is blocked. This can be achieved by turning the knife edge screw (4) one additional turn
anticlockwise.
When working with water immersion objectives with such samples, a weaker reective signal is still
available from the outside of the coverslip. However, when working with oil immersion objectives
with such samples no reective signal will be available to work with. In these scenarios it may still be
possible to use a very weak reective signal from one of the interfaces to hold focus
Specimens in aqueous solution
These are specimens that sit on the inside face of a vessel or coverslip in a solution usually with
refractive index similar to water (n≈1.3). When working with dry objectives with such samples a strong
reective signal is available from the outside the coverslip/vessel and a weak reective signal is
available from the inside of the coverslip/vessel.
By default PureFocus works with the strong outside signal, however it does have capability in certain
conditions to work with the inside signal (See Operation - Section 6). It is recommended to work
with #1.5 coverslips/glass bottoms with PureFocus, although performance is possible with higher
thicknesses depending on the objective being used.
When working with water immersion objectives with such samples, reective signals are still available
from the outside and inside of the coverslip/vessel. However, when working with oil immersion
objectives with such samples there is only a reective signal from the inside of the coverslip/vessel.
Purely reective samples
For samples such as thick glasses, mirrors, metals and silicone wafers there will be a strong isolated
reection signal available.
Other samples with multiple reective layers
In a similar manner to biological samples, some industrial samples may consist of di󰀨erent layers
of reective material/coating, one example being an LCD panel. Depending on the thicknesses and
reectivities involved PureFocus may also be compatible with these samples

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4.3 Types of microscope system
PureFocus works on a range of di󰀨erent microscopes which use innity corrected optics. The
PureFocus head sits in the innity space of the optical path between the objective and tube lens.
Inverted microscopes
On inverted microscopes where the objective moves and the sample is xed the PureFocus head
sits directly behind the objective nosepiece moving with the objective nosepiece as it changes focus
height, this is achieved using an mounting kit for the particular microscope being used.
Upright microscopes
On upright microscopes where the objective is xed and the sample moves the PureFocus head
can sit at any position in the innity space of the optical path, remaining xed to the body of the
microscope using a dovetail kit for the particular microscope being used.
Transmitted light/brighteld 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.
Reected light/epi illumination and uorescence
For reected light illumination and uorescent applications it is benecial to position the PureFocus
head below beam splitters and uorescence lter cubes which could attenuate or prevent the 850nm
PureFocus light reaching the sample
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.

Version issue No. - 1.7
10
Section 5
Installation
5.1 Installing the head
Install the microscope dovetail kit onto the PureFocus head unit. For information on installation for
inverted microscopes please contact a Prior representative.
Mount the PureFocus head unit onto the microscope between the objectives and tube lens, in the
ininity space (there are multiple places it could be mounted in the innity space depending on the
microscope).

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5.2 Initalizing the head
1. Connect cables
2. Insert IR alignment target into nosepiece and mount the setup camera onto the microscope
stage
3. Turn Allen screw labelled Blade (4) clockwise to the limit to remove knife edge from beam path
4. Turn on the control box and run the PureFocus850.exe Graphical User Interface (GUI)*
*Please note you will have to
download the GUI from the
prior.com website.
Downloads are free and
additional infomation is
available on our website on
how to use the GUI.
1. Setup Lens adjustment
2. Dichroic adjustment
(Nominal 45°)
3. Dichroic adjustment
(Nominal 0°)
4. Blade adjustment

Version issue No. - 1.7
12
5. Reset the PureFocus (Settings > Factory Reset) to ensure all settings are at factory defaults
6. Set laser power to 4000 (Laser > Set Power , value 4000)
7. In the main GUI, set the o󰀨set lens (O󰀨set>Goto Limit)
8. Place the Camera alignment tool on the stage and move the IR alignment nosepiece target into
position. Using the camera software, camera.exe* check that the camera is focused onto the
front surface of the IR target. Adjust the camera focus lens if required.
9. For some microscopes an adjustment of the o󰀨set may be required to get a good view of the
laser line across the camera eld of view, similar to the below images
10. Align the laser line with the centre of the target using the dichroic adjustment screws (2 and 3).
Please note that the laser line orientation may vary depending on the orientation of the camera
on the stage.
11. Adjust the setup lens screw (1) to get the sharpest line on the target:
12. Remove the alignment target and put a 10x objective in the nosepiece and position the objective
over the mirror section of the setup slide
13. Set O󰀨set lens back to default position (O󰀨set>Goto Factory Home)
14. Using your camera imaging software and PureFocus controller nob, or microscope manual
Bad Good
*camera.exe can be downloaded from the prior.com website along with the GUI software

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adjustment to focus on the mirror.
15. Set the laser power to 10 (Laser > Set Power , value 10)
16. In the main GUI, go into setup mode (Setup) and observe the graph
17. Finely adjust the focus until the sharpest peak is seen on the sensor
18. Adjust the laser power (Laser > +100/-100) so the peak sits close to or just below 4095
19. Find the centre of the peak (Setup >Auto Set Centre) and when asked ‘Do you wish to globally
apply this now,’ select Yes. A vertical blue line on the graph should now
indicate the centre pixel position. The pinhole centre has now been calibrated and applied to all
objectives.
20. Defocus slightly so the signal on the sensor covers about a quarter of the sensor width and adjust
the laser power again (Laser > +100/-100) so this broader signal is now sitting close to or just
below 4095.

Version issue No. - 1.7
14
21. Introduce the knife edge by turning screw (4) anticlockwise until this signal is blocked up to the
centre pixel
22. Remove the 10x objective and block o󰀨 the beam using a dust cap or other black material.
23. Make a background reading using the Measure Average Background and Set button. When it
asks ‘Do you wish to apply to all objectives?’ click Yes. The background is now applied to all
objectives
24. Click save to ash (Settings > Save to Flash)
25. Close the PureFocus850.exe GUI
26. The unit should now be ready to use
5.3 Controller Motor Connections
The 15 way D connector on the Pure Focus 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:
Pin Number Function Commments
1Motor Phase A+
Minimum motor current rating = 1A.
9 Motor Phase A-
2 Motor Phase B+
10 Motor Phase B-
3Limit Switch +ve
Limit switches should be wired “normally open”11 Limit Switch –ve
4Limit Switch Common
12 Piezo Ground Return
15 Piezo Output (0-10V)

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5.4 Setting up for stepper focus control
1. Turn o󰀨 the control box. If not already connected, connect stepper motor to `Z ’ connector of
control box. Attach stepper motor to focus knob of microscope in preferred location
2. Turn on the control box, run PureFocus850.exe GUI
3. Move to a long working distance objective, such as 4x
4. Change mode to stepper mode (Mode>Stepper)
5. In the Z-position box, set step size to 10000, press ‘Step Up’ and observe if the objective gets
closer to or further away from the sample. If the objective gets further away from the sample, set
the PID sign to negative (PID > Negative > Set Parameters), if the objective gets closer to the
sample, invert the focus direction (Mode>Focus Direction Inverted>Select). Focus direction is
now set such that ‘Up’ takes the objective further from the sample and ‘Down’ takes the objective
closer to the sample, if you wish to invert the denition of `Up’ this can be done done via the
`Mode’ drop down menu (Mode > Focus Direction Inverted).
6. Set the microns per revolution for your microscope focus system using the “Mode” drop down
menu (Mode>Microns per rev of ne focus). This scales the Z position of the PureFocus system
accordingly.The default UPR setting is 100, which is the industry standard for most commercial
microscopes.
WARNING! INCORRECTLY SETTING THE FOCUS DIRECTION CAN LEAD TO COLLISION
AND DAMAGE OF THE SAMPLE AND/OR OBJECTIVE!
5.5 Setting up for piezo focus control
1. Turn o󰀨 the control box, connect optional BNC cable to `Z’ connector of control box. Attach
BNC connector to the desired piezo controller, this BNC outputs 0-10V and
operates nominally centred at 5V
2. Turn on the piezo controller and then the PureFocus control box, run PureFocus850.exe GUI
3. Move to a long working distance objective, such as 4x
4. Change mode to piezo mode (Mode > Piezo)
5. Set step size to 100, press ‘Step Up’ several times and observe if the objective gets closer to
or further away from the sample. If the objective gets further away from the sample, set the PID
sign to negative (PID > Negative), if the objective gets closer to the sample, invert the focus
direction (Mode>Focus Direction Inverted>Select).
6. Focus direction is now set such that ‘Up’ takes the objective further from the sample and ‘Down’
takes the objective closer to the sample, if you wish to invert the denition of `Up’ this can be
done via the `Mode’ drop down menu (Mode > Focus Direction Inverted)
7. Set the travel range of your piezo focus system for a 0 to 10V input signal using the `Mode’
drop down menu (Mode > Piezo travel). This scales the Z position of the PureFocus system
accordingly. The default piezo range setting is 100 microns
WARNING! INCORRECTLY SETTING THE FOCUS DIRECTION CAN LEAD TO COLLISION
AND DAMAGE OF THE SAMPLE AND/OR OBJECTIVE!

Version issue No. - 1.7
16
Section 6
6.1 Controller keypad and digipot
Digipot
Turning adjusts either the Z position or the o󰀨set depending on the mode selected (See O󰀨set/Focus).
If the servo is active then Z position control becomes automated by the PureFocus and the digipot can
no longer adjust the Z position. The o󰀨set can be adjusted with the servo active or inactive, however
changes to the o󰀨set will only be evident with the servo active, when the PureFocus will automatically
control the Z position to give the desired o󰀨set
Obj. Select
Cycles through objectives 1 to 6, OBJ1 to OBJ6 are shown on the display. When an objective is
selected, parameters for that objective are loaded and restored. Objective parameters can be adjusted
and stored using the PureFocus.exe GUI
Servo on/o󰀨
Toggles servo on and o󰀨. When servo is active SRV is shown on the display and Z position control
becomes automated by the PureFocus. When servo is inactive MAN is shown on the display, when in
Focus mode (see O󰀨set/Focus) the Z position can then be manually adjusted using the digipot
O󰀨set/Focus
Toggles between O󰀨set and Focus mode. When O󰀨set mode is selected an OFF is shown on the
display and the digipot adjusts the o󰀨set, when Focus mode is selected a FOC is shown on the display
and the digipot adjusts the Z position
Function
This button is currently not used
6.2 Controller display
The controller displays useful information regarding the state of the PureFocus system
OBJ1
OBJ2
OBJ3
OBJ4
OBJ5
OBJ6
NS
F
MAN
SRV
FOC
OFF

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This table describes each indictor:
OBJ1, OBJ2, OBJ3, OBJ4,
OBJ5, OBJ6
Displays the current working objective, current parameters* are
associated with this objective
NS Appears when no sample is detected (see SAMPLE ag)
FAppears when sample is in focus (see FOCUS ag)
MAN Displayed when in manual Z mode and servo is inactive
SRV Displayed when in servo mode and servo is active
FOC Displayed when in focus mode and digipot controls Z
OFF Displayed when in o󰀨set mode and digipot controls o󰀨set
*For a list of parameters that are uniquely associated with each objective please see the full command set
6.3 Initial setup and operation
Please note, the following operation procedure is for:
*Purely reective samples
*Permanently mounted slides
*Specimens in aqueous solution using dry objectives and the reection from the outside of the coverslip/vessel
*Specimens in aqueous solution using oil objectives and the reection from the inside of the coverslip/vessel
Referencing from the inside of the coverslip/vessel with specimens in aqueous solution with dry or water objectives,
as well as working with other samples with multiple reective layers is a more advanced mode of operation. This will
require full manual setup of the PureFocus parameters to work with the small/multiple reections involved with these
congurations.
PureFocus comes with many pre congured parameter sets for a variety of objectives and sample
types. These are designed to give a starting condition when conguring PureFocus on a new system.
It is highly recommended to begin with the plane mirror sample on the setup slide to verify that
PureFocus is able to hold focus on this simple reective surface using the relevant parameter les.
Afterwards, moving to the life science and/or material science sample is recommended. When using
permanently mounted slides, it is recommended to position the knife edge `past-centre’ so more
than half of the laser line is blocked. This can be achieved by turning the knife edge screw (4) one
additional turn anticlockwise.
6.3.1 Loading objective parameters
Once installed and initialized, to begin operating PureFocus, open the Purefocus.exe GUI and select
`Settings >New Objective Set’. This opens a new dialogue box which allows you to choose six default
starting parameters to associate with six objective positions. Select suitable parameter sets (if not
using six objectives, leave any unused objectives with the default parameter set) and click `Create
Set’. If you have previously calibrated a default pinhole centre and background during installation it
will ask if you wish to apply these to the six objectives, select `Yes’. Finally a `Set Objective Names’
window will appear which allows you to insert customised names for the objective positions that you
are loading.

Version issue No. - 1.7
18
If you have not calibrated a default pinhole centre or background a warning message will be shown
when creating a parameter set, it is recommended that you exit at this point and run through the
installation and initialization.
6.3.2 Focussing on the sample
Move the nosepiece to the objective 1 position and make sure OBJ1 is currently selected on the GUI/
keypad. Place a sample on the stage and through the eye pieces or camera manually focus on the
sample using the digipot, making sure you are in FOC mode, the controller screen should read MAN. If
you are using a piezo, centre the piezo travel by using the `Step up’ and `Step down’ controls until the
Z position is in the middle of the piezo range (Mode > Piezo travel) before manually focussing on the
sample with the microscope ne focus knob.
6.3.3 Setting the o󰀨set
Once in focus, select the `O󰀨sets’ drop down menu followed by `Auto nd’. A pop-up dialogue box
allows a speed to be chosen for the Auto Find feature. For 4x and lower objectives or 60x and higher
objectives, it is recommended that a speed of 20% is used. If an o󰀨set position is found successfully a
message will be shown. The next step is to check that the error signal swings either side of zero when
adjusting the ne focus of the microscope with the digipot. The error signal (TARGET-((A-B)/(A+B)))
can be seen visually and numerically on the GUI main screen. The error signal should become positive
one side of focus and negative the other side of focus. It is this error signal that will ultimately steer the
focus of the microscope automatically.
Please note that for a sample with a single reective surface the o󰀨set will be small and satisfactory
error signal behaviour may be found with the o󰀨set at the factory home or zero position.
If an o󰀨set position was not found this could be because the laser power is too low, if there is available
range try increasing laser power via the `Laser’ menu (Laser > +100/-100). The maximum value
is 4000. If no o󰀨set can be found, even at the maximum laser power, the coverslip/glass bottom
of the sample may be too thick or there could be an alignment or signal problem. At this point it is
recommended that you run through the installation and initialization procedures again to check the
system has been setup correctly.
If this does not solve the issue consider manually setting the o󰀨set by entering `Setup Mode’ and
adjusting the o󰀨set position with the digipot until a single peak is observed on the graph which is
approximately coincident with the centre pixel position, indicated by the blue vertical line on the graph.
If the error signal becomes positive on one side of focus and negative on the other side of focus then
an o󰀨set has been successfully found. The error signal can be seen visually and numerically on the
GUI main screen.
If these methods fail please contact Prior Scientic with details of the problem, the sample or objective
may be incompatible with PureFocus.
6.3.4 Checking for back reections
At this stage it is recommended that you remove the sample and turn o󰀨 all illumination sources to
check how much residual light is reecting back to the PureFocus head from the objective and other
components in the optical path. Open the `Pinhole’ dialogue box and look at the value in the `Average
pixel value’ box. The value should be below 100 for optimum performance. If this number appears
higher, consider reducing the laser power via the `Laser’ dialogue box (Laser>+100/-100). For some