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Deep Brain Image Captured with a Bergamo Microscope Equipped with a PFM450E Piezo Objective Scanner (Courtesy of RIKEN Brain Science Institute, Wako, Japan.)

Applications

• Autofocus System for High Speed Time-Lapse and
  Z-Stack Imaging
• Optical Sectioning for 3D Imaging
• Fine Focus Adjustment
• Scanning Interferometry
• Surface Profilometry and Analysis
• Semiconductor and Wafer Inspection

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Exploded View

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Assembled View

The scanner is installed by threading a brass adapter into the microscope's objective holder with the included spanner wrench and tightening a flexure clamp around the adapter with the included 5/64" (2 mm) hex key. The objective is attached to the scanner using a separate brass adapter and flexure clamp.

Features

• Fast Acquisitions and High Frame Rates Enabled by 25 ms Typical Settling Time
• Maximum Clear Aperture of Ø29.0 mm Supports Large-Field-of-View Objectives
• Capacitive Feedback Sensors Provide Resolution Down to 1 nm and Help User Actively Compensate for Drift
• Quick-Release Flexure Clamps Allow Fast Objective Exchange
• Compatible with Upright, Inverted, and Rotating Microscopes
• Standalone Kinesis^® and APT™ Interfaces, ThorImage^®LS Integration, and Control via External Voltage
• Software Permits Easy Tuning for Different Objective Weights

The PFM450E Piezo Objective Positioner is designed for fine focus adjustment and high-speed Z-stack acquisition. Engineered for compatibility with Thorlabs' Bergamo^® II Multiphoton Microscopes, our Confocal Imaging System, our Cerna^® Microscopy Platform, and third-party microscopes, it bypasses the relatively slow stepper motors built into microscopes in order to quickly obtain scans of 3D volumes. Built-in capacitive feedback sensors allow the module to provide 1 nm resolution in open-loop operation and 3 nm resolution in closed-loop operation, enabling active compensation for short- and long-term drifts.

With a clear aperture of up to Ø29.0 mm and a maximum load of 500 g (1.1 lbs), the piezo objective holder easily accommodates the large-field-of-view objectives commonly used in multiphoton and confocal microscopy. In order to permit easy switching between objectives, the piezo stage is attached to the microscope and objective by independent adapters. This design choice allows the objective to be removed without disconnecting the rest of the assembly. Adapters are available for M32 x 0.75, M27 x 0.75, SM1 (1.035"-40), M26 x 0.706, M25 x 0.75, and RMS (0.800"-36) threads.

Each scanner is shipped with a piezo controller that has been factory calibrated to the specific scanner. Objective positioning is supported through the included standalone Kinesis and APT GUIs, our ThorImageLS image acquisition software, an externally supplied control voltage, or the MZF001 Joystick Console (sold separately). See the Software & External Control tab for more details. The controller offers USB and RS-232 interfaces for computer control; a BNC input for sine, sawtooth, and square wave drive signals; a BNC output that gives either positioning feedback from the scanner's built-in capacitive sensors or a signal proportional to the piezo drive voltage; and a connector for the MZF001 joystick. In addition, a DB15 connector provides signals that can be used for synchronization with external equipment.

Software and External Control

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Z Control Panel from ThorImage^®LS Capture Setup Tab


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Fast Z Panel from ThorImageLS Capture Tab

The scanner can be driven using the included standalone Kinesis^® GUI, the legacy APT™ GUI, our ThorImage^®LS software for Bergamo^® II and confocal microscopes, an externally supplied control voltage, or the MZF001 Joystick Console. In addition, a DB15 connector provides signals that can be used for synchronization with external equipment. Complete details on control are available in the PFM450E manual for Kinesis and APT (PDF links).

Open-Loop Operation vs. Closed-Loop Operation
There are two operating modes for positioning the objective: open loop and closed loop. In open-loop operation, which supports a travel range of 600 µm ± 10%, the user controls the piezo drive voltage (in V). The applied drive voltage corresponds to some amount of objective displacement. For piezoelectric materials, this displacement does not depend linearly on the applied voltage: it exhibits nonlinearity and hysteresis. It is therefore not straightforward to position the objective by choosing the drive voltage. The scanner's built-in capacitive feedback sensors measure the objective displacement with 1 nm resolution in open-loop operation.

In closed-loop operation, which supports a travel range of 450 µm, the user directly controls the objective displacement (in µm). The built-in capacitive feedback sensors measure the objective displacement with 3 nm resolution in closed-loop operation.

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Kinesis^® GUI Panel

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APT™ GUI Panel

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APT Loop Tuning tab. The default settings are for a 150 g objective. As a rule of thumb, for a 2X increase in objective weight, divide all PID parameters by 2 and divide all notch filter settings by the square root of 2. The default settings can be restored in the General tab.

Standalone Kinesis and APT GUIs
The Kinesis and APT GUIs support both open- and closed-loop operation. The piezo drive voltage or objective displacement can be directly typed in, changed by dragging the output knob (APT only), or incremented or decremented in fixed, user-defined amounts.

In addition, the GUIs enable software-based PID loop tuning. The default settings are designed to provide stable operation for most small- and medium-sized objectives, but fine tuning the PID loop helps account for the specific microscope and objective in use in the experiment, reducing positional overshoots and ringing about the commanded objective position.

Screenshots are shown to the left.

ThorImageLS Image Acquisition Software
ThorImageLS supports closed-loop operation. It allows the movement of the objective scanner to be controlled from the same interface as Thorlabs' Bergamo II and confocal microscopes, greatly simplifying experimental integration. For convenience, controls for the piezo objective scanner are found in both the Capture Setup and Capture tabs. Screenshots are shown to the right.

Externally Supplied Control Voltage
In this mode, the scanner supports both open- and closed-loop operation. The voltage is applied using the external input BNC connector on the controller. In open-loop operation, a 1 V change in input voltage results in a 15 V change in drive voltage, while in closed-loop operation, a 1 V change in input voltage results in a 45 µm change in objective displacement. The drive voltage or objective displacement that is determined by the input voltage is added to the value that is set in the APT GUI. If this sum would result in a drive voltage outside the -30 V to +150 V range, those limits will not be exceeded.

The external input BNC connector is typically used with sine, sawtooth, or square drive signals. The maximum frequency of the signal that should be applied depends upon the waveform. For large periodic waveforms (i.e., sine waves that provide >200 µm of total displacement), the recommended drive frequencies are:

• ≤25 Hz for Loads up to 150 g,
• ≤20 Hz for Loads from 150 g to 250 g,
• ≤15 Hz for Loads from 250 g to 400 g, and
• ≤10 Hz for Loads from 400 g to 500 g.

For staircase waveforms with step sizes up to 50 µm, the minimum time between steps can be as small as the 25 ms typical settling time.

MZF001 Joystick Console
The joystick supports closed-loop operation. It provides intuitive, tactile, hand-operated control of the objective position. The rate of change of the position is set by a rate potentiometer knob.

DB15 Connector
For photostimulation, electrophysiology, and optogenetics applications, the DB15 connector on the controller provides several electrical signals that can be used to synchronize the movement of the piezo objective scanner with other equipment, such as multiphoton imaging lasers, patch clamp electrodes, and LED light sources. Details on this connector are available in Appendix A.2 of the manual (PDF link).

External Control Voltage Examples

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We applied a square wave drive signal to our piezo controller to move a 16X physiology objective (Item # N16XLWD-PF) in 10 µm increments. The scanner's built-in capacitive sensors demonstrate that the targeted position is achieved within the 25 ms typical settling time without ringing.

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We used a sawtooth drive signal to acquire a Z-stack with a 16X physiology objective (Item # N16XLWD-PF). As shown by the built-in capacitive sensors, we obtained excellent linearity over the 300 µm scan range, creating evenly spaced image slices.

Piezo Controller Pin Diagrams

Joystick Connector

Female Mini DIN

Pin	Description	Pin	Description
1	RX (Controller Input)	4	+5 V, 100 mA Supply for Joystick
2	Ground	5	TX (Controller Output)
3	Ground	6	Ground

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Items Included with PFM450E (North American Power Cord Shown)

Item # PFM450E consists of the following:

• PFM450 Piezo Objective Scanner
• PPC001 Piezo Controller
• Spanner Wrench for Adapter Installation
• 5/64" (2 mm) Hex Key and Two Extra Cap Screws for Piezo Objective Scanner's Flexure Clamps
• Power Supply with Region-Specific Power Cord
• USB Cable for Computer Connections
• Storage Case with Slots for Piezo Objective Scanner, Spanner Wrench, and Brass Adapters

The brass adapters required to connect the piezo objective scanner to the microscope and the objective are sold separately, as is the optional MZF001 Joystick Console.