Acquisition System Features

Based on the ART time domain platform, the acquisition system uses pulsed lasers to excite fluorophores and a fast detector coupled with a time-correlated single-photon counting system to measure the temporal distribution of fluorescence photons collected from the sample.

Features Summary

  • Automated acquisition to minimize set-up time and maximize efficiency and throughput.
  • Automated calibration processes and built-in tests that support self-diagnostics.
  • A time domain imaging approach that lets you image and quantitate multiple fluorophores in vivo simultaneously.
  • Laser power and integration time automation for improved signal-to-noise and signal-to-background ratios and superior detection.
  • Flexible scanning options—whole body, polygon, line scan, and point scan—within a scanning area of 200 mm by 90 mm.
  • Automated naming and archiving of results data.
  • Animal handling controls that include an interface for inhalation anesthetics and a heated platform that is maintained at a configurable temperature.
  • Multi-modal imaging capabilities.

Mode of Operation

Configured in reflection geometry with adjustable spatial resolution, the illumination and collection points are raster scanned over the region of interest with the use of a 3D translation stage. As shown in the following illustration, excitation light is attenuated and focused on the specimen. Fluorescent light originating from the specimen is then directed to a photomultiplier tube (PMT) through a fluorescent filter. The PMT output is coupled to a time-correlated single-photon counting system that is synched with the laser driver.

System Schematic

The animal is positioned on the scanning plate and a region of interest is selected via a live image capture. The illumination and collections points are then translated automatically for each X, Y, and Z-axis scan point.

NOTE: Contact your Regional Sales Director or Product Distributor for information about the availability of external lasers.

Using the temporal information contained in the time-resolved signal, depth and concentration information can be derived and volumetric 3D images can be obtained. Here the physics of photon migration is incorporated into the model and an accurate recovery of object shape, location, concentration, and fluorescence lifetime can be obtained.

Subsystems and Components

The acquisition system includes the following subsystems and components:

  • An illumination subsystem that provides illumination at a point location on the specimen.
  • A scanning subsystem that determines the location of the illumination and collection point on the specimen.
  • A detection subsystem that captures a TPSF from a point location.
  • A profilometry subsystem that captures the specimen’s 3D shape.
  • Animal support components that provide anesthetic gas connections, animal presence monitoring, and a heated animal platform.
  • Acquisition software that controls the overall scan process and analysis software to process results.

Illumination Subsystem

The illumination subsystem provides a means to select the illumination wavelength and the necessary incident illumination pulse to excite the administered fluorescent compound. It also controls illumination power and laser emission, and provides timing signals to the detection subsystem. The main components of the illumination subsystem include a laser driver rack and laser heads, a controller that sends control signals to the laser driver rack and provides delay lines for timing synchronization between the illumination and detection subsystems, a laser shutter, and attenuators that transmit precise amounts of optical power.

Scanning Subsystem

The role of the scanning subsystem is to move the location of both the illumination and collection points in order to acquire data for all illumination-collection pairs. The apparatus uses back-reflection geometry to scan the animal, which means that the illumination and collection points are located on the same side of the specimen. A distance of approximately 3 mm on the X-axis is maintained between the illumination and collection points at all times, as shown below. The scan step, which determines the resolution, is selectable from 0.5 mm to 3.0 mm. Focal plane adjustments are done automatically for each scan point using the specimen profile as a reference. Regions of interest are defined on the image captured by the camera prior to scanning.

Illumination Collection Points

Detection Subsystem

The signal captured by the scanning subsystem is filtered and collected by a detection subsystem that is based on time-correlated single photon counting (TCSPC) technology. The main components of the detection subsystem include the following:

  • A fluorescence filter changer that can hold up to eight optical filters. The filters mounted in this assembly determine the detection wavelength bandwidth.
  • A detector shutter that protects the photomultiplier tube (PMT) from too intense illumination. The acquisition software controls shutter operation, although interlocks are built into the system to protect the PMT in case of an illumination overload.
  • A PMT that is used in photon-counting mode. It converts the fluorescence signal into an amplified electrical pulse. Each PMT pulse is registered as a photon if its amplitude exceeds a set threshold.
  • A PMT preamplifier that amplifies the PMT signal for the TCSPC board.

Profilometry Subsystem

This subsystem, which includes a profilometry measurement laser, captures the specimen’s 3D shape prior to all in vivo scans. The profile is then used to adjust the focal plane on the surface of the specimen and also provides a spatial reference for the application’s topographic representations of depth and volume views.

Animal Support Components

The animal support provided by the system includes gas anesthesia connections, an animal presence monitoring system, and an embedded heating plate that is maintained within a configurable range up to 42°C. The standard scanning plate includes a nose cone and gas anesthesia fittings.