Multi-Wavelength Photometry System

Plexon’s multi-wavelength photometry system has the capability to deliver multiple wavelengths of excitation light and the ability to detect multiple wavelengths of emitted light. The photometry system offers three excitation ranges centered at 560 nm, 465 nm, and 410 nm and two detection ranges centered at 600 nm and 525 nm. These ranges were chosen to operate with two of the most popular genetically encoded calcium indicators, GCaMP6 and R‑CaMP2.

Features:

  • Record from multiple locations in a single animal or many animals simultaneously with a single system
  • Three excitation ranges centered at 560 nm, 465 nm, and 410 nm
  • Detect fluorescence at 525 nm and 600 nm using two detection cameras
  • Synchronize photometry signals with behavioral events
  • Incorporated LED driver
  • Integrated with video tracking software

Multi-Fiber and Multi-Wavelength Photometry

The new multi-wavelength photometry system builds on the technology of the legacy single wavelength photometry system and incorporates new features based on customer feedback. This camera based system includes three built in LEDs for excitation and two optical sensors which allow for detection of green and red fluorescence simultaneously and a branching patch cable provides the user with the ability to record from multiple brain regions or multiple animals. The system interleaves the 410 nm and 465 nm LEDS and the camera detecting green fluorescence captures light for each of these wavelengths independently, which allows the user to identify the calcium-dependent fluorescence (isobestic control).

The Multi-wavelength Photometry system offers three excitation wavelengths:

  • 465 nm for selectively activating GCaMP6
  • 560 nm for selectively activating RCaMP2
  • 410 nm for use as an isosbestic control to detect calcium-independent signals

System Hardware

  • Photometry Module
    • 3 LEDS (465 nm to excite GCaMP6, 560 nm to excite R‑CaMP2, and 410 nm to serve as an isobestic control)
    • two detection cameras
    • two optic sensors

  • Trigger Box
    • LED driver
    • Digital Input/Output interface

  • Behavioral Camera Kit

Tracking and Data Analysis Software

  • Visualize raw photometry data and ΔF/F in real-time
  • Heatmaps for each individual fiber illustrate changes in fluorescence during recording
  • Create zones and automatically count the number of behavioral events that occur during recording
  • Define photometry events based on ΔF/F threshold crossings and combine with behavioral events to identify if a change in florescence occurs during a specified behavior
  • Interface with External Equipment
  • Track speed and position of subject

Incorporating photometry into the CineLyzer environment provides the information necessary to make correlations between the photometry signal(s) and the subject’s performance on behavioral tasks.

 

Selected technical specifications for Plexon’s Multi-Wavelength Photometry system are provided below. A Plexon Sales Engineer is happy to discuss your needs and how this product could best support your research goals.

Photometry Module

Excitation wavelength410 nm, 465 nm , 560 nm
Detection wavelength505-545 nm, 580-620 nm
Number of FibersUp to 4
LED driverYes, four high current LED drivers
Trigger InterfacesYes, eight camera trigger interfaces
Digital Inputs12
Digital Outputs12
Timing InterfaceYes
SPI InterfaceYes

Behavioral Camera

Resolution656 (H) x 492 (V)
Max frame rate at full resolution60fps

Can I measure neural activity in real time using fiber photometry?

Yes, fiber photometry provides the most sensitive and easiest way to record neuronal activities of the deep brain structures in behaving animals. GCaMP can be used to tag cell-type specific neurons with engineered fluorescent proteins and then measure the levels of fluorescence in response to changes in intracellular calcium concentration. The fluorescence emission of the GCaMP indicator increases with elevating calcium concentration. Fiber photometry enables the researcher to record the neural activity of genetically defined subpopulations of neurons through an optic fiber in freely moving animals.

I am interested in recording from multiple animals simultaneously. Is this possible?

Yes, the branching patch cable terminates in 4 magnetic LC ferrules. Each of these ferrules can be attached to fiber stubs on different animals.

Can I use dLight to detect changes in dopamine and a red-shifted Ca2+ indicator at the same time?

Yes, the 465 nm LED can be used for targeting dLight and the 560 nm LED can be used for activation of the red-shifted Ca2+ indicator. The emitted fluorescence will be collected independently by each detection camera.

Will I be able to view the photometry signal during recording?

Yes, the software included with the Plexon multi-wavelength system will show both raw data and ΔF/F for each fiber and for each detection camera. The software will also show a heat map for each fiber that illustrates changes in florescence for each fiber.

How is Plexon’s photometry system different from traditional systems?

Traditional systems employ a single multimode optical fiber implanted in the brain, a beam splitter that separates the excitation light and the fluorescent signals of neuronal activity indicators returning through the same optical fiber, and a photodetector. This approach allows for imaging of one fiber at a time. Measuring multiple brain regions is crucial to investigate connection between different brain regions to produce behaviors and the neural correlates of social interaction. Plexon’s camera-based approach provides a cost effective means to image from multiple fibers with the same detector.

What types of behavioral tasks can be used with CineLyzer and photometry?

  • T-maze
  • Fear conditioning
  • Operant chamber
  • Elevated plus maze
  • Place preference
  • Barnes maze
  • Novel object recognition
  • Social Interaction
  • Radial arm maze
  • Other behavioral paradigms

How do I know what type of fiber stubs I should use with my photometry system?

There are many options when choosing a fiber stub. Plexon recommends using fiber stubs with a diameter of either 200/225um or 225/245um, which are both larger diameters than the diameter of the fiber used in the branching patch cable. This combination allows for the highest average output in most cases. The diagram below illustrates the power output when various combinations of patch cables and fiber stubs are used. The depiction of the 100um patch cable with the 200um fiber stub is the solution that Plexon recommends for the highest average output.

What if magnetic fiber stubs will not work with my experimental design?

The magnetic branching photometry patch cable is made to easily attach to magnetic LC ferrules. However, this patch cable can also be connected to standard fiber stubs using a ceramic or metal sleeve.