What is Chromatone?

Neurescence’s flagship product – The ChromatoneTM system – provides a powerful integrated approach to observe, manipulate, and dissect a spectrum of neuronal subtypes in multiple brain/spinal cord regions, with single cell resolution. This three-pronged strategy to observe and stimulate activity in defined neuronal subpopulations, while examining behavioral paradigms will reveal accurate underlying mechanisms of physiology and behaviour in awake, freely behaving animals. 

The ChromatoneTM system is the only complete solution for in vivo calcium imaging and optogenetics, that enables simultaneous multicolor imaging and multi-opsin stimulation in up to four brain or spinal cord areas in freely behaving animals.

Services included:

  • Hands-on-surgical training
  • Neuronal trace extraction support
  • Device synchronization with other behavior equipment

Components of the system

The components include the main microscope body, four illumination cubes, control electronics, multimode fibres and imaging fibre bundles. The microscope body is small and light weight, to ensure ease of use during a wide array of behavior paradigms and it integrates cell-type specific calcium imaging and optogenetics.

The illumination cubes contain excitation light sources and optical filters to ensure the correct illumination color arrives at the brain tissue. Active temperature control in the illumination cubes ensures highly stable illumination levels at the brain tissue, throughout a behavior session.

Each illumination cube connects to the microscope body through a multimode fiber and emits three illumination colors:  

  • 470nm +/- 12nm for Green Fluorescent Protein Imaging /ChannelRhodopsin optogenetics 
  • 550nm +/-7.5nm for Red Fluorescent Protein imaging 
  • 632nm +/- 14nm for ChrimsonR Optogenetics

Light from each illumination cube is coupled into an imaging fiber bundle, which carries the light to implanted GRIN lens connectors and then to the brain tissue.

Video 1: GFP imaging ChrismsonR stimulation;
Video 2:  GFP & RFP imaging ChrismsonR stimulation;
Video 3: RFP imaging ChannelRhodopsin stimulation;
Video 4: RFP imaging ChrimsonR stimulation

There are four such imaging fiber bundles, enabling simultaneous multicolor calcium imaging and multi opsin stimulation in up to four brain or spinal cord areas. 

Pictured above is a depicted behavior session using the ChromatoneTM system. 

During an imaging -behavior session, the imaging fibre bundles of the ChromatoneTM are connected to the implanted lens-connectors. Three different illumination colors are available in each implanted region, to enable multicolor imaging (GFP and/or RFP) and multi-opsin optogenetics (ChannelRhodopsin / ChrimsonR). The three illumination colors red, blue and green in each implanted region can be independently controlled using the software.

GRIN lens connectors​

Transgenic animals or viral expression is used to express cell-type specific multicolor fluorescence tags. Graded-refractive-index lenses (GRIN) get implanted with lens-connectors in the brain/spinal cord region of interest. The lens-connectors are designed with the capability of lens-implantation at various angles and a patented focus-lock mechanism. This allows imaging and stimulating hard-to-reach brain/spinal cord regions including nervous system of birds, rodents, juvenile animals, and primates. Lens-connectors are custom-designed for each lab based on research requirements.

Pictured below is a lens-connector next to an American dime for size comparison.

ChromatoneTM uses FOUR highly flexible optical imaging fiber bundles. Each bundle includes 18000 single fibers, 7.5micron diameter each. The imaging fiber bundle enables the implanted lens-connectors to transmit fluorescence signal from neurons to the microscope. Pictured below is a GCaMP tagged brain slice, imaged using the Neurescence MultiscopesTM.

Focus Lock Mechanism

For each imaging session, imaging fibre bundles are connected to the implanted lenses, which transfer images from the tissue to the microscope. The user is required to set the best focus, for the initial imaging session. A patented focus-lock mechanism ensures that the subsequent imaging sessions do not require re-focusing of the lens by default, unless required by the user. The focus is maintained for the duration of multiple behavior sessions thus enabling an accurate longitudinal assessment of neuronal subpopulations.

Software

The ChromatoneTM software provides a flexible and customizable platform that enables optogenetics protocols, live data extraction, visualization and advanced analysis. The software enables the following capabilities:

  • The various color channels (RGB images) can be separated from each other. 
  • Power levels emitted for each illumination color can be set independently in each imaging fiber bundle. 
  • The light delivery of each illumination color can either be set to continuous wave for imaging or pulsed for optogenetics. 
  • Triggered optogenetic stimulation conditioned on specific behavioral tasks.
  • Three level programing enables the most sophisticated optogenetics protocols.

Specifications

Features

Specifications and Options

Illumination colors in each fiber optics

470 +/- 12 nm (GFP, Channelrhodopsin optogenetics)

550 +/- 7.5 nm (RFP)

632 +/- 14 nm (ChrimsonR optogenetics)

Illumination power range at tissue

470 nm: 0 to 6 mW 

550 nm: 0 to 1 mW

632 nm: 0 to 2.5 mW

Illumination mode for each wavelength

Green: CW for imaging

Blue: CW for imaging, 

Pulsed for optogenetics, 

Can get triggered and executes the Optogenetics protocol set by user

Red: CW for imaging,

Pulsed for optogenetics, 

Can get triggered and executes the Optogenetics protocol set by user

Optical Resolution

7 µm

Focusing Range or Working Distance

250 µm in brain tissue

Number of Imaging Probes

4

Diameter of GRIN Lens to Implant

Customizable between 0.5 mm to 1 mm

Available Length of Lens to Enter Brain Tissue

Customizable between 2.5 mm to 8 mm 

Field of View in Each Region

Approximately the same as the lens diameter

Weight of Connector and Lens

< 400 mg

Approximate 

Weight of each Fibre Bundle Carried by Animal

< 1 g

Bending Radius of Fiber Bundles

3 cm

Length of Imaging Fiber Bundles

84 cm

Detection Wavelength

517 +/- 13 nm and 590 +/- 20 nm

Image Size per Frame

3.2 megapixels

Number of Frames per Second

20 in continuous mode, 140 in burst mode

Length of Camera Electric Wire

2 meters

Length of LED Wire

1.5 meter