High-Speed Functional Imaging Across Several Brain Regions Operating in Concert.

Thorlabs' 2-Photon Random Access Mesoscope (2p-RAM, US Patent 10,295,811 and 10,901,194) provides subcellular resolution over an exceptionally large 5 mm x 5 mm field of view. Developed and commercialized in collaboration with Karel Svoboda's research laboratory at HHMI's Janelia Research Campus, this multiphoton mesoscope is designed for in vivo functional imaging of multiple spatially separated brain regions operating in concert. When imaging across user-defined, non-contiguous regions of interest within the field, near-video frame rates are possible; see the video for details. 

Image above: With a large field of view (FOV), the Thorlabs Mesoscope can simultaneously capture multiple brain regions; the black circle represents a  Ø5 mm FOV.*

Our 2p-RAM is capable of two-photon random access scanning; see the image to the upper right. This system features a built-in remote focusing unit, which translates the focal plane over a 1 mm range. The remote focusing unit can be coordinated with the lateral scan unit, which is comprised of virtually conjugated mirrors and a resonant scanner, to enable both lateral and axial translation of the field during the measurement. The lateral scan unit can direct the excitation beam from region to region within the 5 mm x 5 mm field of view in ~6 ms. We offer a 2.7 mm or 5.0 mm WD objective, which both provide a large excitation NA of 0.6 and a collection NA of 1.0. The scan path wavelength range of 900 - 1070 nm was chosen for optimal two-photon excitation of green fluorescent protein (GFP) and red fluorescent protein (RFP).

The mesoscope features motion control systems that permit the mesoscope body to move while the specimen remains fixed. The mesoscope body allows -20° to +20° rotation for the objective, as well as 2" of fine X motion, 6" of fine Y motion, and 2" of fine Z motion; just as with Thorlabs' Bergamo® II multiphoton microscope, X, Y, and Z rotate along with the objective. A multi-jointed periscope maintains the laser alignment over the entire range of motion. Since the study of awake, behaving specimens benefits from large working spaces, the mesoscope's enclosure leaves the surface of the optical workstation free for the experimental apparatus.

Features

  • Enables Functional Imaging within a 5 mm x 5 mm Field of View
  • Scans can be Configured over Whole Field of View or over Multiple Non-Contiguous Regions
  • Microscope Body Enables ±20° Rotation Around Sample and Fine XYZ Motion
  • Remote Focusing Mirror for Fast Axial Control over 1 mm Travel Range
  • Enclosure Provides Large Working Volume for Specimen and Experimental Apparatus
  • Field of View can Move While Specimen Remains Fixed
  • Technology Used Under License from HHMI's Janelia Research Campus
  • Volume Imaging Technique Using Bessel Beams and Dual-Plane Imaging Add-Ons Available;

Range of Motion

  • -20° to +20° Rotation About the Objective Focus
  • 2" of Fine X Motion
  • 6" of Fine Y Motion
  • 2" of Fine Z Motion
  • X, Y, and Z Axes Rotate with the Objective
  • Remote Focusing Mirror Enables Fast Focusing Adjustments over 1 mm Range During Scans

What is a mesoscope?

A mesoscope is a type of microscope with both a fine spatial resolution (<5 μm) for resolving single cells and a large field of view (>5 mm) to detect neurons across a large area.

Random Access Scanning
Our mesoscope creates high-speed images following user-defined scan patterns that translate the field of view laterally and axially. By hopping between regions, coordinated activity across multiple brain regions can be visualized.*

Configurations: 2p-RAM vs 2p-RAM with Dual-Plane Imaging Add-On 

The 2p-RAM (above) contains many optical systems that are specifically optimized to work together, including a built-in remote focusing mirror, which translates the focal plane over a 1 mm range; a lateral scan unit, which comprises virtually conjugated mirrors and a resonant scanner; a multi-jointed periscope that maintains the laser alignment over the entire range of motion; an ancillary path for one-photon imaging and photostimulation; and a custom large-NA objective. The 2p-RAM equipped with the dual-plane imaging add-on (below) includes all the same optical systems in addition to a secondary remote focusing module.

Dual-Plane Imaging Add-On

For the 2p-RAM, we offer the dual-plane imaging add-on, which allows for simultaneous imaging of two independent focal planes in the axial direction. 

In Vivo Calcium Imaging with the Dual-Plane Multiphoton Mesoscope:
Vip Inhibitory Cells in the Primary Visual Cortex (V1), Lateromedial (LM), Anterolateral (AL), and Anteromedial (AM) Areas; Imaged at 11 Hz Frame Rate per Plane, 400 µm FOV. (Courtesy of Dmitri Tsyboulski, Natalia Orlova, Fiona Griffin, Sam Seid, Jersome Lecoq, and Peter Saggau; Allen Institute for Brain Science, Washington, USA.)

Read paper: A Large Field of View Two-Photon Mesoscope with Subcellular Resolution for In Vivo Imaging

Sofroniew, N.J., Flickinger, D., King, J., & Svoboda, K.

*Several images on this webpage are taken from https://elifesciences.org/content/
5/e14472/article-data
 and used here under a Creative Commons Attribution license.

Applications:

Calcium Imaging
Developed and commercialized in collaboration with Karel Svoboda's research laboratory at HHMI's Janelia Research Campus, our 2-Photon Random Access Mesoscope (2p-RAM) is able to capture the activity of neurons across multiple regions of the brain with calcium imaging. Calcium imaging is a common technique used for tracking populations of neurons with calcium indicators. Unlike widefield microscopy, which has high light scattering and low contrast, two-photon microscopy provides the high resolution and improved contrast needed for in vivo calcium imaging. 

Using the 2p-RAM, Svoboda's research team has demonstrated in vivo imaging with a specimen expressing the GCaMP6f calcium indicator. As shown in the video to the right and in the image below, the multiphoton mesoscope can image across user-defined, non-contiguous regions of interest within the field at near-video frame rates. For more details, please see the complete research paper.

Source: Sofroniew NJ, Flickinger D, King J, and Svoboda K. "A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging." ELife. 2016 Jun. 14; 14472.

A low-magnification image from layer 2/3 cortex expressing GCaMP6f under the thy-1 promoter (GP 5.17 line), followed by four fields of view acquired at a higher resolution and frame rate. (Courtesy of Nicholas James Sofroniew, Daniel Flickinger, Jonathan King, and Karel Svoboda; Janelia Research Campus and Vidrio Technologies, Virginia, USA.)

Dual-Plane Imaging
Building upon the work of Karel Svoboda's research laboratory, researchers at the Allen Institute for Brain Science have designed a dual-plane imaging add-on for our multiphoton mesoscope that creates a second excitation path, allowing for simultaneous imaging of two independent focal planes in the axial direction. This module can be added or removed from the system without any adjustments to the original mesoscope. The Allen Institute researchers performed a comparative study between the multiphoton mesoscope with and without the dual-plane imaging add-on, and found that the imaging throughput increased by a factor of 2. The videos below show in vivo calcium imaging with their dual-plane multiphoton mesoscope. For more details on the Allen Institute for Brain Science, please visit their website.

Source: Tsyboulski D, Orlova N, Lecoq J, and Saggau P. "MesoScope Upgrade: Dual Plane Remote Focusing Imaging System for Recording of Ca2+ Signals in Neural Ensembles." Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/OTS), OSA Technical Digest. 2018; JW3A.60.

Excitatory Cells of an Slc17a7-IRES2-Cre;CaMkII-tTa;Ai93 Specimen in the Primary Visual Cortex (V1) and Lateromedial (LM) Areas; Imaged at 11 Hz Frame Rate per Plane, 400 µm FOV.
(Courtesy of Dmitri Tsyboulski, Natalia Orlova, Fiona Griffin, Sam Seid, Jersome Lecoq, and Peter Saggau; Allen Institute for Brain Science, Washington, USA.)

Vip Inhibitory Cells in the Primary Visual Cortex (V1), Lateromedial (LM), Anterolateral (AL), and Anteromedial (AM) Areas; Imaged at 11 Hz Frame Rate per Plane, 400 µm FOV.
(Courtesy of Dmitri Tsyboulski, Natalia Orlova, Fiona Griffin, Sam Seid, Jersome Lecoq, and Peter Saggau; Allen Institute for Brain Science, Washington, USA.)

Volumetric Imaging with Bessel Beams
In partnership with the Howard Hughes Medical Institute and Prof. Na Ji (University of California at Berkeley), Thorlabs offers a Bessel beam module for our multiphoton mesoscope. In vivo volume imaging of neuronal activity requires both submicron spatial resolution and millisecond temporal resolution. While conventional methods create 3D images by serially scanning a diffraction-limited Gaussian beam, Bessel-beam-based multiphoton imaging relies on an axially elongated focus to capture volumetric images. The excitation beam’s extended depth of field creates a 2D projection of a 3D volume, effectively converting the 2D frame rate into a 3D volumetric rate. 

As demonstrated in Ji’s pioneering work, this rapid Bessel beam-based imaging technique has synaptic resolution, capturing Ca2+ dynamics and tuning properties of dendritic spines in mouse and ferret visual cortices. The power of this Bessel-beam-based multiphoton imaging technique is illustrated below, which compares a 300 x 300 μm scan of a Thy1-GFP-M mouse brain slice imaged with Bessel (left) and Gaussian (right) scanning. 45 optical slices taken with a Gaussian focus are vertically stacked to generate a volume image, while the same structural features are visible in a single Bessel scan taken with a 45 μm-long focus. This indicates a substantial gain in volume-imaging speed, making this technique suitable for investigating sparsely labeled samples in-vivo.

Source: Lu R, Sun W, Liang Y, Kerlin A, Bierfeld J, Seelig JD, Wilson DE, Scholl B, Mohar B, Tanimoto M, Koyama M, Fitzpatrick D, Orger MB, and Ji N. "Video-rate volumetric functional imaging of the brain at synaptic resolution." Nature Neuroscience. 2017 Feb 27; 20: 620-628.

A single Bessel scan (above) captures the same structural information obtained from a Gaussian volume scan created by stacking 45 optical sections (below), reducing the total scan time by a factor of 45. The images show a brain slice scanned over a 300 μm x 300 μm area. Scan depth for the Gaussian stack is indicated by the scale bar. Sample Courtesy of Qinrong Zhang, PhD and Matthew Jacobs; the Ji Lab, Department of Physics, University of California, Berkeley.