VIVO Multiphoton

2P and 3P Microscope Systems with Scanless SLM-Based Holography

Highlights

Technology

Software

Data Sets

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Upright, Open & Inverted Imaging Systems with Advanced Photostimulation

Available in upright, open (movable objective), and inverted configurations, VIVO Multiphoton is highly flexible and can be customized to specific imaging needs. VIVO Multiphoton Upright is an intravital imaging system for brain slice and in vivo multiphoton imaging. A flexible and modular design allows for the integration of best-in-class components from platform stages to scanheads to holographic photostimulation. VIVO Multiphoton Open is based on a movable objective microscope platform to provide ultimate flexibility and custom configuration. The system is optimized for mammalian intravital imaging while accommodating all-optical electrophysiology. VIVO Multiphoton Inverted brings 2-photon imaging capabilities to the Marianas live-cell research platform, customizable with an array of stages, cameras, sample autofocus, light sources, photomanipulation devices and environmental control.

Open
Open
Upright
Upright
Inverted
Inverted
Open

RS+ Scanhead

Widefield Camera

Movable Objective Microscope (MOM)

Fast-Gated PMTs

Nouveau Phasor

22mm of objective travel in the x, y, and z axes and motorized path selection

EMCCD and sCMOS cameras for locating labeled neurons or a specific brain area for investigation

Millisecond gating protects GaAsP PMTs during 2P photostimulation paradigms

2P photostimulation via computer-generated holography for all-optical electrophysiology

3-galvo design allows for high-speed resonant scanning and flexible dual-galvo scanning in one system

Upright

Vector RS+

Nouveau Phasor

M-Shaper

Platform Stage

TTL Sync

Kaktus2 Multi-PMT Array

Fully Automated Research Microscope

Nosepiece Detector

Substage Detector

SidePort

Allows for the combination of up to
3 optical paths

Detector array that can accommodate up to 4 PMTs in a combination of types

Motorized z-drive, condenser, optical path selection and PSF-optimized objectives

Dual GaAsP PMTs collect reflected light close to the sample for increased sensitivity

Up to 2 GaAsP PMTs and a 1.2NA lens below the specimen to increase collection of emission signal

Millisecond timing and trigger control of multiple devices

High-speed 30fps full-frame resonant
scanning with rapid switching to spiral & spot photostimulation and ablation

2P photostimulation via computer-generated holography for all-optical electrophysiology

Adaptive optics to dynamically adjust to changing specimen conditions in order to maintain a near-optimal PSF

Accommodates multiple manipulators for electrophysiology or oversized trays for tissue or whole animal presentation

Inverted

Vector2 2P

Fully Automated Research Microscope

Motorized XY Stage

Transmitted IR Detection

Kaktus2 Multi-PMT Array

Widefield Camera

Bialkali PMT to collect transmitted IR light at the top illumination port

Accommodates up to 4 PMTs of any type or combination

EMCCD and sCMOS cameras for epifluorescence imaging

Modular high-speed dual-galvo scanner

Motorized objective and path selection with autofocus (Definite Focus 3) and PSF-optimized objectives

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Highlights

Simultaneous Dual-Plane Multiphoton Imaging

Uncover neuronal connectivity patterns across different layers of the brain with dual plane multiphoton microscopy. Image two disparate planes of the brain in vivo with independent control of power delivery and plane separation using tunable lenses. PMT signals are de-multiplexed, assigned to their respective plane and ready for visualization and analysis in SlideBook software.

Dorsal Plane

Ventral Plane

Merge

 
zebrafish
Transgenic Zebrafish Larvae imaged simultaneously at the dorsal plane and Ventral plane 100µm apart. Tg(elavl3:H2B-GCaMP7f), Elavl3 is a pan-neuronal promoter. Collected at 512×512 at 2Hz.

Nouveau Phasor | All Optical Electrophysiology

Nouveau Phasor is designed for 3D 2-photon stimulation of neurons in vivo. Four distinct modalities enable flexible optogenetics studies involving organisms from C. elegans and Drosophila to zebrafish, mice and larger mammalian species. Nouveau Phasor is able to simultaneously illuminate multiple regions in a 3D volume with the use of SLM-based computer-generated holography (CGH).

Holographic patterns have an axial extent that increases linearly with their lateral extent. This makes it difficult to avoid stimulation above and below the location of interest. The addition of 2D or 3D Temporal Focusing (TF) allows stimulation to be confined along the plane of interest. In 2D-TF the confinement will be in the objective’s plane of focus, while with 3D-TF the confinement can be in any plane in the 3D imaging volume. This allows for selective stimulation of only the cellular components of interest regardless of their spatial location. Nouveau Phasor offers exclusive, patented TF performance in multiple modalities.

Nouveau Phasor Modalities

3D-SHOT

3D Temporal Focusing

3D uniform region photostimulation with axial confinement in the imaging volume
3D flexible region photostimulation via CGH with axial confinement at any axial position in the imaging volume
Nouveau Phasor Modalities

3D Holographic Stimulation

2D Temporal Focusing

3D flexible region photostimulation via CGH
2D flexible region photostimulation via CGH with axial confinement in the plane of focus
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AWAKE ANIMAL BEHAVIOR
2 minute 15 second recording of mouse brain calcium activity in vivo via cranial window expressing ChRmine and GCaMP6m. 16 stimulation events were performed for single-cell activation and pair-of-cells activation. All cells had an increase in calcium activity upon stimulation.

3-Photon | Deep Imaging

Three-photon microscopy (3P) uses longer wavelength NIR light with lower scattering than two-photon microscopy (2P). This allows 3P microscopy to achieve deeper imaging with better signal to noise than traditional 2P microscopy. Whereas 2P has proven to be exceptionally useful to depths of 100µm to 500µm, 3P has increasingly been shown to produce useful data to 1mm and beyond. 3i offers 3P microscopy systems with objectives, adaptive optics, and software to achieve the transmission, pulse compression and point spread function (PSF) integrity needed for successful imaging.

Dual Color Imaging with 3P Excitation

Michael A. Thornton,1 Gregory L. Futia,1 Michael E. Stockton,1 Barish N. Ozbay,2 Karl Kilborn,2 Diego Restrepo,1 Emily A. Gibson,1 Ethan G. Hughes1 Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy. Neurophotonics, 9(3), 031912 (2022).

https://doi.org/10.1117/1.NPh.9.3.031912

1Univ. of Colorado Anschutz Medical Campus (United States)
2Intelligent Imaging Innovations, Inc. (United States)

Simultaneous 3P excitation of EGFP and mScarlet in the primary motor cortex. (A) 3D image volume from an acutely implanted cranial window in an MOBP-EGFP mouse at P65 that was injected with AAV8-hsyn-mScarlet virus at 1000 and 750μm depths in the primary motor cortex. Note large mScarlet-positive layer 5/6 motor output neurons are labeled at the bottom of the image volume and neuronal processes of these cells are labeled throughout the volume. (B) Max projection images of ~30μm volumes in cortical layers 1 (top), 5 (middle), and 6a (bottom).

DeepScan 3P Objective

The DeepScan 3P objective is a high NA long working distance water dipping lens specifically designed for >70% transmission in the 1300nm window and >60% in the 1700nm window. Coupled with the remote focus capability of M-Shaper, it can rapidly capture 3D volumes to depths of well beyond 1mm.

DeepScan 3P Objective

2.0mm Working Distance
0.95NA, f=10mm
Water Dipping

Pulse Compression

Pulse compression is critical to successful three-photon imaging, even more so than in two-photon imaging. 3i engineers design customized beam paths with highly-dispersive mirrors and NIR-optimized optics to deliver sub-50fs pulses to the specimen.

M-Shaper Adaptive Optics Module

When imaging deep into tissue, PSF integrity becomes compromised by the varying optical conditions throughout the specimen. M-Shaper ustilizes adaptive optics to dynamically adjust to those changing conditions in order to maintain a near-optimal PSF. This method of dynamic aberration correction increases signal-to-noise at every plane without increasing acquisition time. Additionally M-Shaper allows for remote focus, enabling high-speed capture of 3D data deep within the specimen without moving the objective.

Without M-Shaper

With M-Shaper

3-photon imaging of MOBP-eGFP (cyan) and THG (yellow) in vivo showing lost signal due to optical aberrations deep within the sample recovered with M-Shaper adaptive optics.
Thornton, M.A., Futia, G.L., Stockton, M.E. et al. Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis. Nat Neurosci 27, 846–861 (2024). https://doi. org/10.1038/s41593-024-01613-7es)

Multi-Channel TCSPC FLIM

VIVO Multiphoton systems are able to perform 2-photon time-correlated single photon counting fluorescence lifetime experiments. SlideBook software controls Becker & Hickl TCSPC boards, GaAsP PMTs or hybrid detectors, and an electronic signal switch to direct PMT signals to either standard A/D conversion for imaging or single photon counting for 2pFLIM. The Becker & Hickl hardware, combined with SlideBook software, provides an effective yet easy to manage tool for conducting FLIM experiments fully integrated with the rest of the system.

Before Uncaging

1 Minute After

2 Minutes After

FLIM
PM-ER interaction in dendritic spines imagined with 2pFLIM, MBL Neurobiology, 2015.

Technology

Scanned Temporal Focusing (sTeFo)

Large volumes of neurons (>500μm3) expressing genetically encoded sensors can now be imaged in an unbiased manner at multiple volumes per second using the new Scanned Temporal Focusing technology for VIVO Multiphoton. sTeFo is a sculpted light approach to ultrafast in vivo volumetric imaging of calcium activity in mice, developed in the Vaziri Lab at The Rockefeller University and published in Nature Methods (2016)1. sTeFo records activity from thousands of neurons at single-cell resolution and physiological rates by scanning a soma-sized ‘sphere’ throughout a volume rather than the diffraction-limited excitation spot of a traditional multiphoton microscope. sTeFo offers a truly volumetric, unbiased approach to interrogating large-scale neuronal activity without making a priori assumptions about neuron locations or discarding axial information.

Diffraction-limited Spot vs. sTeFo

sTeFo relaxes spatial resolution for increased temporal resolution to record neuronal signals from larger populations while maintaining single-cell detection.

Excitation Profile

Shown above is a diffraction-limited (DL) 2P excitation spot profile, expansion of the lateral spot profile, and temporal focusing for axial confinement.

Scan Speed

sTeFo collects large volume datasets at higher speed due to the increased size of the excitation ‘sphere’ that generates fewer and larger voxels.

Simultaneous Volumetric Imaging of Hippocampal CA1-CA3 Regions

Left: Maximum intensity projection (MIP) of 22 CA1 planes, ROIs and example traces. Right: MIP of 15 CA3 planes, ROIs and example traces. Data courtesy Losonczy Lab, The Zuckerman Institute, Columbia University.

Kaktus2 Multi-PMT Array

The Kaktus2 Multi-PMT Array is for experiments where two PMTs are insufficient for signal collection across multiple channels. It is standard for VIVO Multiphoton Inverted systems. It can be mounted in conjunction with mSwitcher to alternate between PMT and high-speed camera detection.

Kaktus2 can accommodate any combination of up to 4 PMTs. Available PMTs include multialkali for high dynamic range, compact GaAsP for high sensitivity, peltier-cooled GaAsP for low light applications, and fast-gated GaAsP for use with photostimulation. Emission signal separation is accomplished by exchangeable drop-in cassettes containing customizable dichroic mirrors and emission filters.

Kaktus2 PMTs

Multialkali

Peltier-Cooled GaAsP

Compact GaAsP

Fast-Gated GaAsP

High dynamic range

High sensitivity and low dark current with active cooling

High sensitivity with a small footprint

PMT of choice when paired with photo stimulation at the millisecond timescale

Blue PMT

Green PMT

Red PMT

Far Red PMT

Convallaria slide imaged with excitation at 920nm showing emission split between four channels.

Patch Clamp Electrophysiology

Traditional patch clamp electrophysiology may be preferred for direct measurement of current and voltage changes taking place across the membrane in neural tissue. Several combinations of stages and micromanipulators can be paired with VIVO Multiphoton systems including existing electrophysiology hardware.

mSwitcher

mSwitcher allows for millisecond switching between a single input and multiple outputs using a high-speed galvo port switcher. This enables the fluorescence emission signal to be projected onto multiple detectors, including cameras, single PMTs, or the Kaktus2 Multi-PMT Array.

SlideBook Software for Acquisition and Analysis

SlideBook software supports research microscopy through the entire experimental process. By managing everything from instrument control to image processing and data analysis, SlideBook allows scientists to focus on investigation rather than instrumentation. SlideBook controls hundreds of instruments in and around the microscope from dozens of manufacturers enabling researchers to integrate their preferred components and upgrade to the latest devices once available.

Slide

User-Selectable App Appearance

Select a color scheme from dozens of options
Switch on-the-fly from dark to light themes

SlideBook Open File Format

Directory-based open file format for big data and high performance computing applications

Volume Rendering

3D and 4D volume view visualization tools support a user- specified bounding box and a storyboard interface where multiple perspectives can be assembled into a single movie

NVIDIA CUDA GPU Acceleration

GPU acceleration of computationally-intensive operations such as deconvolution

Multiwell and Montage

Streamlined multiwell interface
Montaging with a variety of methods

3D Capture Status

Multiphoton Capture Console

Volumetric projection during 4D capture supported across all instruments

Consoles are a single easy-to-use window featuring all frequent controls and status displays. The VIVO Multiphoton scanning console also features an intuitive tool for adjusting laser power delivery at different depths with dynamic signal feedback.

Capabilities

Capture

Control hundreds of devices including microscopes, stages, lasers, wheels, piezos, scanners, shutters and much more.

View

Visualize data through any numbers of portals, from single images to z-stacks, time lapse, color channels and 4D views.

Analyze

Analyze images and extract statistical data via a wide variety of algorithms while maintaining original data integrity.

Scripting

Macro scripting for capture and analysis enhances the flexibility and power available to users.

Communicate

Present and export data easily as 16-bit TIFFs, 3D movies, graphs or spreadsheets. Data is directly portable to MATLAB and Excel and adheres to Open Microscopy Environment (OME) standards.

Partners

MATLAB

Through hierarchical and conditional capture, user-supplied MATLAB programs can control experimental workflows.

Microvolution

Microvolution software delivers nearly instantaneous deconvolution by combining intelligent software programming with the power of a GPU.

Aivia

Aivia is an innovative and complete 2D-to-5D image visualization, analysis and interpretation platform with artificial intelligence-guided image analysis.

Dell

The latest high-power computer workstations control all microscope hardware and enable high-speed processing, segmentation and volume rendering of terabyte (TB) datasets.

Application Data

Mouse
Calcium imaging of odor responses in mice expressing GCaMP6s in mature olfactory sensory neurons. Courtesy of Claire Cheetham, Carnegie Mellon University.
Mouse
Two photon imaging of GCaMP6f responses in a subset of neurons from an intact mouse dorsal root ganglion. Courtesy of Petri Takkala, Prescott lab, University of Toronto.
Mouse
Two photon calcium imaging of a Purkinje neuron filled with Oregon Green BAPTA-1. Courtesy of Meera Pratap, Otis lab, University of California Los Angeles.
 
Drosophila
T4T5 neurons in the Drosophila visual system responding to various directions of visual motion. Courtesy of Dr. Ben Hardcastle, Frye Lab, University of California Los Angeles.
Zebrafish
Z stack of GFP-expressing GABAergic neurons in the zebrafish spinal cord. Courtesy of Jenna Sternberg, Wyart lab, Institut du Cerveau et de la Moelle Épinière, Paris.
Zebrafish
A z-stack projection of ciliated cerebrospinal fluid-contacting neurons (magenta) contacting the central canal in the spinal cord of a larval zebrafish. Cilia are labeled in green. Courtesy of Jenna Sternberg, Wyart lab, Institut du Cerveau et de la Moelle Épinière, Paris.

Additional Resources

Brochures

VIVO Multiphoton

Nouveau Phasor

Publications List

GCaMP imaging of visual responses in adult Drosophila. Frye lab, UCLA Keleş and Frye, 2017. “Object-Detecting Neurons in Drosophila.” Curr Biol. 2017 Jan 30. pii: S0960-9822(17)30012-X. https://www.ncbi.nlm.nih.gov/pubmed/28190726

 

Imaging of electrical activity in an isolated guinea pig heart preparation. Department of Circulation and Medical Imaging, Norwegian University of Science and Technology
Weinberger et al., 2016. “Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells.” Sci Transl Med. 2016 Nov 2;8(363):363ra148.
https://www.ncbi.nlm.nih.gov/pubmed/27807283

 

2-photon stimulation of Chrimson in mouse cortical slices using Phasor 2-Photon, imaging with VIVO Multiphoton on a Sutter Movable Objective Microscope. Adesnik Lab, UC Berkeley
Merel et al., 2016. “Bayseian method for event analysis of intracellular currents.” J Neurosci Methods. 2016 Aug 30;269:21-32.
https://www.ncbi.nlm.nih.gov/pubmed/27208694

 

In vivo imaging in mouse brain through an implanted GRIN lens. Murray Lab, Louisiana Tech University
Voziyanov et al., 2016. “TRIO Platform: A Novel Low Profile In vivo Imaging Support and Restraint System for Mice.” Front Neurosci. 2016 Apr 25;10:169.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842766/

 

GCaMP imaging in spinal motor neurons of awake, behaving zebrafish. Wyart lab, Institut du Cerveau et de la Moelle Epinière (ICM)
Böhm et al., 2016. “CSF-contacting neurons regulate locomotion by relaying mechanical stimuli to spinal circuits.” Nat. Commun. 2015 Mar 7;7:10866.
http://www.nature.com/articles/ncomms10866

 

Calcium imaging in mouse brainstem slices in conjunction with electrophysiology and GABA uncaging using 488 laser line. Kandler lab, University of Pittsburgh
Weisz et al., 2016. “Excitation by Axon Terminal GABA Spillover in a Sound Localization Circuit.” The Journal of Neuroscience, 20 January 2016, 36(3):911-925.
http://www.jneurosci.org/content/36/3/911.long

 

Imaging of cleared mouse spinal cord tissue. Steward Lab, UC Irvine
Willenberg and Steward, 2015. “Nonspecific labeling limits the utility of Cre-Lox bred CST-YFP mice for studies of corticospinal tract regeneration.” J Comp Neurol. 2015 Dec 15;523(18):2665-82. doi: 10.1002/cne.23809.
https://www.ncbi.nlm.nih.gov/pubmed/25976033

 

GCaMP imaging of visual responses in adult Drosophila. Frye Lab, UCLA
Aptekar et al., 2015. “Neurons forming optic glomeruli compute figure-ground discriminations in Drosophila.” The Journal of Neuroscience, 13 May 2015, 35(19): 7587-7599.
http://www.jneurosci.org/content/35/19/7587.long

 

GCaMP imaging in the optic lobe of adult Drosophila during visual stimulation. Frye lab, UCLA
Wasserman et al., 2015. “Olfactory Neuromodulation of Motion Vision Circuitry in Drosophila.” Current Biology , Volume 25 , Issue 4 , 467 – 472.
http://dx.doi.org/10.1016/j.cub.2014.12.012

 

Morphological imaging of labeled neurons in the auditory pathway in mouse brainstem slices. Kandler lab, University of Pittsburgh
Clause et al., 2014. “The precise temporal pattern of prehearing spontaneous activity is necessary for tonotopic map refinement.” Neuron , Volume 82 , Issue 4 , 822 – 835.
http://dx.doi.org/10.1016/j.neuron.2014.04.001

 

2P morphological imaging in rat brain slice combined with 1P ChR excitation and electrophysiology. Otis lab, UCLA
Mathews et al., 2012. “Effects of climbing fiber driven inhibition on Purkinje neuron spiking.” The Journal of Neuroscience, 12 December 2012, 32(50): 17988-17997.
http://www.jneurosci.org/content/32/50/17988.long

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