Sam Tesfai
General Manager,

Thorlabs Imaging Systems

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Bergamo^® II Series Multiphoton Microscopy Platform

Following the principle that the microscope should conform to the specimen, rather than the other way around, we created a completely modular multiphoton imaging platform that adapts to a wide range of experimental requirements. Our multiphoton imaging systems enable simultaneous readout and manipulation of neuron populations at higher speed, greater depth, and higher intensity than with traditional research techniques, such as using microelectrodes. As shown in the Options at a Glance section below and in the Five-Axis Movement of Rotating Bergamo Microscopes video to the lower right, we have developed a rotating body, which allows the microscope to rotate around the sample. We also offer a selection of rigid bodies, which feature an industry-leading 7.74" throat depth for a large three-dimensional working volume around the objective.

The features of Bergamo II listed in the Highlights, Features, and Modules tabs reflect our focus on developing cutting-edge capabilities without compromising usability. Detailed summaries of all the recommended applications for the Bergamo microscope can be found in the Applications tab. To get users started on designing their own microscope, we provide the Configurations tab, which lists a number of example configurations with key features and suggested applications. This platform can be easily modified or upgraded as experimental needs evolve; see the available add-ons and upgrades in the Retrofits tab. 

Bergamo imaging systems have been used to capture impressive sample images (see the Image Gallery tab) and have been featured in numerous publications (see the Publications tab). As shown in the Innovation through Collaboration video to the lower right, the Thorlabs Life Sciences division partnered with Dr. Michael Hausser's research group at the University College London to design a custom Bergamo microscope system to suit their needs. If you have additional questions about our Bergamo imaging systems, please click on the Contact Me button to the right to send us an email.

Highlights

• Rapid Volumetric Imaging Using Bessel Beams
• Three-Photon Imaging
• Spatial Light Modulator for Simultaneous Multi-Site Activation

Rapid Volumetric Imaging Using 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 Bergamo^® multiphoton laser scanning microscope. 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 in the images below, which compare 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.

If you are interested in upgrading your Bergamo microscope to include the Bessel beam imaging modality, please fill out our multiphoton microscope contact form or call (703) 651-1700. For a list of all the upgrades and add-ons available, please see the Retrofits tab.

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.

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A single Bessel scan (left) captures the same structural information obtained from a Gaussian volume scan created by stacking 45 optical sections (right), 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.

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A Thy1-YFP male mouse, 21 weeks old, imaged at 1300 nm, 326 kHz repetition rate, pulse width ~60 fs. At the top of the cortex (0 µm, 1.1 mW laser power), the window was centered at 2.5 mm lateral and 2 mm posterior from the Bregma point over somatosensory cortex. Courtesy of the Chris Xu Group, Cornell University.

Three-Photon Imaging
For our Bergamo multiphoton microscope, we have developed scan path optics for the 800 - 1800 nm range to open the door to three-photon techniques. Three-photon excitation is ideal for deep tissue imaging and requires a high-pulse-energy excitation source, typically around 1300 nm or 1700 nm. Compared to two-photon imaging, three-photon imaging offers less tissue scattering and reduced out-of-focus background, which results in an improved signal-to-background ratio.

Configurations capable of three-photon imaging, such as the one shown below, can include a dichroic mirror to support simultaneous two-photon and three-photon imaging, as well as electronics to support low-repetition-rate lasers with high bandwidth sampling. ThorImage^®LS software has been enhanced with important features for three-photon detection. For instance, users can synchronize the three-photon signal detection to the excitation pulses and control the phase delay for peak signal-to-noise ratio. For more details, please see the ThorImageLS tab.

If you are interested in upgrading your Bergamo microscope to include the three-photon imaging modality, please fill out our multiphoton microscope contact form or call (703) 651-1700. For a list of all the upgrades and add-ons available, please see the Retrofits tab.

Source: Wang T and Xu C. "Three-photon neuronal imaging in deep mouse brain." Optica. 2020; 7 (8): 947-960.

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Two- and Three-Photon Imaging Configuration. For more details, please see the Configurations tab.

Spatial Light Modulator for Simultaneous Multi-Site Activation
Thorlabs’ Spatial Light Modulator (SLM) uses holography patterns to enable photoactivation of multiple locations in a specimen simultaneously. Designed for two-photon excitation with femtosecond pulses, the SLM manipulates the phase across the stimulation laser beam profile to generate hundreds of user-determined focal points.

The diagrams below illustrate the benefits of using the SLM with two-photon activation over two-photon activation alone and single-photon activation. With single-photon activation, unintended nearby cells as well as the target cell become activated because this technique lacks the ability to target a single cell. This problem can be solved with two-photon activation, which allows single-cell resolution targeting; however, only one cell can be targeted at a time. Two-photon activation with SLM overcomes these limitations by generating a number of focal points and allowing multiple target cells to be activated simultaneously. Each beam can be shaped to improve the efficacy of photoactivation, a crucial feature for activating neural populations at varying depths within a single FOV. The SLM phase mask pattern can be rapidly switched, enabling multiple individual focal points to be targeted independently in any sequence. The calibration process, hologram generation, and external hardware synchronization are entirely managed through the ThorImage^®LS software, enabling seamless control. For more details, please see the ThorImageLS tab.

If you are interested in upgrading your Bergamo microscope to include the SLM imaging modality, please fill out our multiphoton microscope contact form or call (703) 651-1700. For a list of all the upgrades and add-ons available, please see the Retrofits tab.

Two-Photon Activation + Spatial Light Modulator

• Generate Multiple Focal Points
• Simultaneously Activate Multiple Target Cells

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Two-Photon Activation

• Single-Cell Resolution Targeting
• Activate Only Target Cell

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Single-Photon Activation

• Lack of Depth Control for 3D Neural Activation
• Activate Target Cell and Unintended Nearby Cells

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Features

Many of these features can also be added to existing Thorlabs microscope systems. For more information, please see the Retrofits tab.

Bergamo^® II Modules

Thorlabs Bergamo^® II microscopes are modular systems that can be customized in the design process to meet the exact needs of the experiment. The modules listed below are displayed in a variety of pre-built examples found on our Configurations tab to help provide a starting point for your design. 

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Figure 2. Fast Switching between the optimal excitation wavelengths of 750 nm and 835 nm provides the high contrast seen in this composite image. The two-channel set was collected at an imaging rate of 7 fps.

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Figure 1.  The above image was acquired using single-wavelength excitation at 788 nm, while the optimum excitation wavelengths for the two tags are 750 nm and 850 nm.

Fast Switching Using a
Tunable Femtosecond Laser

With an industry-leading tuning speed of up to 4000 nm/s and a wide 720 to 1060 nm tuning range, the Tiberius^® Ti:Sapphire Femtosecond Laser is ideal for fast sequential imaging in multiphoton microscopy applications. 

A 25 µm thick sagittal section of an adult rat brain is shown in the images and video to the right. The red channel corresponds to fluorescence from chick anti-neurofilament that is optimally excited at 835 nm, while the green channel corresponds to fluorescence from mouse anti-GFAP that is optimally excited at 750 nm.

Figure 1 shows fluorescence from single-wavelength excitation at 788 nm, which sub-optimally excites the two tags simultaneously. Figure 2 is a composite image of the fluorescence produced by a two-color excitation image sequence acquired at 7 fps where the excitation wavelength was rapidly tuned between 750 and 835 nm. The video in Figure 3 shows the fast-switching used to create the composite image in Figure 2 at 1/16^th of the actual speed. When compared to single-wavelength excitation at 788 nm with the same intensity, fast switching offers much higher image contrast as it provides optimal excitation of both fluorophores.

This immunofluorescence sample was prepared by Lynne Holtzclaw of the NICHD Microscopy and Imaging Core Facility, a part of the National Institutes of Health (NIH) in Bethesda, MD.

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Figure 2. Close-Up of a Gaussian Beam

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Figure 1. Close-Up of a Bessel Beam

Volumetric Imaging Technique Using Bessel Beams

Thorlabs is excited to offer a new ultra-fast imaging technique that uses a Bessel beam to provide video-rate volumetric functional imaging of neuronal pathways and interactions in vivo. These unique beams are non-diffractive and self-healing, which allows them to maintain a tight focus and even reform as they pass through tissue. This technique is offered for Thorlabs' Bergamo II multiphoton microscopes and Thorlabs' Multiphoton Mesoscope. 

The images to the right depict a Bessel beam and a Gaussian beam, respectively. As you can see in the images, the Gaussian beam has a singular point of focus that progressively becomes weaker as it diverges from the central point, whereas the Bessel beam has a beam annulus that maintains its focus.

To read the press release about this new technique, click here.

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Rotating Bergamo II systems are outfitted with multi-joint articulating periscopes. This periscope's design offers the enhanced flexibility needed to allow the entire scanning system to be tilted with respect to the sample.

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Upright Bergamo II systems are equipped with periscopes that permit the microscope's full travel range in X, Y, and Z to be used without compromising the optical performance.

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Emission filters and dichroic cubes are held behind magnetically sealed doors on the front of the PMT detection module.

Collection Optics of Example Configurations^a

B243:	Multi-Target Photoactivation (Rotating)	Epi: 10°
B242:	Two- and Three-Photon Imaging (Rotating)	Epi: 14°
B251:	Random Access Scanning (Rotating)	Epi: 14°
B241:	In Vivo Two-Photon Imaging (Rotating)	Epi: 14°
B252:	Dual-Path Random Access Scanning (XYZ)	Epi: 14° Transmitted: 13°
B262:	Dual-Path Confocal Imaging (XYZ)	Epi: 14°
B231:	Simple Imaging (XYZ)	Epi: 8° Transmitted: 13°
B211:	Video and High-Speed Imaging (Z-Only)	Epi: 8°
B201:	Simple Imaging (Z-Only)	Epi: 8°

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A Quantulux sCMOS Camera and 1.4 Megapixel Scientific Camera

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Trans-Illumination Module, Motorized Condenser Stage, and
Rigid Stand Sample Holder Underneath the Objective

Example Configurations

Explore the details of example Bergamo II rotating, XYZ, and Z-axis system configurations by clicking on the expandable sections below. The modular nature of our multiphoton microscopy platform allows us to modify configurations to meet individual experimental needs or adjust the functionality of a microscope after installation; more information on modules featured in the systems below can be found on the Modules tab.

Configuration	System Highlights
Rotating Body
Configuration B243: Multi-Target Photoactivation	Click for Details	Key Features	Suggested Applications
		Spatial Light Modulator (SLM) in Secondary Beam Path, Providing Holographic, All-Optical Multi-Target Stimulation Large Throat Depth for In Vivo Animal Studies Separate Lasers for Multiphoton Imaging and Photoactivation Galvo-Resonant Scanning for High-Speed Image Acquisition	Synapses and Circuits Ion Channels, Transporters, and Neurotransmitter Reporters Functional and Molecular Imaging Neurological Disorders
Additional Specifications for Multi-Target Photoactivation Configuration Configuration Specifications Highlighted System Specifications FOV 12 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Photoactivation Targeted: Holographic, Simultaneous Multi-Site Full Field: During Scanner Flyback Microscope Components Microscope Body -45 to +45° Rotation Primary Scan Path (Imaging Scanners) Galvo-Resonant (8 or 12 kHz) Secondary Scan Path Spatial Light Modulator Scan Path Wavelength Range 450 - 1100 nm or 680 - 1600 nm Pockels Cell Slow (250 kHz) on Primary Path Variable Attenuator Motorized Multiphoton Detection 2 Non-Cooled GaAsP PMTs Collection Optics Module 10° with Shutter Objective Holder Single Objective Nikon 16X (with Piezo Objective Scanner) Sample Stage None Widefield Imaging Single Cube Epi-Illuminator Module Single-Channel Mounted LED 4 MP CCD Scientific Camera with GigE Interface Laser Multiphoton Imaging: Tiberius® Tunable fs Laser Photoactivation: Menlo Systems' BlueCut fs Fiber Laser Our spatial light modulator (SLM) manipulates the phase of the stimulation laser beam to generate hundreds of user-determined focal points. The SLM phase mask pattern can be rapidly switched, enabling multiple individual focal points to be targeted independently of each other. Each beamlet can be shaped to improve the efficacy of photoactivation; a crucial feature for activating neural populations at varying depths within a single FOV. This configuration utilizes two separate lasers for imaging and photostimulation; in addition to the highly configurable nature of the Bergamo microscope system, the scanning paths can be adjusted for a dual-output laser source. Click to Enlarge The SLM is installed on the secondary scan path, enabling photostimulation simultaneous with multiphoton imaging. Click to Enlarge The Tiberius fs tunable laser is used for multiphoton imaging, while Menlo Systems' BlueCut fiber laser is used for photostimulation.
Configuration B242: Two- and Three-Photon Imaging	Click for Details	Key Features	Suggested Applications
		Dual-Channel Paths for Simultaneous 2P and 3P Imaging, or 3P Imaging with Photoactivation Beam Conditioning with Variable Beam Expander, Pockels Cell, and Variable Attenuator Simultaneous 2P or 3P Imaging with Widefield Epi-Fluorescence Large Working Space and Rotating Microscope Body for In Vivo Large Animal Studies	Structural Neurobiology Neurological Disorders
Additional Specifications for 2P / 3P Imaging Configuration Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed Galvo-Resonant Scanner: 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Galvo-Galvo Scanner: 3 fps at 512 x 512 Pixels Photoactivation Targeted: Simultaneous IR, Sequential Visible Microscope Components Microscope Body -5 to +95° Rotation Primany Scan Path Galvo-Resonant (8 or 12 kHz) Secondary Scan Path Galvo-Galvo (Ø4 mm or Ø5 mm) Scan Path Wavelength Range 680 - 1600 nm on Primary Path 800 - 1800 nm on Secondary Path Variable Beam Expander Primary Path Pockels Cell Fast (1 MHz) on Primary Path Slow (250 kHz) on Secondary Path Variable Attenuator Motorized Beam Stabilization Primary and Secondary Paths Multiphoton Detection 2 Cooled GaAsP PMTs Collection Optics Module 14° with Shutter Objective Holder Single Objective Nikon 25X (with Piezo Objective Scanner) Sample Stage None Widefield Imaging Single-Cube Epi-Illuminator Module Four-Channel LED Source Quantalux™ sCMOS Scientific Camera Laser Primary Path: 3P Pulsed Laser Secondary Path: Tiberius® fs Tunable Laser This dual-path configuration uses both galvo-resonant and galvo-galvo scanners and infrared wavelength scanning optics to image second- and third-harmonic generation (SHG and THG). Click to Enlarge The primary and secondary scan path optics are optimized for 2P and 3P imaging, respectively. The beam paths are co-registered for simultaneous multi-channel imaging. Click to Enlarge View your subject at angles up to +95° with our Rotating Microscope Bases. The body rotation is centered on the focal point of your objective, allowing free movement without the need to refocus.
Configuration B251: Random Access Scanning	Click for Details	Key Features	Suggested Applications
		Resonant-Galvo-Galvo Scanner (US Patent 10,722,977) for High-Resolution, Fast Acquisition of Multiple Regions within a Single FOV Integrate with Soundbox for Sound-Sensitive Experiments Edge Blanking with Fast Pockels Cell Simultaneous Multi-Channel Epi-Fluorescence Tiberius fs Ti:Sapphire Tunable Laser	Synapses and Circuits Ion Channels, Transporters, and Neurotransmitter Reporters Functional and Molecular Imaging Neurological Disorders
Additional Specifications for Random Access Scanning Configuration This high-speed random access scanning configuration uses a resonant-galvo-galvo scanner to take multiple high-resolution images within a single field of view. This scanner provides all the speed of a resonant-galvo scanner while enabling multiple user-defined regions to be chosen. This imaging functionality is ideal for correlating neural responses in multiple regions of the brain. Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Photoactivation Targeted: Sequential Full Field: During Scanner Flyback Microscope Components Microscope Body -5 to +95° Rotation Imaging Scanners Resonant-Galvo-Galvo (8 or 12 kHz) Scan Path Wavelength Range 450 - 1100 nm Pockels Cell Fast (1 MHz) Variable Attenuator Motorized Multiphoton Detection 2 Non-Cooled GaAsP PMTs Collection Optics Module 14° Objective Holder Single Objective Olympus 20X (with Piezo Objective Scanner) Sample Stage Rigid Stand with Breadboard Insert Widefield Imaging Single-Cube Epi-Illuminator Module Single-Channel Mounted LED Quantalux™ sCMOS Scientific Camera Laser Tiberius® Tunable fs Laser Click to Enlarge The microscope can be placed within a soundbox for sensitive auditory studies, as shown here.
Configuration B241: In Vivo Two-Photon Imaging	Click for Details	Key Features	Suggested Applications
		Large Working Space with Fully Rotatable Microscope Integrate with Soundbox for Sound-Sensitive Experiments Galvo-Resonant Scanner for High-Speed Imaging Edge Blanking with Fast or Standard Pockels Cell Simultaneous Multi-Channel Epi-Fluorescence Tiberius® fs Ti:Sapphire Tunable Laser	Ion Channels, Transporters, and Neurotransmitter Reporters Functional and Molecular Imaging Synapses and Circuits Auditory Functional Imaging
Additional Specifications for In Vivo 2P Imaging Configuration This rotating Bergamo microscope configuration is compact enough to be placed in an enclosure, enabling sound or light-sensitive in vivo studies to be performed. Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Photoactivation Fixed Spot at the Center of the Field of View Microscope Components Microscope Body -5 to +95° Rotation Imaging Scanners Galvo-Resonant (8 kHz or 12 kHz) Scan Path Wavelength Range 450 - 1100 nm or 680 - 1600 nm Pockels Cell Fast (1 MHz) or Slow (250 kHz) Variable Attenuator Motorized Multiphoton Detection 2 Non-Cooled GaAsP PMTs Collection Optics Module 14° with Shutter Objective Holder Single Objective Olympus 20X (with Piezo Objective Scanner) Sample Stage Rigid Stand with Breadboard Insert Widefield Imaging Six-Filter-Turret Epi-Illuminator Module Solis™ High-Powered White LED Quantalux™ sCMOS Scientific Camera Laser Tiberius® fs Tunable Laser Click to Enlarge 2P Rotating Microscope Encased Inside a Soundbox
Upright XYZ Body
Configuration B252: Dual-Path Random Access Scanning	Click for Details	Key Features	Suggested Applications
		Light-Tight Enclosure for Laser Conditioning Modules and Components Resonant-Galvo-Galvo Scanner (US Patent 10,722,977) for High-Speed, High-Resolution Imaging of Multiple FOVs PMT Options for Scanned or De-Scanned Multiphoton Detection Gibraltar Breadboard Platform for Samples and Supplementary Equipment Dual-Output Spectra Physics InSight Laser High-Speed, High-Resolution Volumetric Imaging Using Bessel Beams (Optional)	Ion Channels, Transporters, and Neurotransmitter Reporters Drug Discovery Ex Vivo Neurobiological Studies Electrophysiology and Patch-Clamp Recordings In Vivo Neurobiological Studies using Bessel Beam Volumetric Imaging Technique (Optional)
Additional Specifications for Dual-Path Random Access Scanning Configuration Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed Resonant-Galvo-Galvo Scanner: 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Galvo-Galvo Scanner: 3 fps at 512 x 512 Pixels Scan Resolution Bi-Directional: 8 kHz RGG and GG: Up to 2048 x 2048 Pixels 12 kHz RGG: Up to 1168 x 1168 Pixels Uni-Directional: 8 kHz RGG and GG: Up to 4096 x 4096 Pixels 12 kHz RGG: Up to 2336 x 2336 Pixels Photoactivation Targeted: Simultaneous IR, Sequential Visible Full Field: During Scanner Flyback Microscope Components Microscope Body XYZ Motion Primary Scan Path Resonant-Galvo-Galvo (8 or 12 kHz) Secondary Scan Path Galvo-Galvo (Ø4 mm or Ø5 mm) Scan Path Wavelength Range Primary Path: 680 - 1600 nm Secondary Path: 450 - 1100 nm Pockels Cell Slow (250 kHz) on Primary Path Variable Attenuator Motorized Multiphoton Detection 2 Cooled GaAsP PMTs 2 Non-Cooled GaAsP PMTs Collection Optics Module 14° with Shutter Objective Holder Single Objective Nikon 25X (with Piezo Objective Scanner) Sample Stage Gibraltar Platform Widefield Imaging Transmitted Light Module with Laser Scanning Dodt Six-Filter-Turret Epi-Illuminator Module Fiber-Coupled 4-Channel LED Quantalux™ sCMOS Scientific Camera Laser Spectra Physics InSight Tunable Laser Fully equipped for random access scanning, this configuration features both a resonant-galvo-galvo scanner on the primary path and a galvo-galvo scanner on the secondary path. The system uses a dual-output laser for multiphoton imaging. Click for Details The large Gibraltar stage shown above allows both samples and supplementary equipment to be aligned in the focal plane of the objective. Here, a motorized micromanipulator is mounted onto the breadboard to manipulate cells for patch-clamp recordings. Click to Enlarge A custom enclosure can be added to house beam conditioning modules and other components, creating a light-tight region in which parts may be altered without affecting the rest of the beam path. Click to Enlarge The resonant-galvo-galvo scanner on the primary path allows multiple regions of interest to be quickly scanned in a single field of view, while the galvo-galvo scanner on the secondary path can be used for simultaneous photoactivation.
Configuration B262: Dual-Path with Confocal Imaging	Click for Details	Key Features	Suggested Applications
		Dual-Paths for Multiphoton Imaging with Confocal Imaging or Photoactivation Galvo-Resonant Scanner for High-Speed Imaging and Galvo-Galvo Scanner for Custom Geometric Scans Four-Channel Confocal Fiber Laser and Tiberius® fs Ti:Sapphire Tunable Laser	Structural Neurobiology Ion Channels, Transporters, and Neurotransmitter Reporters Neural Development and Plasticity Functional and Molecular Imaging
Additional Specifications for Dual-Path Confocal Configuration Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed Galvo-Resonant Scanner: 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Galvo-Galvo Scanner: 3 fps at 512 x 512 Pixels Scan Resolution Bi-Directional: 8 kHz Galvo-Resonant and Galvo-Galvo: Up to 2048 x 2048 Pixels 12 kHz Galvo-Resonant: Up to 1168 x 1168 Pixels Uni-Directional: 8 kHz Galvo-Resonant and Galvo-Galvo: Up to 4096 x 4096 Pixels 12 kHz Galvo-Resonant: Up to 2336 x 2336 Pixels Photoactivation Targeted: Simultaneous IR, Sequential Visible Full Field: During Scanner Flyback Microscope Components Microscope Body XYZ Motion Primary Scan Path (Multiphoton) Galvo-Resonant (8 or 12 kHz) Secondary Scan Path Galvo-Galvo (Ø4 mm or Ø5 mm) Scan Path Wavelength Range 450 - 1100 nm or 680 - 1600 nm on Primary and/or Secondary Path Pockels Cell Slow (250 kHz) (High-Speed and Wide-Wavelength-Range Options Available) Variable Attenuator Motorized Multiphoton Detection 2 Non-Cooled GaAsP PMTs Confocal Detection 4-Channel Multialkali PMTs with Variable-Size Pinhole Wheel Collection Optics Module 14° Objective Holder Single Objective Nikon 25X (with Piezo Objective Scanner) Sample Stage Rigid Stand with Breadboard Insert Widefield Imaging Six-Filter-Turret Epi-Illuminator Module Broadband Light Source Quantalux™ sCMOS Scientific Camera Laser Multiphoton: Tiberius® fs Tunable Laser Confocal: 4-Channel Viper Laser Source This multi-path, multi-modality configuration allows for a range of experimental conditions to be observed using any combination of multiphoton, confocal, and epi-fluorescence imaging, along with photoactivation. Multiphoton imaging with photoactivation is enabled by two beam paths; the primary with a galvo-resonant scanner for high-speed imaging, and the secondary with a galvo-galvo scanner for area-specific photoactivation. The four-channel confocal laser connects through a fiber bulkhead to the secondary scan path, and the four-channel PMT detection module uses an additional port at the back focal plane of the objective. Epi-fluorescence is enabled by a six-filter-turret epi-illuminator module and sCMOS Quantalux camera. Click to Enlarge Thorlabs provides a variety of sample mounting options. The breadboard insert on the rigid stand provides the space and stability for a VR fly theater and supplementary cameras to be mounted below the objective, as shown.
Configuration B231: Simple XYZ Imaging	Click for Details	Key Features	Suggested Applications
		High-Speed Galvo-Resonant Scanning High-Sensitivity GaAsP PMT Free-Space Photodetector Removable Transmitted Light Module with Laser Scanning Dodt Functionality Epi-Fluorescence with Quantalux™ sCMOS Camera Tiberius fs Ti:Sapphire Tunable Laser	Structural Neurobiology Functional and Molecular Imaging Drug Discovery Fixed Stage Experiments Neurogenetics Cell Biology of Neurons, Muscles, and Glia
Additional Specifications for Simple XYZ Configuration Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 8 kHz Scanner: 30 fps at 512 x 512 Pixels or 12 kHz Scanner: 45 fps at 512 x 512 Pixels Imaging Resolution Bi-Directional: 8 kHz Scanner: Up to 2048 x 2048 Pixels 12 kHz Scanner: Up to 1168 x 1168 Pixels Uni-Directional: 8 kHz Scanner: Up to 4096 x 4096 Pixels 12 kHz Scanner: Up to 2336 x 2336 Pixels Photoactivation Full Field: 4-Channel Microscope Components Microscope Body XYZ Motion Imaging Scanners Galvo-Resonant (8 or 12 kHz) Scan Path Wavelength Range 450 - 1100 nm Pockels Cell Slow (250 kHz) (High-Speed and Wide-Wavelength-Range Options Available) Variable Attenuator Motorized Detection 2 Non-Cooled GaAsP PMTs Collection Optics Module 8° with Shutter Objective Holder Manual Dual Objective Nikon 16X (with Piezo Objective Scanner) Nikon 40X Sample Stage Rigid Stand Slide Holder with Translation Stage Widefield Imaging Transmitted Light Module with Laser Scanning Dodt Single-Cube Epi-Illuminator Module Four-Channel LED Source Quantalux™ sCMOS Scientific Camera Laser Tiberius® fs Tunable Laser This configuration is well-balanced for both in vitro and fixed stage in vivo microscopy research. The modular system with a removable trans-illumination module provides high versatility for multiple experimental techniques, imaging modalities, and sample subjects. Click to Enlarge The ample space under the microscope enables large setups with supplemental equipment to be easily arranged under the objective. Here, we show a Drosophila VR theater stage along with two additional cameras. The setup can be synchronized with image acquisition using ThorSync, an add-on to our ThorImageLS software. Click to Enlarge Any one of our PMTs can be replaced with a free-space photodetector for signal detection of other imaging modalities. This image shows a variation of the microscope where one PMT is replaced with the DET10A2 for reflected signal detection.
Upright Z-Axis Body
Configuration B211: Video and High-Speed Imaging	Click for Details	Key Features	Suggested Applications
		High-Speed Galvo-Resonant Scanning Pockels Cell and Motorized Variable Attenuator for Remote Laser Adjustment 2-Channel PMT Detection Small Footprint with Large Throat Depth Tiberius fs Ti:Sapphire Tunable Laser	Neural Development and Plasticity Neurogenetics Ion Channels, Transporters, and Neurotransmitter Reporters Functional and Molecular Imaging Cell Biology of Neurons, Muscles, and Glia
Additional Specifications for Video Imaging Configuration Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 8 kHz: 30 fps at 512 x 512 Pixels or 12 kHz: 45 fps at 512 x 512 Pixels Imaging Resolution Bi-Directional: 8 kHz Scanner: Up to 2048 x 2048 Pixels 12 kHz Scanner: Up to 1168 x 1168 Pixels Uni-Directional: 8 kHz Scanner: Up to 4096 x 4096 Pixels 12 kHz Scanner: Up to 2336 x 2336 Pixels Photoactivation Full Field: 4-Channel Microscope Components Microscope Body Z-Axis Motion Imaging Scanners Galvo-Resonant (8 kHz or 12 kHz) Scan Path Wavelength Range 450 - 1100 nm Pockels Cell Slow (250 kHz) Variable Attenuator Motorized Detection 2 Multialkali PMTs (GaAsP PMTs and Cooled PMTs Available) Collection Optics Module 8° with Shutter Objective Holder Single Objective Olympus 20X Sample Stage Rigid Stand Slide Holder with XY Translation Stage Widefield Imaging Single-Cube Epi-Illuminator Module Four-Channel LED Source Quantalux™ sCMOS Scientific Camera Laser Tiberius® fs Tunable Laser This configuration enables video-rate, sequential two-photon imaging to study fast dynamic biological and chemical processes. Click to Enlarge Rigid stands provide stability for numerous sample mounting options, including the multi-camera VR theater shown here for Drosophila studies. Click to Enlarge Galvo-resonant scanners are available at 8 kHz and 12 kHz resonant frequencies.
Configuration B201: Simple Z-Axis Imaging	Click for Details	Key Features	Suggested Applications
		Galvo-Galvo Scanning 2-Channel Detection with High-Sensitivity GaAsP PMTs Small Footprint with Large Throat Depth 930 nm Menlo YLMO Pulsed Laser	Neurogenetics Neural Development and Plasticity
Additional Specifications for Simple Z-Axis Imaging Configuration This configuration provides the simplest microscopy system for studies that require multiphoton imaging at the sample plane without unnecessary features. Please note that this configuration requires a power attenuator (not pictured). Configuration Specifications Highlighted System Specifications FOV 20 mm Diagonal Square (Max) at the Intermediate Image Plane Imaging Speed 3 fps at 512 x 512 Pixels Scanning Resolution Bi-Directional: Up to 2048 x 2048 Pixels Unidirectional: Up to 4096 x 4096 Pixels Microscope Components Microscope Body Z-Axis Motion Imaging Scanners Galvo-Galvo (Ø4 mm or Ø5 mm) Scan Path Wavelength Range 680 - 1600 nm or 450 - 1100 nm Variable Attenuator Manual Detection 2 Cooled GaAsP PMTs Collection Optics Module 8° Objective Holder Single Objective Nikon 16X Sample Stage Rigid Stand Slide Holder with Translation Stage Laser Menlo YLMO 930 nm Laser Click to Enlarge Menlo Systems' YLMO 930 nm laser is shown here as the primary laser source. Imaging optics may be tuned for short or long-wavelength multiphoton imaging, and the galvo-galvo scanner switched to accommodate a larger or smaller beam diameter.

Sam Tesfai
Imaging Systems General Manager

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Trans-Illumination Add-On for Rotating Bergamo II Systems

Retrofit Options for Existing Multiphoton Systems

• Upgrades to the Functionality of Existing Microscope Infrastructure and Components
• Add-On Components to Increase Capabilities

Thorlabs' modular design enables our microscopes to continually evolve with experimental needs. Customers with previous model microscopes and product lines for multiphoton microscopy have the flexibility to update their infrustructure or add to their existing components as their microscopy needs change. See the expandable tables below for options available for each of our multiphoton system lines, and contact us for additional information about incorporating an upgrade or add-on into your system.

Please note that certain upgrades and add-ons require an on-site visit by one of our specialists for installation.

Images Taken Using Bergamo^® Systems

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Selected Publications Using Thorlabs' Imaging Systems

2019

Ceriani F, Johnson SL, Sedlacek M, Hendry A, Kachar B, Marcotti W, and Mammano F. "Dynamic coupling of cochlear inner hair cell intrinsic Ca²⁺ action potentials to Ca²⁺ signaling of non-sensory cells." bioRxiv. 2019 Aug 10; 731851.

Brawek B and Garaschuk O. "Single-Cell Electroporation for Measuring In Vivo Calcium Dynamics in Microglia." Microglia Methods in Molecular Biology. 2019 Aug 8; 2034: 231-241.

Brawek B, Olmedillas del Moral M, and Garaschuk O. "In Vivo Visualization of Microglia Using Tomato Lectin." Microglia Methods in Molecular Biology. 2019 Aug 8; 2034: 165-175.

Liang Y and Garaschuk O. "Labeling Microglia with Genetically Encoded Calcium Indicators." Microglia Methods in Molecular Biology. 2019 Aug 8; 2034: 243–265.

Royzen F, Williams S, Fernandez FR, and White JA. "Balanced synaptic currents underlie low-frequency oscillations in the subiculum." Hippocampus. 2019 July 13; 23131.

Rathore APS, Mantri CK, Aman SAB, Syenina A, Ooi J, Jagaraj CJ, Goh CC, Tissera H, Wilder-Smith A, Ng LG, Gubler DJ, and St. John AL. "Dengue virus-elicited tryptase induces endothelial permeability and shock." J Clin Invest. 2019 July 2; JCI128426.

Stringer C, Pachitariu M, Steinmetz N, Carandini M, and Harris KD. "High-dimensional geometry of population responses in visual cortex." Nature. 2019 June 26; 571: 361–365.

Ziraldo G, Buratto D, Kuang Y, Xu L, Carrer A, Nardin C, Chiani F, Salvatore AM, Paludetti G, Lerner RA, Yang G, Zonta F, and Mammano F. "A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function." Front Physiol. 2019 June 11; 10: 392.

Romero S, Hight AE, Clayton KK, Resnik J, Williamson RS, Hancock KE, and Polley DB. "Cellular and widefield imaging of sound frequency organization in primary and higher-order fields of the mouse auditory cortex." bioRxiv. 2019 June 6; 663021.

Díaz-García CM, Lahmann C, Martínez-François JR, Li B, Koveal D, Nathwani N, Rahman M, Keller JP, Marvin JS, Looger LL, and Yellen G. "Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor." J Neuro Res. 2019 May 20; 97: 946–960.

Philip V, Newton D, Oh H, Collins S, Bercik P, and Sibille E. "The Effect of Gut Microbiota on Glutamatergic/GABAergic Gene Expression in Adult Mice." Biological Psychiatry. 2019 May 15; 85: S127–S128.

Bowen Z, Winkowski DE, Seshadri S, Plenz D, and Kanold PO. "Neuronal avalanches in input and associative layers of auditory cortex." bioRxiv. 2019 Apr 29; 620781.

Burgold J, Schulz-Trieglaff EK, Voelkl K, Gutiérrez-Ángel S, Bader JM, Hosp F, Mann M, Arzberger T, Klein R, Liebscher S, and Dudanova I. "Cortical circuit alterations precede motor impairments in Huntington’s disease mice." Sci Rep. 2019 Apr 29; 9: 1–13.

Matovic S, Ichiyama A, Igarashi H, Salter EW, Wang XF, Henry M, Vernoux N, Tremblay ME, and Inoue W. "Stress-induced neuronal hypertrophy decreases the intrinsic excitability in stress habituation." bioRxiv. 2019 Mar 31; 593665.

Weissenberger Y, King AJ, and Dahmen JC. "Decoding mouse behavior to explain single-trial decisions and their relationship with neural activity." bioRxiv. 2019 Mar 5; 567479.

Ceriani F, Hendry A, Jeng JY, Johnson SL, Stephani F, Olt J, Holley MC, Mammano F, Engel J, Kros CJ, Simmons DD, and Marcotti W. "Coordinated calcium signaling in cochlear sensory and non-sensory cells refines afferent innervation of outer hair cells." The EMBO Journal. 2019 Feb 25; 38: e99839.

Lee KS, Vandemark K, Mezey D, Shultz N, and Fitzpatrick D. "Functional Synaptic Architecture of Callosal Inputs in Mouse Primary Visual Cortex." Neuron. 2019 Feb 6; 101: 421-428.e5.

2018

Marvin JS, Scholl B, Wilson DE, Podgorski K, Kazemipour A, Müller JA, Schoch S, Quiroz FJU, Rebola N, Bao H, Little JP, Tkachuk AN, Cai E, Hantman AW, Wang SSH, DePiero VJ, Borghuis BG, Chapman ER, Dietrich D, DiGregorio DA, Fitzpatrick D, and Looger LL. "Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR." Nat Methods. 2018 Oct 30; 15: 936–939.

Moeyaert B, Holt G, Madangopal R, Perez-Alvarez A, Fearey BC, Trojanowski NF, Ledderose J, Zolnik TA, Das A, Patel D, Brown TA, Sachdev RNS, Eickholt BJ, Larkum ME, Turrigiano GG, Dana H, Gee CE,
Oertner TG, Hope BT, and Schreiter ER. "Improved methods for marking active neuron populations." Nat Commun. 2018 Oct 25; 9: 1–12.

Chen CL, Hermans L, Viswanathan MC, Fortun D, Aymanns F, Unser M, Cammarato A, Dickinson MH, and Ramdya P. "Imaging neural activity in the ventral nerve cord of behaving adult Drosophila." Nat Commun. 2018 Oct 25; 9: 1–10.

Corns LF, Johnson SL, Roberts T, Ranatunga KM, Hendry A, Ceriani F, Safieddine S, Steel KP, Forge A, Petit C, Furness DN, Kros CJ, and Marcotti W. "Mechanotransduction is required for establishing and maintaining mature inner hair cells and regulating efferent innervation." Nat Commun. 2018 Oct 1; 9: 1-15.

Saleem AB, Diamanti EM, Fournier J, Harris KD, and Carandini M. "Coherent encoding of subjective spatial position in visual cortex and hippocampus." Nature. 2018 Sept 10; 562: 124-127.

Gillet SN, Kato HK, Justen MA, Lai M, and Isaacson JS. "Fear Learning Regulates Cortical Sensory Representations by Suppressing Habituation." Front. Neural Circuits. 2018 Jan 10; 11: 112.

2017

Scholl B, Wilson DE, and Fitzpatrick D. "Local order within global disorder: synaptic architecture of visual space." Neuron. 2017 Dec 6; 95 (5): 1127-1138.

Klapoetke NC, Nern A, Peek MY, Rogers EM, Breads P, Rubin GM, Reiser MB, and Card GM. "Ultra-selective looming detection from radial motion opponency." Nature Neuroscience. 2017 Nov 09; 551: 237-241.

Kato HK, Asinof SK, and Isaacson JS. "Network-level control of frequency tuning in auditory cortex." Neuron. 2017 Jul 19; 95 (2): 412-423.

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.

2016

Mongeon R, Venkatachalam V, and Yellen G. "Cytosolic NADH-NAD⁺ Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging." Antioxid Redox Signal. 2016 Oct 1; 25 (10): 553-563.

Pachitariu M, Stringer C, Schröder S, Dipoppa M, Rossi LF, Carandini M, and Harris KD. "Suite2p: beyond 10,000 neurons with standard two-photon microscopy." bioRxiv. 2016 Jun 30; 061507.

Dipoppa M, Ranson A, Krumin M, Pachitariu M, Carandini M, and Harris KD. "Vision and locomotion shape the interactions between neuron types in mouse visual cortex." bioRxiv. 2016 Jun 11; 058396.

Rose T, Jaepel J, Hübener M, and Bonhoeffer T. "Cell-specific restoration of stimulus preference after monocular deprivation in the visual cortex." Science. 2016 Jun 10; 352 (6291): 1319–22.

Strobl MJ, Freeman D, Patel J, Poulsen R, Wendler CC, Rivkees SA, and Coleman JE. "Opposing Effects of Maternal Hypo- and Hyperthyroidism on the Stability of Thalamocortical Synapses in the Visual Cortex of Adult Offspring." Cereb Cortex. 2016 May 26; pii: bhw096 (epub ahead of print).

Lee KS, Huang X, and Fitzpatrick D. "Topology of ON and OFF inputs in visual cortex enables an invariant columnar architecture." Nature. 2016 May 5; 533 (7601): 90-4.

Monai H, Ohkura M, Tanaka M, Oe Y, Konno A, Hirai H, Mikoshiba K, Itohara S, Nakai J, Iwai Y, and Hirase H. "Calcium imaginq reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain." Nat Comm. 2016 Mar 22; 7 (11100): 1-10.

Ganmor E, Krumin M, Rossi LF, Carandini M, and Simoncelli EP. "Direct Estimation of Firing Rates from Calcium Imaging Data." arXiv. 2016 Jan 4; 1601.00364 (q-bio.NC): 1-34.

2015

Roth MM, Dahmen JC, Muir DR, Imhof F, Martini FJ, and Hofer SB. "Thalamic nuclei convey diverse contextual information to layer 1 of visual cortex." Nat Neurosci. 2015 Dec 21; 19 (2): 299-307.

Barnstedt O, Keating P, Weissenberger Y, King AJ, and Dahmen JC. "Functional Microarchitecture of the Mouse Dorsal Inferior Colliculus Revealed through In Vivo Two-Photon Calcium Imaging." J Neurosci. 2015 Aug 5; 35 (31): 10927-39.

Chen SX, Kim AN, Peters AJ, and Komiyama T. "Subtype-specific plasticity of inhibitory circuits in motor cortex during motor learning." Nat Neurosci. 2015 Jun 22; 18: 1109-15.

Jia Y, Zhang S, Miao L, Wang J, Jin Z, Gu B, Duan Z, Zhao Z, Ma S, Zhang W, and Li Z. "Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620." Oncol Rep. 2015 Jun; 33 (6): 2681-8.

Lu W, Tang Y, Zhang Z, Zhang X, Yao Y, Fu C, Wang X, and Ma G. "Inhibiting the mobilization of Ly6C^high monocytes after acute myocardial infarction enhances the efficiency of mesenchymal stromal cell transplantation and curbs myocardial remodeling." Am J Transl Res. 2015 Mar 15; 7 (3): 587-97.

Boyd AM, Kato HK, Komiyama T, and Isaacson JS. "Broadcasting of cortical activity to the olfactory bulb." Cell Rep. 2015 Feb 24; 10 (7): 1032-9.

Cossell L, Iacaruso MF, Muir DR, Houlton R, Sader EN, Ko H, Hofer SB, and Mrsic-Flogel TD. "Functional organization of excitatory synaptic strength in primary visual cortex." Nature. 2015 Feb 19; 518 (7539): 399-403.

2014

Partridge JG, Lewin AE, Yasko JR, and Vicini S. "Contrasting actions of group I metabotropic glutamate receptors in distinct mouse striatal neurones." J Physiol. 2014 Jul 1; 592 (Pt 13): 2721-33.

Peters AJ, Chen SX, Komiyama T. "Emergence of reproducible spatiotemporal activity during motor learning." Nature. 2014 Jun 12; 510 (7504): 263-7.

Ehmke T, Nitzsche TH, Knebl A, and Heisterkamp A. "Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light." Biomed Opt Express. 2014 Jun 12; 5 (7): 2231-46.

Liu J, Wu N, Ma L, Liu M, Liu G, Zhang Y, and Lin X. "Oleanolic acid suppresses aerobic glycolysis in cancer cells by switching pyruvate kinase type M isoforms." PLoS One. 2014 Mar 13; 9 (3): e91606.

Palmer LM, Shai AS, Reeve JE, Anderson HL, Paulsen O, and Larkum ME. "NMDA spikes enhance action potential generation during sensory input." Nat Neurosci. 2014 Feb 2; 17 (3): 383-90.

Cai F, Yu J, Qian J, Wang Y, Chen Z, Huang J, Ye Z, and He, S. "Use of tunable second-harmonic signal from KNbO₃ nanoneedles to find optimal wavelength for deep-tissue imaging." Laser & Photon Rev. 2014; 8: 865-874.

2013

Kato HK, Gillet SN, Peters AJ, Isaacson JS, and Komiyama T. "Parvalbumin-expressing interneurons linearly control olfactory bulb output." Neuron. 2013 Dec 4; 80 (5): 1218-31.

Takata N, Nagai T, Ozawa K, Oe Y, Mikoshiba K, and Hirase H. "Cerebral blood flow modulation by Basal forebrain or whisker stimulation can occur independently of large cytosolic Ca²⁺ signaling in astrocytes." PLoS One. 2013 Jun 13; 8 (6): e66525.

Brochures

The buttons below link to PDFs of printable materials for Bergamo^® II microscopes.