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.
Options at a Glance
Innovation through Collaboration
Five-Axis Movement of Rotating Bergamo Microscopes
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.
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.
Click to Enlarge 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.
Click to Enlarge 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 with SLM (right) allows simultaneous excitation of multiple target cells, which is not possible with single-photon (left) or two-photon (middle) activation.
Features
Many of these features can also be added to existing Thorlabs microscope systems. For more information, please see the Retrofits tab.
Laser Scanning, Widefield Imaging, and Transmitted Light Imaging
Scan Paths
Resonant-Galvo-Galvo
8 kHz Scanner: Image at 2 fps (4096 x 4096 Pixels), 30 fps (512 x 512 Pixels), or 400 fps (512 x 32 Pixels)
12 kHz Scanner: Image at 45 fps (512 x 512 Pixels) or 600 fps (512 x 32 Pixels)
Design Registered Under US Patent 10,722,977
Galvo-Resonant
8 kHz Scanner: Image at 2 fps (4096 x 4096 Pixels), 30 fps (512 x 512 Pixels), or 400 fps (512 x 32 Pixels)
12 kHz Scanner: Image at 45 fps (512 x 512 Pixels) or 600 fps (512 x 32 Pixels)
Galvo-Galvo
User-Defined Scan Geometries: Squares, Rectangles, Circles, Ellipses, Lines, and Polylines
Capture Weak Signals with Long Dwell Time Integration
Consistent Dwell Times Across Field of View
48 fps at 512 x 32 Pixels and 70 fps at 32 x 32 Pixels
Spatial Light Modulator
Simultaneous Multi-Site Photostimulation using Holographic Beam Control
Entirely Controlled Through ThorImage®LS Software
Precise Z-Axis Control of Photoexcited Locations
Widefield Viewing
Compatible with Thorlabs or Third-Party C-Mount-Threaded Scientific Cameras
Locate Areas of Interest without Unnecessary Laser Excitation
Epi-Illumination
Single-Filter-Cube or Six-Filter-Turret Epi-Illuminator Modules
Visualize Samples with Fluorescence or Reflected Light
Variety of Available Sources for Brightfield Illumination
Rigid Stands for Slides, Recording Chambers, or Platforms
Minimal Footprint Conserves Space Around Objective and the Microscope
Slim Profile Leaves Room for Dodt or DIC Imaging Modules
Excellent Long-Term Stability
Easily Rotate Samples Into and Out of the Beam Path
XY Platforms for Micromanipulators
Large Working Space that Surrounds the Objective on Three Sides
Ideal for Setups Where the Sample and Apparatus Need to Move in Unison, Such as Patch Clamping
2" Travel in X and Y; 0.5 µm Encoder Resolution
Gibraltar Platform
Large, Stable Working Space for Sample and Supplementary Equipment
Honeycomb Breadboard for Vibration Stability
Open Design Allows for Unrestricted Instrument Operation
Thorlabs Support
Fully Designed and Manufactured In-House
Engineers Work Under One Roof to Lower Your Costs and Create Seamless Solutions
Expertise in Every System Component
Modular System Construction
As Your Experimental Needs Evolve, Upgrade Your Microscope Without Sacrificing Existing Capabilities
Professional Installation
Thorlabs Technician Visits Your Lab to Assemble, Test, and Demonstrate Use of Your Microscope
Quick Support
Thorlabs Technicians and Application Specialists Available for Videoconferencing
Communicate with Our Support Staff Faster than an Engineer Could Travel to Your Location
Thorlabs Will Ship You a Camera with a Microphone to Facilitate the Conversation
With Permission, Thorlabs Will Remote Desktop in to Address Software Issues
Thorlabs recognizes that each imaging application has unique requirements. If you have any feedback, questions, or need a quotation, please use our multiphoton microscopy contact form or call (703) 651-1700.
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.
Galvo-Resonant Scanners, Galvo-Galvo Scanners, and Spatial Light Modulators
Bergamo® II microscopes can be configured with one or two co-registered scan paths to propagate, condition, and direct an input laser beam. Each path can utilize a resonant-galvo-galvo scanner, galvo-resonant scanner, galvo-galvo scanner, and/or a spatial light modulator (SLM). These choices allow the user to optimize each experiment as needed for high frame rates, high sensitivity, and/or targeted exposure of the regions of interest.
Resonant-Galvo-Galvo Scanners for Random Access Scanning Thorlabs offers 8 kHz and 12 kHz resonant-galvo-galvo (RGG) scanners. The design of our RGG scanners is registered under US Patent 10,722,977. These scanners enable multiple areas within a single FOV to be imaged at high speed in quick succession. Our 8 kHz scanners utilize the entire field of view and offer a maximum frame rate of 400 fps, while our 12 kHz scanners provide an increased frame rate of 600 fps.
Galvo-Resonant Scanners for High-Speed Imaging Thorlabs offers 8 kHz and 12 kHz galvo-resonant scanners. Our 8 kHz scanners utilize the entire field of view and offer a maximum frame rate of 400 fps, while our 12 kHz scanners provide an increased frame rate of 600 fps.
Galvo-Galvo Scanners for User-Defined ROI Shapes Galvo-galvo scanners support user-drawn scan geometries (lines, polylines, squares, and rectangles) and also support custom photoactivation patterns (circles, ellipses, polygons, and points). They offer consistent pixel dwell times for better signal integration and image uniformity.
Spatial Light Modulator for Simultaneous Targeting Unlike scanners, which physically move from point to point, spatial light modulators (SLMs) use holography to diffract the beam and shape it in a user-defined pattern. This includes general beam shaping as well as the creation of multiple focal points at the FOV; the latter allows multiple sites in a sample to be photoexcited simultaneously.
Figure 3. Wavelength Switching Using Tiberius Ti:Sapphire Laser Shown at 1/16th Actual Speed
Click to Enlarge 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.
Click to Enlarge 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 1/16th 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.
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.
Click to Enlarge 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.
Click to Enlarge 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.
Periscopes
Most lasers used in multiphoton microscopy are delivered by a free-space beam. The Bergamo II's ability to translate the objective around the focal plane in up to four axes (X, Y, Z, and θ) also requires the beam path to translate along the same axes while maintaining alignment. Bergamo II systems overcome this engineering challenge using multi-jointed periscopes.
Click to Enlarge Emission filters and dichroic cubes are held behind magnetically sealed doors on the front of the PMT detection module.
Super Broadband Scan Optics
Bergamo II microscopes feature proprietary scan optics that are optimized and corrected for excitation wavelengths within the 450 - 1100 nm, 680 - 1600 nm, or 800 - 1800 nm wavelength range, ideal for photostimulation, two-photon imaging, and three-photon imaging, respectively. These broad ranges, extending from the visible well into the near infrared, were chosen to support the latest widely tunable Ti:Sapphire lasers and OPO systems, as well as dual-output lasers such as the Chameleon Discovery.
Our optics take full advantage of the optical designs used in the low-magnification, high-numerical-aperture objectives by filling the back aperture of the objective up to Ø20 mm. This creates an exceptional scan area that lets you find a region of interest more quickly or simply image more cells at once.
Large-Angle Signal Collection Optics
Deriving the most signal from limited photons is the fundamental goal of any detection system. By positioning the PMTs immediately after the objective (a "non-descanned" geometry), light that is scattered by the sample, which therefore appears to originate outside the objective's field of view, still strikes the PMTs and adds to the collected signal. This is a benefit unique to multiphoton microscopy. Collecting beyond the objective's design field of view greatly enhances overall detection efficiency when imaging deep in tissue.
In the epi direction, we offer signal collection anglesa of 8°, 10°, or 14°, while in the transmitted direction, we offer a signal collection anglea of 13°. Our collection modules can optionally be outfitted with mechanical shutters for photoactivation experiments.
Easy-to-Reach Emission Filters and Dichroic Holders
Bergamo II systems are fully compatible with industry-standard fluorescence filter sets that include Ø25 mm fluorescence filters and 25 mm x 36 mm dichroic mirrors. Unlike competing designs, Thorlabs' detector modules have magnetic holders that make it simple and quick to exchange filters for different measurements.
We also offer detection modules for large-area Ø32 mm fluorescence filters and 32 mm x 42 mm dichroics, which support greater collection angles for increased signal.
Detectors in Epi and Transmitted Directions
We employ high-sensitivity GaAsP PMTs in our multiphoton systems, which can offer high quantum efficiency, aiding in imaging weakly fluorescent or highly photosensitive samples. Our PMTs can either be thermoelectrically cooled for improved sensitivity toward weak signals or non-cooled for a smaller package size and greater numerical aperture. Multialkali PMTs are also available.
All Bergamo® II microscopes can be equipped with either two or four detection channels in the epi direction, and/or two detection channels in the forward direction. The user can configure the forward-direction channels to detect the same fluorescent tags as the epi-direction PMTs, raising the microscope's sensitivity toward thin, weakly fluorescent specimens.
A maximum of four channels can be controlled by the software at a given time.
Angles Quoted for an Objective with a Ø20 mm Entrance Pupil
This controller is specifically designed for rotating Bergamo II microscope bodies. It uses knobs to control up to five motorized axes. On rotating systems, a rocker switch changes between fine objective focusing and translation of the elevator base. Each axis can be disabled on an individual basis in order to maintain a location along the desired direction.
The integrated touchscreen lets two spatial locations be saved and retrieved locally. Up to eight spatial locations can be saved on the computer running ThorImage®LS. The touchscreen also reads out the position of every motor.
Bergamo II microscopes accept infinity-corrected objectives with M34 x 1.0, M32 x 0.75, M25 x 0.75, or RMS threads. Together, these options encompass the majority of low-magnification, high-NA objectives used in multiphoton microscopy. With a large field number of 20, our scan optics completely utilize the optical designs of these specialized objectives, offering enhanced light-gathering ability compared to competing microscopes using the same objectives.
Thorlabs' Rigid Stands are rotatable, lockable, low-profile platforms for mounting slides, recording chambers, our Z-axis piezo stages, and custom experimental apparatuses. Each fixture is supported by a solid Ø1.5" stainless steel post for passive vibrational damping, which is in turn held to the workstation by the red post holder.
A locking collar maintains the height of the platform, allowing it to easily rotate into and out of the optical path, and a quick-release mechanism holds the post in place once the desired position is achieved.
Click to Enlarge A Quantulux sCMOS Camera and 1.4 Megapixel Scientific Camera
Scientific Cameras
Our low-noise, scientific-grade CCD, sCMOS, and CMOS cameras were designed for full compatibility with Thorlabs’ multiphoton microscopy systems. Useful for widefield and fluorescence microscopy, they are capable of visualizing in vitro and in vivo samples using reflected light and fluorescence emission. They work in conjunction with the epi-fluorescence module to help locate fiducial markers, and they also enable imaging modalities that do not require laser exposure.
Thorlabs' cameras are driven by our internally developed ThorCam software package. CCD cameras are available in 1.4 MP, 4 MP, 8 MP, and fast-frame-rate versions, the sCMOS camera is available with a 2.1 MP sensor, and our CMOS cameras are available with a 1.3 MP, 2.3 MP, 5 MP, 8.9 MP, or 12.3 MP sensors. Generally speaking, cameras with lower resolution offer higher maximum frame rates. These cameras also feature a separate auxiliary port that permits the image acquisition to be driven by an external electrical trigger signal.
Bergamo® II microscopes are also directly compatible with any camera using industry-standard C-mount or CS-mount threads.
Click to Enlarge Trans-Illumination Module, Motorized Condenser Stage, and Rigid Stand Sample Holder Underneath the Objective
User-Installable Dodt Contrast and DIC Imaging Modules
The modular construction of the Bergamo® II makes it exceptionally easy for the user to convert the microscope between in vitro and in vivo applications. Our user-installable trans-illumination modules for Dodt contrast, laser-scanned Dodt contrast, and differential interference contrast (DIC) take less than 5 minutes to attach or remove from the microscope body. These modules are available for both rotating and upright bodies.
Each option is paired with our basic 3-axis controller, which optimizes the illumination conditions by translating our motorized condenser stage over a 1" range. This versatile design is compatible with air and high-NA oil immersion condensers designed by Nikon.
To complement these modules, we manufacture slim-profile rigid stand sample holders that are ideal for positioning slides between the transmitted light module and the objective.
Thorlabs recognizes that each imaging application has unique requirements. If you have any feedback, questions, or need a quotation, please use our multiphoton microscopy contact form or call (703) 651-1700.
Structural Neurobiology
Neurological Disorders
Neural Development and Plasticity
Neurogenetics
Functional and Molecular Imaging
Synapses and Circuits
Ion Channels, Transporters, and Neurotransmitter Reporters
1.2 mm In Vivo Deep Brain 3D Image Stack, Courtesy of Dr. Hajime Hirase and Katsuya Ozawa, RIKEN Brain Science Institute, Wako, Japan
Application Summary
Scientists investigate the structure of the brain to understand functions of neuronal proteins as well as the causes of neurological diseases. Due to the difficulty of imaging through brain tissue caused by light scattering, this study often requires a multi-modality configuration allowing for a range of experimental conditions using any combination of multiphoton, confocal, and epi-fluorescence imaging. The three example multiphoton microscope configurations in the table below are designed to accommodate the needs of Structural Biology. Each configuration features fast-Z power ramping to accomplish high-resolution imaging deep within a sample. Our Two- and Three-Photon Imaging configuration uses both galvo-resonant and galvo-galvo scanners and infrared wavelength scanning optics to image second- and third-harmonic generation (SHG and THG). Alternatively, our Dual-Path Multiphoton Microscope with Confocal Imaging is outfitted with a confocal path that accommodates up to 4 laser lines and a 4-channel PMT detection module. The addition of a six-cube epi-illuminator module and sCMOS Quanatlux camera allows this system to perform epi-fluorescence imaging. Our Simple XYZ Imaging configuration is well-balanced for both in vitro and fixed stage in vivo microscopy research. With a removable transmitted illumination module, this versatile system can support a wide variety of experimental techniques, imaging modalities, and sample subjects.
Stitched Confocal Fluorescence Image of Rat Retina Stained with DAPI, Alexa Fluor® 555 and Alexa Fluor® 633, Courtesy of Dr. Jennifer Kielczewski, National Eye Institute, National Institutes of Health, Bethesda, MD
Application Summary
Researching neurological disorders involves measuring neural function using two-photon calcium imaging. This research requires fast image acquisition and photostimulation. We recommend three of our multiphoton microscope configurations for this application (see the table below). Each configuration offers a large working space and rotating microscope body, making it ideal for in vivo animal studies. Our Multi-Target Photoactivation configuration features a spatial light modulator (SLM), which allows the activation of groups of neurons at varying depths within a single field of view. For increased penetration of samples with scattering tissues, the three-photon capability of our Two- and Three-Photon Imaging configuration is recommended. Our 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 fluorescence activation regions for correlating neural responses in multiple regions of the brain.
Two-Photon Image of Neurons Expressing Thy1-YFP in a Cleared Region of the Hippocampus, Courtesy of the 2017 Imaging Structure and Function in the Nervous System Course at Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Application Summary
Scientists interested in this application focus on tracing neuronal pathways and researching dendritic spine plasticity. Imaging systems used in this type of research are capable of two-photon calcium imaging and/or confocal fluorescence imaging. High-resolution and high-sensitivity are crucial features for these systems. We offer three multiphoton configurations that are ideal for this application (see the table below). Each configuration may be used in photon-limited environments as they feature sensitive detection modules, in addition to our 10° or 14° full field-of-view collection optics with ultrasensitive GaAsP PMTs. Our Dual-Path with Confocal Imaging configuration allows for a range of experimental conditions to be observed using any combination of multiphoton, confocal, and epi-fluorescence imaging, along with photoactivation. Alternatively, the Video and High-Speed Imaging and Simple Z-Axis Imaging configurations provide a small footprint and large throat depth for in vivo two-photon imaging.
Laser-Scanned Two-Photon SHG+Dodt Gradient Contrast of Zebrafish Embryo
Application Summary
Neurogenetic studies require a wide variety of experimental setups for in vivo research. Multiphoton imaging is ideal for studying live organisms, especially embryos, as it reduces the occurrence of photobleaching and phototoxicity that is common with other light microscopy techniques. There are three multiphoton configurations we suggest for Neurogenetic applications (see the table below). The small footprint and large throat depth of each system provide ample room for sample mounts and experimental apparatus, such as the large setup used for Drosophilia studies by Chen et al. (click here for supplementary videos). Additionally, these systems provide video-rate, sequential two-photon imaging to study fast dynamic biological and chemical processes in vivo without damaging the sample.
Imaging Visually Evoked Synaptic Calcium Transient In Vivo, Using Dendrite Labels with GCAMP6, 200 µm Deep in Layer 2/3 Visual Cortex, Courtesy of David Fitzpatrick, Max Plank Institute for Neurobiology, Jupiter, FL, USA
Application Summary
In vivo functional imaging of organisms and the individual neurons linked to specific organism behaviors requires high-speed and high-sensitivity imaging. We offer six configurations for this application (see the table below). These configurations are equipped with an 8 or 12 kHz galvo-resonant imaging scanner and our 10° or 14° wide-angle collection optics to enable fast, high-resolution imaging. Each microscope system features a large throat depth, 5” of vertical travel, and smooth movement along the Z-axis to create a large working space ideal for in vivo volume imaging deep into highly scattering samples of neural tissue. For an improved range of movement around a sample, we recommend a configuration with a rotating body.
Simultaneous Photostimulation of 100 Cells Co-Expressing GCaMP6f (Green) and C1V1 (Red), Courtesy of Lloyd Russell, Dr. Adam Packer, and Professor Michael Häusser, University College London, United Kingdom
Application Summary
By studying synapses and circuits, researchers are able to understand neuronal activity. Imaging synapses and circuits often requires simultaneous stimulation of populations of neurons. To achieve this, we offer three configurations of our multiphoton microscope capable of fast image acquisition of multiple regions within a single field of view (see table below). Our Multi-Target Photoactivation configuration features a spatial light modulator (SLM), which allows multiple sites in a sample to be photoexcited simultaneously. With the SLM, each beamlet can be shaped to improve the efficacy of photoactivation, a crucial feature for activating neural populations at varying depths within a single field of view (FOV). Our 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 photoactivation regions. Lastly, our In Vivo Two-Photon Imaging configuration uses a galvo-resonant scanner for high-speed imaging.
Cochlear Organotypic Culture Loaded with Fluo4-AM and DM-Nitrophen AM, Calcium Release Triggered Using Galvo-Galvo Uncaging Pathway in Outer Hair Cell Indicated by Red Box After ~13 Seconds, Courtesy of Federico Ceriani and Walter Marcotti, University of Sheffield
Application Summary
In this application, scientists are interested in neural connections and intercellular movement. With multiphoton imaging, they are able to trace the direction and speed of ions moving through channel membrane proteins or neurotransmitters moving from one neuron to another. This research requires a microscopy system that has high-resolution and high-speed imaging of multiple fields of view (FOVs). We recommend six of our multiphoton configurations for this application (see the table below). This area of research often requires both in vivo and in vitro imaging within the same study, so within each configuration, our transmitted light modules can be installed or removed by the user in just a few minutes, making it exceptionally easy to switch between the two imaging modalities. These configurations are capable of high-frame-rate imaging and targeted laser activation for photostimulation, making them ideal for correlating neural responses in multiple regions of the brain.
Top: Astrocytes are labelled with SR101 (red). Arrows point to astrocytes that had Ca2+ elevations during tDCS. The numbers correspond to the cells and neurogliopil regions plotted in the graphs below. Bottom: Fluorescent intensity (ΔF/F) traces of astrocytes (orange), neurons (green) and neurogliopil (brown). Figure Courtesy of Monai H. et al. (See Below)
Application Summary
The study of cell biology, muscles, and glia often involves imaging in vitro or in vivo samples tagged with multiple fluorophores. Through multiphoton imaging with these fluorescent markers, researchers are able to observe gene expression related to neural function in different areas of the brain. We recommend two of our configurations for this application, see the table below. With two to four channel detection modules and fast sequential imaging using our Tiberius® Tunable fs laser, both of these configurations offer the flexibility necessary for experiments in this field. In addition, each configuration has a small footprint and a large throat depth to provide ample room for numerous sample mounting options, including in vivo imaging of mammalian brains via transcranial windows.
Drug discovery research is rapidly expanding and often requires a wide variety of experimental setups and imaging techniques. Two-photon imaging is frequently used to measure the characteristics of drug applications, including depth of drug penetration and the area of its spread throughout the cortex. We offer two configurations that are suitable for this application, see the table below. These configurations are are well-balanced for both in vitro and fixed stage in vivo microscopy research. The modularity of the Bergamo systems' removable trans-illumination module provides versatility in regard to experimental techniques, imaging modalities, and sample subjects. The Gibraltar breadboard platform line is ideal for mounting samples and supplementary equipment within these 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.
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.
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.
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.
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
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
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
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
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.
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)
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.
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.
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.
Thorlabs recognizes that each imaging application has unique requirements. If you have any feedback, questions, or need a quotation, please use our multiphoton microscopy contact form or call (703) 651-1700.
Sam Tesfai Imaging Systems General Manager
Questions? Need a Quote?
Click to Enlarge 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.
Bergamo II Compatible Upgrades and Add-Ons
Upgrade
Benefit
On-Site Installation Required
Scanning
SLM Holographic Photoactivationa
Multi-Site Holographic Two-Photon Photoactivation
Yes
Switch Scanning Functionality to Galvo-Galvo or Galvo-Resonant
Galvo-Galvo
Enable Control of Pixel Dwell Time Add Arbitrary Scanning and Photostimulation Capabilities
Yes
Galvo-Resonant
High-Speed Imaging Capability
12 kHz Resonant Scanner
Increase Resonant Scanning Speed
Yes
Beam Conditioning
High-Speed Pockels Cellb
ROI Masking Using Galvo-Resonant Scanner
Yes
Wide-Wavelength-Range Pockels Cell
Increase Operating Range to 680 - 1300 nm
Yes
Detection
10° Collection Module
Increase Emitted Signal Collection Angle Add Capability for up to 4 Channels of PMT Detection
Yes
14° Collection Module
Increase Emitted Signal Collection Angle Increase Microscope Clearance for Electrophysiology Studies
Yes
Mechanical Shutter for 8°, 10°, or 14° Collection Module
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.
The full source code for ThorImage®LS is available for owners of a Bergamo®, Cerna®, Veneto™ or confocal microscope. Click here to receive your copy.
ThorImage®LS Software
ThorImageLS is an open-source image acquisition program that controls Thorlabs' microscopes, as well as supplementary external hardware. From prepared-slice multiphoton Z-stacks to simultaneous in vivo photoactivation and imaging, ThorImageLS provides an integrated, modular workspace tailored to the individual needs of the scientist. Its workflow-oriented interface supports single image, Z-stacks, time series, and image streaming acquisition, visualization, and analysis. See the video to the lower right for a real-time view of data acquisition and analysis with ThorImageLS.
ThorImageLS is included with a Thorlabs microscope purchase and open source, allowing full customization of software features and performance. ThorImageLS also includes Thorlabs’ customer support and regular software updates to continually meet the imaging demands of the scientific community.
Image Acquisition Synced with Hardware Inputs and Timing Events
Live Image Correction and ROI Analysis
Independent Galvo-Galvo and Galvo-Resonant Scan Areas and Geometries
Tiling for High-Resolution Large-Area Imaging
Independent Primary and Secondary Z-Axis Control for Fast Deep-Tissue Scans
Automated Image Capture with Scripts
Compatible with ImageJ Macros
Multi-User Settings Saved for Shared Workstations
Individual Colors for Detection Channels Enable Simple Visual Analysis
Seamless Integration with Experiments
Simultaneous Multi-Point Photoactivation and Imaging with Spatial Light Modulator
Fast Z Volume Acquisition with PFM450E or Third-Party Objective Scanners
Electrophysiology Signaling
Wavelength Switching with Tiberius® Laser or Coherent Chameleon Lasers
Pockels Cell ROI Masking
Power Ramped with Depth to Minimize Damage and Maximize Signal-to-Noise
New Functionality: Version 4.0 - October 15, 2019 (Click to Expand for More Details)
Please contact ImagingTechSupport@thorlabs.com to obtain the latest ThorImageLS version compatible with your microscope. Because ThorImageLS 4.0 adds significant new features over 3.x, 2.x and 1.x versions, it may not be compatible with older microscopes. We continue to support older software versions for customers with older hardware. See the full web presentation for functionality of previous versions.
New Hardware Support
Added Support for Windows® 10 OS
Added Support for CS895MU and CS505MU Monochrome Cameras (Requires ThorCAM 3.2)
Allows for Hot Pixel Correction
Added Support for CSN210 Motorized Dual-Objective Nosepiece
The buttons below link to PDFs of printable materials for Bergamo® II microscopes.
Laser Scanning
Scan Path Wavelength Range
450 - 1100 nm, 680 - 1600 nm, or 800 - 1800 nm
Scan Paths
Resonant-Galvo-Galvo Scanner, Galvo-Resonant Scanners, Galvo-Galvo Scanners, or Spatial Light Modulator; Single or Dual Scan Paths
Scan Speed
8 kHz Resonant-Galvo-Galvo or Galvo-Resonant
2 fps at 4096 x 4096 Pixels 30 fps at 512 x 512 Pixels 400 fps at 512 x 32 Pixels
12 kHz Resonant-Galvo-Galvo or Galvo-Resonant
4.4 fps at 2048 x 2048 Pixels 45 fps at 512 x 512 Pixels 600 fps at 512 x 32 Pixels
Galvo-Galvo
3 fps at 512 x 512 Pixels 48 fps at 512 x 32 Pixels 70 fps at 32 x 32 Pixels Pixel Dwell Time: 0.4 to 20 µs
Galvo-Galvo Scan Modes
Imaging: Line, Polyline, Square, or Rectangle Non-Imaging: Circle, Ellipse, Polygon, or Point
Field of View
20 mm Diagonal Square (Max) at the Intermediate Image Plane [12 mm Diagonal Square (Max) for 12 kHz Scanner]
Scan Zoom
1X to 16X (Continuously Variable)
Scan Resolution
Up to 2048 x 2048 Pixels (Bi-Directional) [Up to 1168 x 1168 Pixels for 12 kHz Scanners] Up to 4096 x 4096 Pixels (Unidirectional) [Up to 2336 x 2336 Pixels for 12 kHz Scanners]
Compatible Objective Threadings
M34 x 1.0, M32 x 0.75, M25 x 0.75, and RMS
Multiphoton Signal Detection
Epi-Detection
Up to Four Ultrasensitive GaAsP PMTs, Cooled or Non-Cooled
Forward-Direction Detection
Two Ultrasensitive GaAsP PMTs
Maximum of Four PMTs Controlled by the Software at a Given Time
Collection Optics
8°, 10°, or 14° Collection Angle (Angles Quoted When Using an Objective with a 20 mm Entrance Pupil) Easy-to-Exchange Emission Filters and Dichroic Mirrors
Confocal Imaging
Motorized Pinhole Wheel with 16 Round Pinholes from Ø25 µm to Ø2 mm Two to Four Laser Lines (488 nm Standard; Other Options Range from 405 nm to 660 nm) Standard Multialkali or High-Sensitivity GaAsP PMTs Easy-to-Exchange Emission Filters and Dichroic Mirrors
Widefield Viewing
Manual or Motorized Switching Between Scanning and Widefield Modes Illumination Provided via LED or Liquid Light Guide C-Mount Threads for Scientific Cameras
Transmitted Light Imaging
Differential Interference Contrast (DIC) or Dodt Gradient Contrast Widefield or Laser Scanned Illumination Provided by Visible and/or NIR LEDs Compatible with Air or Oil Immersion Condensers
Three-Photon Imaging
Scan Optics for 900 - 1900 nm Range Achieve Reduced Background Scatter for Greater Sensitivity in Deep Tissue Imaging
Volume Imaging Using Bessel Beams
3D Volumetric Functional Imaging at Video Frame Rates Enhanced Temporal Resolution for Studying Internal Systems at Cellular Lateral Resolution In Vivo
Translation
Microscope Body Rotation (Rotating Bodies Only)
-5° to +95°, -50° to +50°, or -45° to +45° Around Objective Focus 0.1° Encoder Resolution
Coarse Elevator Base Z (Rotating Bodies Only)
5" (127 mm) Total Travel; 1 µm Encoder Resolution
Fine Microscope Body X and Y
2" (50.8 mm) Total Travel; 0.5 µm Encoder Resolution
Fine Microscope Arm Z
1" (25.4 mm) Total Travel; 0.1 µm Encoder Resolution
Thorlabs recognizes that each imaging application has unique requirements. If you have any feedback, questions, or need a quotation, please use our multiphoton microscopy contact form or call (703) 651-1700.
Thorlabs' sales engineers and field service staff are based out of eight offices across four continents. We look forward to helping you determine the best imaging system to meet your specific experimental needs. Our customers are attempting to solve biology's most important problems; these endeavors require matching systems that drive industry standards for ease of use, reliability, and raw capability.
Thorlabs' worldwide network allows us to operate demo rooms in a number of locations where you can see our systems in action. We welcome the opportunity to work with you in person or virtually. A demo can be scheduled at any of our showrooms or virtually by contacting ImagingSales@thorlabs.com.
Customer Support Sites (Click Each Location for More Details)
gaiqing Wang
 (posted 2020-05-07 11:13:33.577)
I am looking for a cheap way to do confocal imaging in vivo. Is this Bergamo II Series Multiphoton Microscope my best option? Can you send me a quote?
YLohia
 (posted 2020-05-07 09:45:11.0)
Thank you for contacting Thorlabs. We will reach out to you directly to discuss your requirements.
jfpena
 (posted 2016-12-19 18:15:55.003)
I am looking for a cheap way to do confocal imaging in vivo. Is this Bergamo II Series Multiphoton Microscope my best option? Can you send me a quote?
tfrisch
 (posted 2016-12-22 11:44:31.0)
Hello, thank you for contacting Thorlabs. A member of our Imaging Team will reach out to you directly to discuss this system and your application.
birech
 (posted 2016-11-17 06:33:49.463)
I asked for a price quote for this product, Bergamo II Series Multiphoton Microscopes three days ago. I am working at the University of Nairobi in Kenya and would wish to order one.
Regards,
Birech
tfrisch
 (posted 2016-11-17 06:56:23.0)
Hello, thank you for contacting Thorlabs. I have forwarded this request to our Imaging Sales Team. I apologize for the delay.