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.
Bergamo IIシリーズ顕微鏡は、M34 x 1.0、M32 x 0.75、M25 x 0.75ならびにRMSネジ付きの無限遠補正対物レンズに対応します。これにより多光子顕微鏡に使用される多くの低倍率、高NA対物レンズを装置に取り付けることが可能になります。当社の走査用光学素子は視野数が20と大きく、これらの多光子イメージング用対物レンズの光学設計を利用することで、同じ対物レンズを使用した他社製顕微鏡と比べても集光性能が高くなっています。
1.2 mm In Vivo Deep Brain 3D Image Stack, Courtesy of Dr. Hajime Hirase, Katsuya Ozawa,RIKEN Brain Science Institute, Wako, Japan
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
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
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
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
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
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
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)
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.
20 mm Diagonal Square (Max) at the Intermediate Image Plane
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
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
Targeted: Simultaneous IR, Sequential Visible Full Field: During Scanner Flyback
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
Slow (250 kHz) (High-Speed and Wide-Wavelength-Range Options Available)
2 Non-Cooled GaAsP PMTs
4-Channel Multialkali PMTs with Variable-Size Pinhole Wheel
Collection Optics Module
Nikon 25X (with Piezo Objective Scanner)
Rigid Stand with Breadboard Insert
Six-Filter-Turret Epi-Illuminator Module Broadband Light Source Quantalux™ sCMOS Scientific Camera
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.
Resonant-Galvo-Galvo Scanner, Galvo-Resonant Scanners, Galvo-Galvo Scanners, or Spatial Light Modulator; Single or Dual Scan Paths
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
45 fps at 512 x 512 Pixels 600 fps at 512 x 32 Pixels
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]
1X to 16X (Continuously Variable)
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
Up to Four Ultrasensitive GaAsP PMTs, Cooled or Non-Cooled
Two Ultrasensitive GaAsP PMTs
Maximum of Four PMTs Controlled by the Software at a Given Time
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
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
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
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
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