Tunable Femtosecond Ti:Sapphire Laser for Two-Photon Microscopy

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Structural Insight with
Two-Photon Imaging

Multiphoton microscopy takes advantage of the NIR transparency windows in living tissue and highly localized excitation to generate multi-channel fluorescence images of 3D volumes. Compared to visible light, which is used in conventional widefield microscopy and confocal microscopy, NIR light offers significantly reduced scatter and absorption by biological compounds, resulting in deeper images below the surface.

The image of a fruit fly eye to the right demonstrates the Tiberius' ability to resolve morphological features. This two-channel image contains GFP-labeled photoreceptors and unlabeled regions that exhibit multiphoton autofluorescence. The excitation wavelength was 770 nm and a 25X, NA 1.05 Olympus objective was used.

Peter Fendel
Director, Laser Division

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Femtosecond Ti:Sapphire Laser for Multiphoton Imaging

• Wide Tuning Range of 720 nm to 1020 nm
• Industry-Leading Tuning Speed: Up to 200 nm in <50 ms
• High Output Power: >2.0 W at 800 nm
• Ultrafast <120 fs Pulses Help Minimize Pulse Broadening
• Compact Footprint Uses Half the Table Space of Competing Lasers
• High Long-Term Reliability for Exceptionally Low Cost of Ownership
• Integrated Spectrometer for Real-Time Diagnostics
• Pure Air Circulator Unit Purges Laser Cavity for Smooth Tuning Through Water Absorption Lines

Typical Applications

• Multi-Channel Fluorescence Images of 3D Volumes
• Photostimulation and Uncaging
• Label-Free Imaging via Multiphoton Autofluorescence and SHG

The Tiberius^® Femtosecond Tunable Ti:Sapphire Laser was designed in close collaboration with Thorlabs' life science application specialists. Manufactured in-house, it leverages the company's extensive expertise in optical design and precision manufacturing. Please refer to the Design and Manufacturing tab for more detail.

This multiphoton imaging laser offers an average power of more than 2.0 W at 800 nm and a center wavelength that is tunable from 720 nm to 1020 nm. This 300 nm wide tuning range allows the user to target specific compounds for multiphoton fluorescence imaging and photostimulation / uncaging. A tuning curve is shown in the Specs tab, and a demonstration of the speed of the Tiberius' wavelength tuning and the improved image contrast it provides is given in the Fast Tuning tab.

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Included GUI for Control of the Tiberius

The Tiberius laser emits pulses that are 120 fs in duration. The relatively narrow spectral bandwidth of these pulses was selected in order to reduce the pulse broadening caused by Pockels cells and other dispersive elements while still providing high peak intensity for two-photon excitation.

Since tabletop space is often at a premium, the Tiberius laser has been designed with a vertical cavity construction that minimizes the footprint on the optical table. At 746 mm x 175 mm (29.4" x 6.9"), the Tiberius' footprint is about half that of competing designs, preserving valuable workspace for the rest of your experimental setup. Each laser also comes with a laser controller, pump laser controller, chiller, and pure air circulator unit.

For laser operation, the Tiberius includes an intuitive GUI. Shown above, the GUI reports the center wavelength and output power of the laser, using the built-in spectrometer to provide real-time diagnostics of the spectral position and shape. User-programmable buttons provide single-click access to commonly used excitation wavelengths, and an SDK is included that features closed-loop tuning and supports C, LabVIEW™, and MATLAB^®. In addition, the Tiberius is wholly integrated with ThorImage^®LS, enabling seamless and synchronized control for photoactivation experiments and live high-speed imaging.

In-House Expertise in Design and Manufacturing

The Tiberius^® is designed and manufactured entirely in-house, leveraging our multi-disciplinary team of design engineers and the substantial infrastructure of a vertically integrated company. Thorlabs' Laser Division tightly controls every aspect of the manufacturing, assembly, and testing process of the Tiberius in order to guarantee the laser's stability and reliability.

The laser's design represents the culmination of complex theoretical cavity simulations combined with "old-fashioned" prototyping. A sound understanding of the intracavity laser dynamics proved fundamental to optimizing the laser for the specific needs of our nonlinear imaging customers.

Precision Optomechanics Manufacturing
The Tiberius benefits from Thorlabs' 25+ years of experience in manufacturing precision photonics components and assemblies. For example, it makes extensive use of the high-performance, ultrastable Polaris^® designs that the company has developed for custom OEM needs and industrial-grade applications. These expert designs minimize thermally induced drift and help ensure stable long-term alignment.

2D Numerical Model of Tiberius Laser Cavity

Our high degree of vertical integration lowers costs for our customers and ensures that every aspect of the laser performs as intended, delivering superior value and return on investment.

Optimized Ultrafast Laser Optics
To maximize the Tiberius' optical performance, it was critical to optimize the laser cavity geometry and optics together as a single unit. The optical coatings were therefore designed by Thorlabs and are precisely tuned for our cavity's proprietary design, enabling the long-term stability and broad tuning range that multiphoton microscopy requires.

To manufacture these high-performance coatings, we selected ion beam sputtering (IBS), which provides the most precise layer control and the most dense coatings among all coating methods. These characteristics result in coatings with high damage thresholds, minimal dependence on environmental factors, and excellent consistency from run to run. Thorlabs operates a number of IBS machines to produce these critical components for the Tiberius.

Improved Image Contrast with Fast Tuning

With an industry-leading tuning speed of up to 200 nm in <50 ms, the Tiberius^® is ideal for fast sequential imaging. The Tiberius' fast-tuning capability provides high-contrast images when used in multi-color, multiphoton microscopy applications.

Quickly switching between two optimized excitation wavelengths has several benefits over single-wavelength excitation. These include the much higher image contrast provided by fast switching and being able to maximize fluorescence at lower excitation powers, which reduces the risk of photobleaching.

Figures 1 and 2 illustrate the increased contrast enabled by imaging multiple fluorophores in a sample using fast sequential imaging.^† The sample is a 25 µm thick sagittal section of an adult rat brain. 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 from a two-color excitation imaging sequence at 7 fps by fast tuning between 750 nm and 835 nm, which excites both tags optimally.

The video in Figure 3 shows the fast switching between the red and green fluorescence in both real time and at 1/16^th the imaging rate, which makes it easier to see the details of each. The two-channel set was collected at an imaging rate of 7 fps with a resolution of 512 x 512 pixels. The Tiberius' fast tuning functionality integrates seamlessly into ThorImage^®LS software, enabling synchronized control for photoactivation experiments and live high-speed imaging on millisecond timescales using the same laser.

^†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. 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.^†

Laser Safety and Classification

Safe practices and proper usage of safety equipment should be taken into consideration when operating lasers. The eye is susceptible to injury, even from very low levels of laser light. Thorlabs offers a range of laser safety accessories that can be used to reduce the risk of accidents or injuries. Laser emission in the visible and near infrared spectral ranges has the greatest potential for retinal injury, as the cornea and lens are transparent to those wavelengths, and the lens can focus the laser energy onto the retina. 

Safe Practices and Light Safety Accessories

• Thorlabs recommends the use of safety eyewear whenever working with laser beams with non-negligible powers (i.e., > Class 1) since metallic tools such as screwdrivers can accidentally redirect a beam.
• Laser goggles designed for specific wavelengths should be clearly available near laser setups to protect the wearer from unintentional laser reflections.
• Goggles are marked with the wavelength range over which protection is afforded and the minimum optical density within that range.
• Blackout Materials can prevent direct or reflected light from leaving the experimental setup area.
• Thorlabs' Enclosure Systems can be used to contain optical setups to isolate or minimize laser hazards.
• A fiber-pigtailed laser should always be turned off before connecting it to or disconnecting it from another fiber, especially when the laser is at power levels above 10 mW.
• All beams should be terminated at the edge of the table, and laboratory doors should be closed whenever a laser is in use.
• Do not place laser beams at eye level.
• Carry out experiments on an optical table such that all laser beams travel horizontally.
• Remove unnecessary reflective items such as reflective jewelry (e.g., rings, watches, etc.) while working near the beam path.
• Be aware that lenses and other optical devices may reflect a portion of the incident beam from the front or rear surface.
• Operate a laser at the minimum power necessary for any operation.
• If possible, reduce the output power of a laser during alignment procedures.
• Use beam shutters and filters to reduce the beam power.
• Post appropriate warning signs or labels near laser setups or rooms.
• Use a laser sign with a lightbox if operating Class 3R or 4 lasers (i.e., lasers requiring the use of a safety interlock).
• Do not use Laser Viewing Cards in place of a proper Beam Trap.

Laser Classification

Lasers are categorized into different classes according to their ability to cause eye and other damage. The International Electrotechnical Commission (IEC) is a global organization that prepares and publishes international standards for all electrical, electronic, and related technologies. The IEC document 60825-1 outlines the safety of laser products. A description of each class of laser is given below:

Class	Description	Warning Label
1	This class of laser is safe under all conditions of normal use, including use with optical instruments for intrabeam viewing. Lasers in this class do not emit radiation at levels that may cause injury during normal operation, and therefore the maximum permissible exposure (MPE) cannot be exceeded. Class 1 lasers can also include enclosed, high-power lasers where exposure to the radiation is not possible without opening or shutting down the laser.
1M	Class 1M lasers are safe except when used in conjunction with optical components such as telescopes and microscopes. Lasers belonging to this class emit large-diameter or divergent beams, and the MPE cannot normally be exceeded unless focusing or imaging optics are used to narrow the beam. However, if the beam is refocused, the hazard may be increased and the class may be changed accordingly.
2	Class 2 lasers, which are limited to 1 mW of visible continuous-wave radiation, are safe because the blink reflex will limit the exposure in the eye to 0.25 seconds. This category only applies to visible radiation (400 - 700 nm).
2M	Because of the blink reflex, this class of laser is classified as safe as long as the beam is not viewed through optical instruments. This laser class also applies to larger-diameter or diverging laser beams.
3R	Lasers in this class are considered safe as long as they are handled with restricted beam viewing. The MPE can be exceeded with this class of laser, however, this presents a low risk level to injury. Visible, continuous-wave lasers are limited to 5 mW of output power in this class.
3B	Class 3B lasers are hazardous to the eye if exposed directly. However, diffuse reflections are not harmful. Safe handling of devices in this class includes wearing protective eyewear where direct viewing of the laser beam may occur. In addition, laser safety signs lightboxes should be used with lasers that require a safety interlock so that the laser cannot be used without the safety light turning on. Class-3B lasers must be equipped with a key switch and a safety interlock.
4	This class of laser may cause damage to the skin, and also to the eye, even from the viewing of diffuse reflections. These hazards may also apply to indirect or non-specular reflections of the beam, even from apparently matte surfaces. Great care must be taken when handling these lasers. They also represent a fire risk, because they may ignite combustible material. Class 4 lasers must be equipped with a key switch and a safety interlock.
All class 2 lasers (and higher) must display, in addition to the corresponding sign above, this triangular warning sign