OPTIMIZED FOR 3P IMAGING
- High average and peak power for 3P imaging up to 4.3 MHz repetition rate
- Fast image acquisition rate for viewing dynamic processes in-vivo
- High flexibility based on Spirit 1030-70/100 platform
- Computer controlled and user-friendly operation
- Dual pulse length option available for applications in optogenetics
The new Spirit-NOPA®-VISIR, pumped with a Spirit® 1030-70/100 industrial femtosecond laser, is Spectra-Physics’ leading platform for three-photon (3P) imaging, enabling breakthrough applications in neuroscience. 3P excitation fluorescence microscopy allows imaging at unprecedented resolution and tissue depth. Utilizing the unique capability of this Spectra-Physics’ imaging source for 3P, very high acquisition rates are possible. While 3P requires higher peak powers compared to two-photon (2P) excitation, it yields better optical confinement and signal-to-background ratio in deep tissue imaging, leading to improved image resolution and contrast. Spectra-Physics’ 3P imaging platform enables fast image acquisition due to the very high repetition rates which are available from the source. This enables viewing of dynamic processes, such as calcium transients of GCaMP6s-labeled neurons in-vivo to record spontaneous activity on populations of neurons within the intact mouse brain . Additionally, imaging through the intact skull within the transmission windows centered at approximately 1300 nm and 1700 nm is made possible . 3P excitation of GFP- and RFP-like markers has been shown to be most successful at 1300 and 1700 nm, respectively.
For standard imaging techniques, 1300 and 1700 nm wavelengths have to date been uncommon, and microscope transmission is typically not optimized at these wavelengths. Spectra-Physics’ Spirit-NOPA-VISIR pumped with up to 70 W provides sufficient peak power, even at high repetition rates, to compensate for losses in the microscope optics and other scattering effects in the sample, thereby enabling researchers to fully reap the benefits of 3P imaging. It was recently shown that small features such as dendritic spines could be imaged at a depth of 120 µm with outstanding levels of resolution through a thinned mouse skull using 3P imaging, showing an improved signal-to-background ratio in direct comparison to a conventional 2P setup (Fig. 1). Deep tissue, high resolution imaging down to the hippocampus in a YFP-H tg mouse was also demonstrated at a depth exceeding 1 mm, and compared to a conventional 2P setup, deeper imaging was achieved (Fig. 2). Using the third harmonic generation signal (THG), imaging of small blood vessels at a depth of 600 µm inside a mouse brain (layer V) was demonstrated with high contrast and image resolution (Fig. 3).
Figure 1:Dendritic spines 120 µm deep in cortex imaged through a thinned skull. 3P imaging was performed at 2 MHz using a Spirit 1030-70 and Spirit-NOPA-VISIR (right) in comparison to a conventional 2P setup (left) (Image courtesy M. Fuhrmann, DZNE, Bonn).
Spectra-Physics’ Spirit-NOPA-VISIR in combination with a Spirit 1030-70 or Spirit 1030-100 enables unprecedented performance for 3P imaging. At a repetition rate of 2 MHz and a pump power of 70 W, a typical average power of >2.5 W at 1300 nm and a resulting peak power up to 15 MW is achieved for the compressed output (compressors are optionally available). This outstanding performance headroom compensates for typical losses in microscopes to maximize fluorescence intensity and imaging depth. The Spirit 1030-70 or Spirit 1030-100 laser can also be used to pump multiple OPAs at high power levels and varying repetition rate requirements. This power capability can be used for combined multiphoton imaging and photo-activation applications in multi-user facilities.
Figure 2:Cortex to Hippocampus in YFP-H tg mouse. 3P imaging was performed at 2 MHz using a Spirit 1030-70 and Spirit-NOPA-VISIR (right) in comparison to a conventional 2P setup (left) (Image courtesy M. Fuhrmann, DZNE, Bonn).
The Spirit-NOPA-VISIR is fully computer controlled to enable user-friendly operation. To cover 2P excitation imaging and applications in optogenetics as well, the Spirit-NOPA-VISIR can be configured with a dual pulse length option. With this option, narrow bandwidth, uncompressed laser pulses are available over an extended tuning range of the Spirit-NOPA-VISIR, closing the gap between signal and idler. The dual pulse length option is ideally suited for photostimulation of neurons in optogenetics and excitation of fluorescent dyes in the 900 nm to 1200 nm spectral range.
Figure 3:Third harmonic generation (THG) imaging of blood vessels. Imaging performed using a Spirit 1030-70 and Spirit-NOPA-VISIR. (Image courtesy M. Fuhrmann, DZNE, Bonn).
 D.G. Ouzounov, T. Wang, M. Wang, D.D. Feng, N.G. Horton, J.C. Cruz-Hernández, Y.-T. Cheng,
J. Reimer, A. S. Tolias, N. Nishimura, C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain” Nat. Methods 14, 388–390 (2017).
 T. Wang, D.G. Ouzounov, C. Wu, N.G. Horton, B. Zhang, C.-H. Wu, Y. Zhang, M.J. Schnitzer, C. Xu “Three-photon imaging of mouse brain structure and function through the intact skull” Nat. Methods 15, 789–792 (2018).
|Light sheet microscopy|
|Spirit-NOPA-VISIR Pump Parameters||1300 nm Compressed Output, typical||1700 nm Compressed Output, typical|
|70 W at 4.3 ±0.3 MHz||>2.5 W||>1 W|
|70 W at 3 MHz||>2.5 W||>1 W|
|70 W at 2 MHz||>2.5 W||>1.5 W|
|70 W at 1 MHz||>2.5 W||>1.5 W|
|Tuning Range||650–900 nm (signal)
1200–2500 nm (idler)
|Conversion Efficiency||>10% at peak, when pumped at >20 µJ
(uncompressed signal + idler combined)
|Pulse Width2||<350 fs uncompressed2
<70 fs at 650–900 nm with signal compressor;
<100 fs at 1200–2000 nm with idler compressor
|Compressor Transmission||Signal prism compressor: >65% at 650–900 nm
<70 fs at 650–900 nm with signal compressor;
Idler bulk compressor: >80% at 1200–2000 nm
|Optional SHG Output|
|Tuning Range||325–450 nm (SH of signal)
600–700 nm (SH of idler)
|Pulse Energy||>10% of signal/idler at peak|
|Optional Dual Pulse Length Configuration|
|Tuning Range in Long Pulse Mode||650–1025 nm (signal)
1040–2500 nm (idler)
|Pump Requirements3 from Spirit 1030-70 or Spirit 1030-100|
|Repetition Rate||Single shot to 4.3 ±0.3 MHz|
|Pulse Energy4||up to 100 µJ|
|Average Power||up to 70 W|
1. Due to our continuous product improvement program, specifications are subject to change without notice.
2. Compressors for signal and/or idler are options.
3. Spirit-NOPA-VISIR pump parameters are determined at the time of order and are set at the factory.
4. Spirit-NOPA-VISIR is optimized for one pump pulse energy only.
Typical Table Layout: Spirit 1030-70/100 and Spirit-NOPA-VISIR
Spirit 1030-70/100 pumping Spirit-NOPA-VISIR Dimensions (without Signal compressor, top view)
Spirit-NOPA-VISIR Module Dimensions (front view)
|Spirit-NOPA-VISIR Data Sheet|
|Spirit Component Selection Guide|
Automated collinear optical parametric amplifiers (OPAs)
Automated optical parametric amplifiers (OPAs)