NetNotes
Broadband Femtosecond Laser for Multiphoton Microscopy Confocal Listserver Anyone out there with an opinion on use of the Fyla broadband
femtosecond laser for multiphoton microscopy (https://www.fyla .com/product/sch/)? I am wondering if this is the way the market will go considering the cost of purchasing and maintaining Ti:Sapph tun- able lasers. All the best, Peter Owens
peter.owens@nuigalway.ie
Hi Peter, I’ve worked with ultrabroadband systems like this be-
fore (8fs Menlo), and while they are good for certain applications, they add a great deal of complexity to a system. Aſter extensive “playing” I concluded these systems are only worth it if performing advanced techniques such as coherent control. Potential issues: – Ultrabroadband pulses disperse greatly, and usually in a non- linear fashion, so pulse fragmentation can be an issue.
– Due to dispersion, a spatial light modulator (phase) system is required to keep the pulse near the temporal width it pos- sessed directly out of the laser.
– Te dispersion of the microscope on such a broadband pulse is very high, so oſten a 2-stage compression system is neces- sary to compensate for the group velocity dispersion/group delay dispersion (GVD/GDD).
– Broader-band lasers exist (I’ve worked with a system with nearly 300nm BW), but dispersion control becomes increas- ingly complex as spectral bandwidth increases.
– Te power is distributed over the entire spectrum of the pulse, so without optimal dispersion control there is almost no signal.
– If pulses are compressed down to their minimum at the sam- ple, the pulse is so short multiphoton phototoxic effects are a risk. I found my 8fs 85Mhz pulse laser bleached everything pretty rapidly.
Advantages: – If the laser provides enough spectral power density, multicol- or 2P with broadly separated dyes can be performed. A nor- mal 2P system can do this for some dyes, but ultrabroadband lasers let you take this further.
– Te two photons do not need to be the same wavelength; with good dispersion control, nearly the entire pulse can contribute to signal generation. A photon that is too red meets a photon that is too blue, and the pair add up to the energy you need to excite the fluorophore cross correlation (2P).
– If using a sufficiently sophisticated dispersion control scheme, a computer science student can be hired to use al- gorithmic approaches to manipulate the phase of the pulse components for coherent control of the fluorescence. Tis allows “hitting” the fluorophore in a way that is optimized in wavelength and phase to get the best signal, and to even toggle different fluorophores on or off to minimize bleaching and phototoxic effects, etc., by guiding the energy through the system via phase control.
– Very short pulses are good for other nonlinear effects, CARS, SHG, XFG, etc., and this can also be tweaked through coher- ent control of the pulse.
So, in short, investment in tight control of the dispersion
and machine learning can get amazing imaging and some neat tricks. Otherwise, the extra complexity is much, much more trouble than it is worth. As a general comment, my money is on lower- repetition-rate, higher pulse energy systems. Tese typically have repetition rates in the 5–10MHz range, so performing fast video rate imaging with them is not possible, but the high energy- per-pulse yet low *average* power allows very deep imaging, which is the best use-case for 3P/2P in the first place. For video rate, the Ti:Sapph still continues to give the most
bang-for-the-buck, although wavelength extension systems that push out to 1300nm (for 3P) are very useful as well. Some of these extended wavelength sources are also low-repetition and these are common for deep 3P imaging at 1200–1300nm. Craig Brideau
craig.brideau@
gmail.com
I haven’t used the SCH, so the below is pure speculation (as
usual). I’ve worked with Ti:Sapphs a lot, and we mostly use the 700–900 nm range (things like NADH, GFP, etc.). Of course, longer wavelengths are great for three-photon and orange fluorophores, but the Ti:Sapph dies out at 1000 nm. Power is important. While we oſten used 1% of the available power, in some cases of deep imaging tens of milliwatts out of the lens may be needed. 15 fs pulses are not an advantage. With this huge bandwidth, even slight dispersion will lead to longer pulses than the standard “narrow band” 100–200 fs. And the offered range of GVD compensation seems quite nar- row. Te spectrum is centered around 1064 nm. Remember, with the logarithmic plot in the datasheet, every 10 dB is a factor of 10 power difference! With the very broad spectrum it’s hard to pre- dict what will happen to the pulse length if part of the spectrum is removed with a bandpass filter. A lot of power will be lost for sure.
2022 July •
www.microscopy-today.com
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