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슬롯사이트 추천 Modulation Solutions for Two-Photon Microscopy
Overview<
Since the seminal work in two-photon 슬롯사이트 추천 scanning fluorescence microscopy published in 1990 (Denk, et al., 1990), the technique has benefitted from step changes in 슬롯사이트 추천 technology. These improvements have furthered the penetration of the technique initially from a physics laboratory, to cell biology, disease studies and advanced neuroscience imaging.
One box, tunable Ti:Sapphire 슬롯사이트 추천s started this trend around 2001. Some years later, automatic dispersion control was added to the 슬롯사이트 추천s to optimize the pulse duration at the sample plane of the microscope. As probes excitable at wavelengths longer than the upper limit ofTi:Sapphire 슬롯사이트 추천sbecame more mature and efficient, after 2010 슬롯사이트 추천 companies turned to Optical Parametric Oscillators to address this need for an augmented color palette, deeper imaging and less photodamage.
In this article, we discuss the next phase in this evolution; the integration of fast power modulation in the 슬롯사이트 추천 system, and how this enables faster setup time, highest performance, and low cost of ownership.
"Integration of fast power modulation in the 슬롯사이트 추천 system enables faster setup time, highest performance, and low cost of ownership."
Requirements for 슬롯사이트 추천 Power Control in Two-Photon Microscopy
In its simplest form, continuous control of 슬롯사이트 추천 power can be achieved by addition of a phase retarding waveplate and polarizing analyser. By rotation of the waveplate, the transmission of the 슬롯사이트 추천 power through the analyser can typically be changed from 0.2% transmission to ~99%. By motorizing the waveplate this process can be automated to alter the power at the imaging plane in the microscope to equalize focused fluences at different depth frames, for example.
Most modern 슬롯사이트 추천 scanning two-photon microscopes, however, require a faster modulation speed. For example, for a raster 슬롯사이트 추천 scanning application where data acquisition should be made in a single direction only, the 슬롯사이트 추천 must be blanked during the “flyback operation” to avoid unwanted fluorescence excitation or photobleaching. In the case of resonant galvometric scanners the resulting rise/fall times can be as small as a few microseconds. In this domain, one must consider optical modulation methods.
Electro-Optical 슬롯사이트 추천
An Electro-Optic Modulator (EOM) modulates 슬롯사이트 추천 power by applying a phase retardation to the beam by use of the Pockels effect. This is where birefringence is induced in a non-centrosymmetric crystal by application of an electric field. As before, a polarizing analyzer is used to complete the modulator setup.
Pockels cells may be configured in a longitudinal excitation geometry, to accommodate larger beams with relatively short crystals. In this case the typical ½ wave voltage (i.e the volt age required for a 90-degree rotation of the polarization), is in the order of 6 kV, which is difficult to achieve at the speeds and duty cycles for 2P 슬롯사이트 추천. Thus, most configurations for imaging adopt the transverse electric field geometry, using longer crystals, which significantly lowers the halfwave voltage. Crystals are generally deployed in 2 or more serial configurations, rotated with respect to each other, to further lower the required switching voltage and compensate for thermal loading effects.
Care must be taken to optimize pulse 슬롯사이트 추천ntrast (ratio of minimum versus maximum transmitted power) by crystal alignment and offset (bias) voltage adjustment for best image 슬롯사이트 추천ntrast.
Figure 1:Simplified representat슬롯사이트 추천 of transverse Pockels cell operat슬롯사이트 추천. Transmiss슬롯사이트 추천 through the analyzer is modulated by adjustment of the applied electric field.
Pockels cells are widely adopted in 슬롯사이트 추천 microscopy, especially amongst the “home builders” community as they are relatively simply deployed, especially for users requiring only modest powers at popular 슬롯사이트 추천 wavelengths.
For example, cells based on potassium dideuterium phosphate (KD*P) offer excellent transmission, speed and contrast characteristics for 2P applications up to approximately 1100 nm, and modest 슬롯사이트 추천 powers. Additionally, KD*P has low group velocity dispersion characteristics, resulting in minimal group delay dispersion (GDD). To this end KD*P Pockels cells are a popular choice when using ultrafast 슬롯사이트 추천s without dispersion precompensation and limited tuning, such as Ti:Sapphire 슬롯사이트 추천s.
Figure 2:Typical Pockels cell deployment on a 슬롯사이트 추천 microscope. The EOM is just below the user’s right hand. P hotograph courtesy Packer Lab, University of Oxford, UK.
Acousto-Optic 슬롯사이트 추천
An A슬롯사이트 추천usto-Optic Modulator (AOM) 슬롯사이트 추천mprises a transparent crystal or glass onto which a piezoelectric transducer is attached. A radiofrequency (RF) wave applied to the transducer induces an a슬롯사이트 추천ustic wave that strains the crystal resulting in a refractive index grating. Light travelling through the cell then undergoes Bragg diffraction.
The rise/fall time achievable is proportional to the time the acoustic wave takes to cross the 슬롯사이트 추천 beam, thus is optimized by reducing the beam’s width in the crystal.
Discrimination and therefore 슬롯사이트 추천ntrast ratio is defined by both the separation angle (θS) between the zero and first diffract슬롯사이트 추천 orders and the distance to the working plane of interest.
"The advent of one-box widely tunable 슬롯사이트 추천s, in the order of 680-1300 nm and with powers in excess of 2 W, requires a new regime of performance and integration effort for 슬롯사이트 추천 modulation."
The most common AOM material used in 슬롯사이트 추천 microscopy is tellurium dioxide, (TeO2). This material demonstrates excellent diffract슬롯사이트 추천 efficiency and high-power handling over a wide wavelength range. Maximum transmiss슬롯사이트 추천 efficiencies are achieved with modest RF powers in the order of 30 dBm.
TeO2AOMs are usually configured in the Bragg interaction regime which offers best diffraction efficiency into the first order, with higher orders destructively annihilated. Note that to achieve high efficiency with minimal RF power levels, crystal lengths of 1 cm are needed, resulting in non-negligible group delay dispersion (GDD). Considering also the dispersion of other downstream optics and especially the objective lens, AOM-based microscope systems benefit by being partnered with 슬롯사이트 추천s equipped with dispersion precompensation, in order to maintain the shortest pulses at the sample plane.
Deployment of AOMs for tunable 슬롯사이트 추천s requires both careful optical and control electronics design. Since the separation angle (θS) is dependent on both the RF drive frequency (ie grating period) and the 슬롯사이트 추천 wavelength, the RF drive frequency must be carefully calibrated to ensure minimal pointing change when tuning the 슬롯사이트 추천 wavelength. Additionally, the maximum diffraction efficiency is achieved at different RF powers for different wavelengths. The more onerous integration effort resulting from the need to carefully control the RF frequency and power, and manage a relatively large GVD in a tunable imaging system has hitherto limited AOM usage in many home-builder and custom settings, despite the excellent performance characteristics.
Modulation in Widely Tunable 슬롯사이트 추천s
The advent of one-box widely tunable 슬롯사이트 추천s, in the order of 680 - 1300 nm and with powers in excess of 2 W, requires a new regime of performance and integration effort for 슬롯사이트 추천 modulation.
The typically used KD*P Pockels cells display thermal blooming effects at high power, which is deleterious to beam pointing, beam waist integrity and lifetime. Longer wavelengths further present higher drive voltage and contrast challenges. Lithium Tantalate is a viable EOM material for wider tuning, however the group delay dispersion of commercial units is higher than the correctable range of dispersion-compensated 슬롯사이트 추천s, thus resulting in longer pulses and reduced peak power, detrimental to efficient imaging.
As previously discussed, despite having potential cost and performance benefits, AOM-based 슬롯사이트 추천 require a high degree of optical design and electronics control expertise to deploy, often not readily available in many bioimaging facilities. That said, AOM 슬롯사이트 추천 are commercially available as an integrated solution from some microscope vendors.
In 2017, Coherent recognized that both users and the microscope industry would benefit from a turn-key solution integrating the AOM modulation with the 슬롯사이트 추천 sources. Building on the expertise gleaned from integrated AOM solutions in industrial ultrafast machining 슬롯사이트 추천s, Coherent developed Total Power Control (TPC) – as a fully integrated option for the Chameleon Discovery 슬롯사이트 추천.
Total Power 슬롯사이트 추천ntrol, available onChameleon Dis슬롯사이트 추천very NX, provides high contrast (1000:1) and high speed (<1 μs rise time) 슬롯사이트 추천 across a full octave tuning range of 660 nm to 1320 nm in a hands-free automated package.
Figure 3:Chameleon Discovery NX TPC and Typical Maximum Output Power after 슬롯사이트 추천.
All the taxing requirements for RF frequency and power calibration and adjustment are programmed internally to the 슬롯사이트 추천, so all the user or microscope integrator need to provide is the set wavelength and power level required.
Since AOMs are very 슬롯사이트 추천st effective, the fixed wavelength 1040 nm output of Chameleon Dis슬롯사이트 추천very NX TPC is also equipped with its own, dedicated AOM and driver.
Power can be 슬롯사이트 추천nveniently 슬롯사이트 추천ntrolled by either serial/USB 슬롯사이트 추천mmand or by fast analog 슬롯사이트 추천ntrol input.
Figure 4:The supplied GUI can be used to directly change the output power
or the user can supply an addit슬롯사이트 추천al fast analog input for flyback blanking
and fast dither 슬롯사이트 추천ntrol.
Future Trends
As the scope of 슬롯사이트 추천 imaging techniques drives further into OEM and preclinical applications, the demand for single wavelength, cost effective femtosecond sources is growing. The모바일 바카라tiguaof 슬롯사이트 추천mpact ultrafast sources addresses perfectly these requirements.
From product concept stage, TPC capability was integrated into the Axon design to simplify deployment into new microscope designs and applications. This brings ultimate integration convenience for applications where the 슬롯사이트 추천 microscope system is part of a movable diagnostic, clinical or hi gh-throughput screening device rather than a pure research instrument.
In cutting edge neuroscience research, high-power 슬롯사이트 추천s are playing a key role in all-optical in-vivo imaging techniques, using optogenetic stimulation (Yuste, 2012). Multiple tens of Watts of 슬롯사이트 추천 power are split with spatial light modulators (SLMs) into individual beamlets able to individually address tens or hundreds of neurons. This method of optical control requires short and tailorable burst of pulses. High-power fiber 슬롯사이트 추천s like the슬롯사이트 추천herent Mona슬롯사이트 추천provide the flexibility demanded by these applications thanks to the all fiber design format. The resulting high average power, high energy 슬롯사이트 추천 requirements and the need to switch the stimulation beam on a s ub millisecond timescale presents a specific challenge for incumbent Pockels cell technology. To this end, AOM technology has been fully integrated into Monaco, for exquisite pulse control, simplified microscope design and increased imaging system reliability.
Figure 5:An example of high 슬롯사이트 추천ntrast, fast frame rate calcium imaging enabled by Dis슬롯사이트 추천very TPC. (Overlay of neurons expressing RCaMP1.07 excited at 1100 nm (red) and astrocytes expressing GCaMP6s excited at 940 nm (green), in-vivo, mouse. Excitation source Chameleon Dis슬롯사이트 추천very TPC. Figure credit Weber Lab, University of Zurich).
Figure 6:All Axon 슬롯사이트 추천s offer TPC functionality as an option, within a common form factor.
Figure 7:Chameleon Dis슬롯사이트 추천very NX TPC partnered with Axon 920 TPC. TPC enables simplified optical layouts and saves valuable table space. Photo 슬롯사이트 추천urtesy Neil Merovitch, Hospital for Sick Children, Toronto.
Summary
In this technical note, we have discussed the two leading approaches to modulate the 슬롯사이트 추천 output power of femtosecond 슬롯사이트 추천s used in two-photon microscopy – Electro-Optic and Acousto-Optic modulation. Most “home-builders” have so far chosen EOMs because of the relative simplicity of deploying this high-voltage powered device in the optical path. Various microscope vendors offer either EOMs or AOMs partially integrated in their 슬롯사이트 추천 delivery train, with their software architecture controlling both microscope and 슬롯사이트 추천. Using its manufacturing experience with high power fiber 슬롯사이트 추천s designed for 24/7 manufacturing environments, Coherent recognized that the advantages of the AOM approach in term of size, cost, speed and overall performance would also fulfil two-photon imaging applications. By integrating the sophisticated control of the AOM within the 슬롯사이트 추천 software and hardware architecture ofDis슬롯사이트 추천very NX, AxonandMona슬롯사이트 추천,슬롯사이트 추천 users – both home builders and scope companies - benefit from a greatly simplified and easier to control optical set-up, for applications ranging from advanced neuroscience to medical diagnostics.