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바카라 꿀팁 Modulation Solutions for Two-Photon Microscopy

Overview<

Since the seminal work in two-photon 바카라 꿀팁 scanning fluorescence microscopy published in 1990 (Denk, et al., 1990), the technique has benefitted from step changes in 바카라 꿀팁 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 바카라 꿀팁s started this trend around 2001. Some years later, automatic dispersion control was added to the 바카라 꿀팁s to optimize the pulse duration at the sample plane of the microscope. As probes excitable at wavelengths longer than the upper limit ofTi:Sapphire 바카라 꿀팁sbecame more mature and efficient, after 2010 바카라 꿀팁 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 바카라 꿀팁 system, and how this enables faster setup time, highest performance, and low cost of ownership.

바카라 꿀팁 Modulation
바카라 꿀팁 Microscopy

"Integration of fast power modulation in the 바카라 꿀팁 system enables faster setup time, highest performance, and low cost of ownership."

 

Requirements for 바카라 꿀팁 Power Control in Two-Photon Microscopy

In its simplest form, continuous control of 바카라 꿀팁 power can be achieved by addition of a phase retarding waveplate and polarizing analyser. By rotation of the waveplate, the transmission of the 바카라 꿀팁 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 바카라 꿀팁 scanning two-photon microscopes, however, require a faster modulation speed. For example, for a raster 바카라 꿀팁 scanning application where data acquisition should be made in a single direction only, the 바카라 꿀팁 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 바카라 꿀팁

An Electro-Optic Modulator (EOM) modulates 바카라 꿀팁 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 바카라 꿀팁nfigured in a longitudinal excitation geometry, to ac바카라 꿀팁mmodate 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 micros바카라 꿀팁py. Thus, most 바카라 꿀팁nfigurations 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 바카라 꿀팁nfigurations, rotated with respect to each other, to further lower the required switching voltage and 바카라 꿀팁mpensate for thermal loading effects.

Care must be taken to optimize pulse 바카라 꿀팁ntrast (ratio of minimum versus maximum transmitted power) by crystal alignment and offset (bias) voltage adjustment for best image 바카라 꿀팁ntrast.

Electro-Optical 바카라 꿀팁 (EOM)

Figure 1:Simplified representat바카라 꿀팁 of transverse Pockels cell operat바카라 꿀팁. Transmiss바카라 꿀팁 through the analyzer is modulated by adjustment of the applied electric field.

Pockels cells are widely adopted in 바카라 꿀팁 microscopy, especially amongst the “home builders” community as they are relatively simply deployed, especially for users requiring only modest powers at popular 바카라 꿀팁 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 바카라 꿀팁 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 바카라 꿀팁s without dispersion precompensation and limited tuning, such as Ti:Sapphire 바카라 꿀팁s.

바카라 꿀팁 Modulation Solutions

Figure 2:Typical Pockels cell deployment on a 바카라 꿀팁 microscope. The EOM is just below the user’s right hand. P hotograph courtesy Packer Lab, University of Oxford, UK.

 

Acousto-Optic 바카라 꿀팁

An A바카라 꿀팁usto-Optic Modulator (AOM) 바카라 꿀팁mprises a transparent crystal or glass onto which a piezoelectric transducer is attached. A radiofrequency (RF) wave applied to the transducer induces an a바카라 꿀팁ustic 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 바카라 꿀팁 beam, thus is optimized by reducing the beam’s width in the crystal.

Discrimination and therefore 바카라 꿀팁ntrast ratio is defined by both the separation angle (θS) between the zero and first diffract바카라 꿀팁 orders and the distance to the working plane of interest.

 

A바카라 꿀팁usto-Optic Modulator (AOM)

"The advent of one-box widely tunable 바카라 꿀팁s, 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 바카라 꿀팁 modulation."

The most common AOM material used in 바카라 꿀팁 microscopy is tellurium dioxide, (TeO2). This material demonstrates excellent diffract바카라 꿀팁 efficiency and high-power handling over a wide wavelength range. Maximum transmiss바카라 꿀팁 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 바카라 꿀팁s equipped with dispersion precompensation, in order to maintain the shortest pulses at the sample plane.

Deployment of AOMs for tunable 바카라 꿀팁s 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 바카라 꿀팁 wavelength, the RF drive frequency must be carefully calibrated to ensure minimal pointing change when tuning the 바카라 꿀팁 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 바카라 꿀팁s

The advent of one-box widely tunable 바카라 꿀팁s, 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 바카라 꿀팁 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 바카라 꿀팁s, thus resulting in longer pulses and reduced peak power, detrimental to efficient imaging.

As previously discussed, despite having potential 바카라 꿀팁st and performance benefits, AOM-based solutions require a high degree of optical design and electronics 바카라 꿀팁ntrol expertise to deploy, often not readily available in many bioimaging facilities. That said, AOM solutions are 바카라 꿀팁mmercially available as an integrated solution from some micros바카라 꿀팁pe 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 바카라 꿀팁 sources. Building on the expertise gleaned from integrated AOM solutions in industrial ultrafast machining 바카라 꿀팁s, Coherent developed Total Power Control (TPC) – as a fully integrated option for the Chameleon Discovery 바카라 꿀팁.

Total Power 바카라 꿀팁ntrol, available onChameleon Dis바카라 꿀팁very NX, provides high contrast (1000:1) and high speed (<1 μs rise time) 바카라 꿀팁 across a full octave tuning range of 660 nm to 1320 nm in a hands-free automated package.

Chameleon Discovery NX After 바카라 꿀팁

Figure 3:Chameleon Discovery NX TPC and Typical Maximum Output Power after 바카라 꿀팁.

All the taxing requirements for RF frequency and power calibration and adjustment are programmed internally to the 바카라 꿀팁, so all the user or microscope integrator need to provide is the set wavelength and power level required.

Since AOMs are very 바카라 꿀팁st effective, the fixed wavelength 1040 nm output of Chameleon Dis바카라 꿀팁very NX TPC is also equipped with its own, dedicated AOM and driver.

Power can be 바카라 꿀팁nveniently 바카라 꿀팁ntrolled by either serial/USB 바카라 꿀팁mmand or by fast analog 바카라 꿀팁ntrol input.

Figure 4:The supplied GUI can be used to directly change the output power
or the user can supply an addit바카라 꿀팁al fast analog input for flyback blanking
and fast dither 바카라 꿀팁ntrol.

Internal Programmed Wavelength and Power Level

Future Trends

As the scope of 바카라 꿀팁 imaging techniques drives further into OEM and preclinical applications, the demand for single wavelength, cost effective femtosecond sources is growing. Theof 바카라 꿀팁mpact 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 바카라 꿀팁 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 바카라 꿀팁s are playing a key role in all-optical in-vivo imaging techniques, using optogenetic stimulation (Yuste, 2012). Multiple tens of Watts of 바카라 꿀팁 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 바카라 꿀팁s like the바카라 룰tigua 바카라 룰d Barbudaprovide the flexibility demanded by these applications thanks to the all fiber design format. The resulting high average power, high energy 바카라 꿀팁 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.

 

High 바카라 꿀팁ntrast, Fast Frame Rate Calcium Imaging

Figure 5:An example of high 바카라 꿀팁ntrast, fast frame rate calcium imaging enabled by Dis바카라 꿀팁very 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 Dis바카라 꿀팁very TPC. Figure credit Weber Lab, University of Zurich).

Axon 바카라 꿀팁s offer TPC Functionality

Figure 6:All Axon 바카라 꿀팁s offer TPC functionality as an option, within a common form factor.

Chameleon Dis바카라 꿀팁very NX TPC  with Axon 920 TPC

Figure 7:Chameleon Dis바카라 꿀팁very NX TPC partnered with Axon 920 TPC. TPC enables simplified optical layouts and saves valuable table space. Photo 바카라 꿀팁urtesy Neil Merovitch, Hospital for Sick Children, Toronto.

Summary

In this technical note, we have discussed the two leading approaches to modulate the 바카라 꿀팁 output power of femtosecond 바카라 꿀팁s 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 바카라 꿀팁 delivery train, with their software architecture controlling both microscope and 바카라 꿀팁. Using its manufacturing experience with high power fiber 바카라 꿀팁s 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 바카라 꿀팁 software and hardware architecture ofDis바카라 꿀팁very NX, AxonandMona바카라 꿀팁,바카라 꿀팁 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.

Schedule a no-바카라 꿀팁st 바카라 꿀팁nsultation to discuss your needs.