Laser Scanning Multiphoton Microscopes
[2,3]. Detection is possible—up to 4 channels to collect SHG, THG, and fluorescence signals.
Widefield Epi-Fluorescence/ Brightfield Modes Widefield and brightfield modes
Figure 4: The MPX-1040 easily allows imaging of samples from the micro to macro scale and from 2D to 3D. A) Autofluorescence collected by epi-widefield fluorescence of a human lung tissue cryosection. Scale bar = 1 mm. B) Two-photon maximum intensity projection of a 3D z-stack of cellular spheroids stained for actin (red) and cell nuclei (blue). Scale bar = 100 μm. C) Two-photon maximum intensity projection of a 3D z-stack of a stable H2B-mCherry (yellow) expressing dividing cell embedded in a fluorescent bead (green) containing hydrogel, stained for actin (red). Scale bar = 10 μm. D) Live-mouse multiphoton imaging of GCamp-expressing neurons in the cortex. E) 3D deep tissue multiphoton imaging of a mouse kidney (left) and colon (right) mounted in a 2-well ibidi dish embedded in an agarose gel. F) Imaging of standard 2D samples mounted on an objective slide.
footprint eases integration into a microscope setup. Fiber lasers are rugged and operate in variable environmental conditions, mitigating concerns about misalignment or performance deg- radation over time. Fiber lasers have been used frequently and proven well-suited for nonlinear imaging applications [8]. Te MPX consists of a dual-wavelength femtosecond fiber
laser engineered with a fixed output at 1040 nm (> 500 mW) and a second tunable laser in the range of 750–960 nm and 1150– 1300 nm (> 200 mW). Tis combination covers the commonly used wavelengths for multiphoton research. Imaging Ca2+
in
neuroscience is a very popular application for LSMM and can be achieved at 920 nm using green fluorescent protein (GFP) and a second wavelength at 1040 nm. A built-in acousto-optical modulator (AOM) can be used for fast power control and galvo flyback blanking. Dispersion compensation for pulse broad- ening can correct timing from 0 fs2
to -40,000 fs2 , allowing
optimal pulse shaping for penetrating thick tissue. Te pulse duration at the sample is <140 fs when operating at 80 MHz repetition rate. Tese parameters are all measured at the sample position and are sufficient for sectioning > 1 mm. Fiber lasers have very good power stability, a diffraction limited output beam (typically M2
< 1.1), and are compact, rug-
ged, and energy-efficient for almost any indoor workspace. Te ultra-wide tuning range allows access to many fluorophores and endogenous markers for many nonlinear optical methods not accessible by fixed wavelength sources. Te capability for further tuning in the infrared is particularly important for new imaging techniques, for example, three- and four-photon excitation, yielding deeper penetration depth and higher res- olution due to less light scattering and higher power density
20
are enabled by a fully integrated high-performance multi-channel continuous wave (CW) excitation source ranging from 395–747 nm with >300 mW per color provided at the sample. Te lines are separated by two individual manually change- able filter sets (dichroic + emission) for quadband and petaband illu- mination and imaging. Te signals are captured by a high-performance widefield sCMOS f luorescence camera with very low-noise and quantum efficiency up to 80%. Epi- brightfield illumination is achieved with a quasi-white light source mimicked from the same CW laser set, with predefined programmable brightness. Te acquisition scheme consists of a filter set with 50% reflection and transmission filters across the visible spectrum. Te
modular design of the MPX allows other cameras, depending on the specific needs, to be integrated.
Fast Imaging and Analysis Scanning speed improves the efficiency of the imaging pro-
cedure, minimizes motion blur, and optimizes workflow, while maintaining submicron resolution to identify cellular and fibril- lar structures. For general beam scanning across the sample, galvo-galvo mirrors are used at 4.6 fps at a resolution of 512 × 512 pixels. Fast imaging is achieved with galvo-resonance scanners with 8 kHz resonance for 30 fps over the same area array. Te maximum field-of-view (FOV) is a 30 mm diagonal square at the intermediate image plane. Sectioning and z-stacks can also be acquired by using a high-precision objective piezo z-stage. Te MPX can tailor and streamline the workflow for faster
data acquisition and analysis, even for 3D and real-time video imaging, with an efficient and user-friendly interface. Investiga- tors can freely and rapidly choose between the built-in modali- ties manually or through the soſtware. Tis process makes it easy to overlay and correlate images and to process multidi- mensional and multimodal results. Customized contrast and comparison algorithms provide dynamic control and analysis used for correlative and digital microscopy.
Software Te in-house developed Chromogazer™ microscope soſt-
ware controls all system functions, including the femtosecond laser for multiphoton imaging and continuous wave illumina- tion light engines used for epi-fluorescence imaging. Chro- mogazer provides dynamic control, detection monitoring, and automatic error logging and reporting for the whole system.
www.microscopy-today.com • 2022 May
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