Multiphoton Plane Illumination

• Multiphoton plane illumination is a nonlinear optical technique that confines multiphoton excitation to a thin, extended plane, enabling rapid volumetric imaging with high spatial resolution.
• It employs methods like SLM-based complex illumination, oblique-plane geometry, and Airy beam shaping to minimize photodamage and ensure uniform fluorescence generation.
• Quantitative advances demonstrate improved axial and lateral resolution while suppressing illumination artifacts, supporting applications from subcellular dynamics to large-scale tissue imaging.

Multiphoton plane illumination refers to a class of nonlinear optical microscopy modalities where excitation light is spatially confined to a thin, extended plane, enabling simultaneous activation of multiphoton processes over entire fields of view. Plane illumination drastically increases volumetric imaging speed compared to conventional point-scanning multiphoton techniques, mitigates photodamage by distributing excitation over large areas, and enables uniform two-photon or higher-order fluorescence generation critical for imaging in scattering biological tissues. Core implementations include amplitude/phase-modulated wide-field approaches, oblique-plane geometry with high-numerical-aperture single-objective detection, and propagation-invariant beam shaping (such as Airy and Bessel light sheets). Recent research demonstrates quantitative gains in imaging speed, resolution, and photodamage suppression using multiphoton plane illumination across diverse biological applications ranging from subcellular dynamics to single-molecule tracking.

1. Theoretical Frameworks for Plane Illumination in Nonlinear Microscopy

Multiphoton absorption exhibits an intensity-square ($I^2$) dependence, necessitating both spatially homogeneous excitation and sufficiently high instantaneous power density. In wide-field nonlinear microscopy, phase-only spatial light modulators (SLMs) are used to synthesize arbitrary complex fields $U(x,y) = A(x,y)\, e^{i\phi(x,y)}$ with prescribed amplitude and phase profiles. The complex illumination method (CIM) analytically encodes any $U(x,y)$ as the sum of two “carrier” waves $B\,e^{i\varphi_0(x,y)}$ and $B\,e^{i\varphi_1(x,y)}$, with $B = A_{\max}/2$ and carrier phases $\varphi_{0,1}$ defined by the target amplitude and phase. Checkerboard-multiplexed phase patterns, imaged and filtered to select only the zero diffraction order, yield the exact prescribed field at the sample, enabling amplitude- and phase-controlled nonlinear plane excitation. This analytic construction avoids speckle and the approximation errors inherent in iterative Fourier transform algorithms (IFTAs), which suffer from residual intensity nonuniformity and limited multilevel fidelity (Carbonell-Leal et al., 2019).

Oblique-plane and Airy beam light-sheet modalities leverage the convolution of excitation sheet thickness and detection point-spread function (PSF) to deliver volumetric sectioning. In two-photon oblique-plane microscopy (2P-OPM), the probability of multi-photon excitation at each point obeys $P \propto I_{\rm peak}^n$ ($n=2$ for TPE), and axial and lateral resolution are described by

$$\Delta x \simeq 0.61\,\frac{\lambda}{\mathrm{NA}_{\rm det}}, \qquad \Delta z \simeq 2n\,\frac{\lambda}{\mathrm{NA}_{\rm det}^2}$$

where $\lambda$ is the emission wavelength, NA$_{\rm det}$ is the detection numerical aperture, and $n$ is the refractive index (Keomanee-Dizon et al., 12 Nov 2025).

Planar Airy beams are constructed by rotating the cubic phase mask, thereby confining the otherwise parabolic main lobe to the focal plane and forming a symmetric sheet with well-defined thickness ($\Delta z \simeq 3.7\,\mu$m) and extended field of view ($\sim0.6$ mm), as the Airy propagation invariance allows uniform intensity distribution across hundreds of microns (Hosny et al., 2020).

2. Optical Architectures and Plane-Generation Techniques

Major multiphoton plane-illumination architectures include:

• CIM wide-field SLM-based system: Utilizes HOLOEYE PLUTO SLM (1920×1080 pixels, 8 μm pitch) for phase multiplexing, combined with a 4f common-path interferometer (CFEM) and spatial filtering. Enables simultaneous amplitude and phase control over $>50\times50\,\mu$m$^2$ regions. The excitation source is an ultrafast Ti:Sapphire laser (800 nm, 30 fs, 1 kHz, 0.8 mJ/pulse). Downstream demagnification optics project the modulated plane to the sample and a high-NA objective collects fluorescence (Carbonell-Leal et al., 2019).
• 2P-OPM (single-objective, oblique-plane): Employs a single high-NA water-immersion objective (O1, NA=1.49) for both excitation and detection. Excitation is a near-infrared femtosecond sheet (typical NA$_{\rm exc}=0.3$–0.5) launched at $45^\circ$ via a dichroic mirror, forming an oblique plane in the specimen. Remote-focus and tube lens optics relay fluorescence to three sCMOS cameras for multicolor imaging. Excitation sheet is “painted” by a galvo scan; a resonant galvo “wobbles” to reduce shadowing artifacts (Keomanee-Dizon et al., 12 Nov 2025).
• Planar Airy beam light-sheet microscope: Femtosecond Ti:Sapphire (930.9 nm, 140 fs, 80 MHz) illumination is shaped by a cubic phase mask (reflective or via SLM), positioned on a motorized rotation stage. A 4f relay follows, with beam expansion and galvo scanning to synthesize the sheet. Orthogonal water-dipping objectives are used for fluorescence detection. Precise mask rotation (by $45^\circ$) produces a true planar sheet matched to the detection focal plane (Hosny et al., 2020).
• Reverberation multiphoton microscopy (MPM): A 50:50 non-polarizing beamsplitter and time-delay cavity (“reverberation loop”) produce a pulse train focused at successively deeper planes ($\Delta z$ per loop controlled by relay misalignment $\delta d$), with depth-indexed detection via GHz-bandwidth photomultiplier and FPGA time-bin sorting. Integration requires minimal add-on hardware to conventional point-scanning MPM (Beaulieu et al., 2018).

3. Quantitative Performance Metrics

Performance parameters from recent literature are tabulated below.

Modality	Axial ($\Delta z$)	Lateral ($\Delta x$)	FOV	Uniformity/Fluctuation
CIM SLM-wide-field	∼3–5 μm	∼3–5 μm feature res.	60×60 μm²	±12% (linear); ±23% (SHG)
Planar Airy 2PE sheet	3.69 μm	0.81 μm / 0.85 μm	0.6 mm	±5% over 0.4 mm FOV
2P-OPM (oblique-plane)	653±84 nm	292±40 nm / 331±40 nm	10 μm sheet	2× higher contrast depth
Reverberation MPM	1.25 μm	435 nm	N×68 μm	<9% plane crosstalk

CIM achieves root-mean-square error (RMSE) of 7%–10% for amplitude fidelity over 60×60 μm². Planar Airy light-sheets maintain ∼3.7 μm axial thickness and ±5% 2PE uniformity over 0.4 mm FOV. 2P-OPM attains 653 nm axial and <0.35 μm lateral resolution, acquiring volumes at $3.25\times10^6$ voxels/s. Reverberation MPM attains 1.25 μm axial resolution across up to 7–8 planes per pulse train, with effective volumetric frame rates scaling up to N-fold compared to point scanning (Carbonell-Leal et al., 2019, Keomanee-Dizon et al., 12 Nov 2025, Hosny et al., 2020, Beaulieu et al., 2018).

4. Suppression of Illumination Artefacts: Speckle, Nonuniformity, and Photodamage

CIM-based plane illumination eliminates speckle by optically conjugating the SLM output and employing spatial filtering to reject non-zero diffraction orders, ensuring deterministic field generation. Analytical construction yields exact (up to diffraction limit) amplitude and phase, compared to IFTA/CGH approaches that produce up to ±62% intensity-profile fluctuations. Since multi-photon processes amplify any nonuniformity ($I^2$ for TPA, $I^m$ for higher-order), CIM’s ±12% linear fluctuation corresponds to ±23% in second harmonic generation (SHG), while CGH amplifies to ±77% (Carbonell-Leal et al., 2019).

In 2P-OPM and planar Airy approaches, low excitation NA ($0.3$–$0.5$) ensures broad sheet thickness and minimizes $I_{\rm peak}$, reducing photodamage mechanisms ($\propto I^m$ with $m>2$) and enabling longer dwell times for improved SNR. Planar Airy sheet rotation confines excitation to the focal plane, minimizing out-of-focus bleaching, and avoids asymmetric side lobes or the need for deconvolution. In reverberation MPM, distributed pulse energy among focal spots further reduces photodamage in upper layers, and effective SNR is preserved so long as total sample power remains within biological limits (Keomanee-Dizon et al., 12 Nov 2025, Hosny et al., 2020, Beaulieu et al., 2018).

5. Application Domains and Demonstrated Imaging Scenarios

Multiphoton plane illumination has enabled high-throughput, volumetric imaging in several biological settings:

• Wide-field nonlinear microscopy: CIM allows parallel two-photon excitation and emission across cellular-sized ROIs ($>50\times50\,\mu$m$^2$), enabling millisecond-scale functional imaging without mechanical scanning. Adaptive wavefront correction, structured illumination, and multifocal multi-level excitation are feasible by programmatically varying amplitude/phase (Carbonell-Leal et al., 2019).
• Oblique-plane multiplex imaging: 2P-OPM supports single-molecule sensitivity in vivo ($\sim10^2$ photons/spot, 30–60 nm localization precision), optogenetic stimulation, subcellular control, and multicolor transcription factor dynamics in epithelia, gastruloids, and Drosophila embryos. Contrast in scattering tissue is doubled over 1P, and depth-integrated resolving power is maintained (Keomanee-Dizon et al., 12 Nov 2025).
• Planar Airy beam light-sheet: Uniform two-photon excitation over 0.6 mm FOV enables rapid volumetric imaging (20 frames/s per plane) in acute brain slices, organoid preparations, and cleared tissue, with negligible photobleaching and direct 3D accurate reconstructions (Hosny et al., 2020).
• Reverberation MPM multiplane imaging: Yields near-instantaneous 3D imaging at the speed of 2D frame acquisition; demonstrated in scattering samples (rodent brain slices) with up to 8 planes sampled per pulse train, maintaining diffraction-limited resolution. Plane-by-plane intensity equalized via loop focal spacing and software (Beaulieu et al., 2018).

6. Implementation Parameters, Calibration, and Limitations

Successful deployment of multiphoton plane illumination requires attention to several calibration and alignment factors:

• SLM spectral calibration and spatial filtering (CIM): Precise adjustment ensures amplitude/phase fidelity and suppression of higher diffraction orders. Checkerboard multiplexing cell size is typically 4×4–6×6 SLM pixels.
• Mask alignment and rotation (Airy light-sheet): Manual rotation of cubic phase mask ($0^\circ$ for conventional, $45^\circ$ for planar sheet) ensures main lobe planarization; galvo amplitude matches swept width to FOV; sample focus co-aligned with excitation plane.
• Optical demagnification and detection NA: Combination of achromats and high-NA objectives sets both excitation sheet geometry and detection field.
• Volumetric extension: Current CIM demonstrations are 2D; 3D light shaping requires stacking focal planes and managing tissue-induced aberrations (e.g., Zernike compensation terms in phase profile).
• Power scaling and photodamage: In multiphoton plane modalities, the excitation power must be tuned to balance sectioning thickness, SNR, and photobleaching constraints. For reverberation MPM, laser repetition rate and cavity length control number of planes and axial separation.

Trade-offs include slight reduction in lateral resolution for larger field-of-view sheets, reduced efficiency for sparsely filled SLM patterns, and requirement for high-bandwidth, multi-channel detection electronics in multiplane systems.

7. Future Directions and Implications

Multiphoton plane illumination methodologies are rapidly advancing optical microscopy’s ability to probe subcellular and molecular dynamics at high speed, low photodamage, and high volumetric fidelity. Potential extensions include 3D light shaping via stacked phase masks, adaptive aberration correction embedded in field encoding, and integration with Bessel or Airy beams for extended-volume sectioning. Emerging applications include fast Ca$^{2+}$/voltage activity imaging, connectomics, single-molecule trafficking, and optogenetic control across large specimen volumes, all in standard mounting protocols compatible with glass coverslips and minimally modified biological samples (Carbonell-Leal et al., 2019, Keomanee-Dizon et al., 12 Nov 2025, Hosny et al., 2020, Beaulieu et al., 2018).

A plausible implication is that, as hardware and algorithmic control improve, multiphoton plane illumination will continue to supplant point-scanning methods for functional, high-throughput, and multiplexed imaging in complex tissues.