Two-Photon Oblique Plane Microscope

• Two-photon oblique plane microscopy is a fluorescence imaging modality that creates an oblique light sheet via two-photon excitation for volumetric imaging in scattering samples.
• It employs a single high-NA objective for both excitation and detection, using a relay system to transform the oblique plane onto a sensor for distortion‐free, rapid 3D capture.
• Optimized for deep tissue studies, the technique reduces photodamage and enhances contrast, enabling high-speed imaging with single-molecule sensitivity.

Two-photon oblique plane microscopy (2P-OPM), including its implementation as two-photon scanned oblique plane illumination (SOPi) microscopy, refers to a class of single-objective, high–numerical aperture (NA) imaging modalities that achieve volumetric, high-resolution fluorescence microscopy deep within scattering specimens. By optically generating and scanning an obliquely oriented light sheet via two-photon (2P) excitation, these systems enable distortion-free, rapid 3D imaging with strong optical sectioning, deep penetration, and single-molecule sensitivity, all within standard glass-based sample mounting formats (Kumar et al., 2018, Keomanee-Dizon et al., 12 Nov 2025).

1. Optical System Architecture

2P-OPM systems merge multiphoton plane illumination with high-NA detection and single-objective configurations. The excitation and detection paths are generally co-axial, using a high-NA primary objective (O1), typically oil immersion (e.g., Olympus 60×/NA 1.49), to both deliver the scanned light sheet and collect fluorescence (Keomanee-Dizon et al., 12 Nov 2025). The fluorescence is relayed via a remote-focusing relay—incorporating secondary (O2, 40×/NA 0.95, air) and tertiary (O3, 40×/NA 1.0, glass) objectives—that reimages the oblique focal plane onto a scientific CMOS (sCMOS) sensor. The final detection NA is limited primarily by the smallest aperture in the relay (effective detection NA ≈ 1.44 × 1.29) (Keomanee-Dizon et al., 12 Nov 2025). Emission splitting into multiple spectral channels for multicolor imaging is realized via a dichroic tree.

For SOPi implementations, excitation is launched through a single, front-facing water immersion objective (Olympus XLUMPLFLN20XW, 20×, NA 1.0) (Kumar et al., 2018). A galvo-based scan mirror system de-scans the emission path, stabilizing the oblique plane image and minimizing geometric distortion.

Key parameters (example values):

Parameter	2P-OPM (Keomanee-Dizon et al., 12 Nov 2025)	SOPi (Kumar et al., 2018)
Primary Objective	60×/NA 1.49 (oil)	20×/NA 1.0 (water)
Illumination NA (2P)	0.3–0.5 (adjustable)	0.06–0.08
Detection NA	1.44 × 1.29 (effective)	0.34 (overall system)
Obliquity angle	45°	45°
Emission Camera	sCMOS	sCMOS (11 μm pixels)
Field of View (2P)	~100×115×30 µm³	~750×270×500 µm³

The 45° oblique illumination geometry is realized by laterally displacing the excitation beam at the objective’s back focal plane, yielding a scanned sheet intersecting the focal volume (Kumar et al., 2018, Keomanee-Dizon et al., 12 Nov 2025).

2. Two-Photon Plane Illumination and Scanning

Two-photon excitation uses near-infrared ultrafast lasers (Ti:Sapphire or ytterbium fiber), typically in the 680–1300 nm range, with pulse widths of <140 fs and repetition rates ranging from 11–80 MHz (Keomanee-Dizon et al., 12 Nov 2025, Kumar et al., 2018). The light sheet is created by rapidly scanning a tightly focused 2P beam in the oblique plane via a high-speed galvo (GM-x), while an orthogonal galvo (GM-y) steps the focal plane for volumetric data acquisition. In more advanced variants, a resonant galvo “wobbles” the light sheet along the y′-axis, reducing shadowing artifacts by time-averaging the sheet position (mSPIM technique).

Bessel-like or Gaussian light sheets can be realized using “layer-cake” phase masks, increasing the depth of focus (e.g., from ~2.5 μm to ~10 μm for NA 0.5) (Keomanee-Dizon et al., 12 Nov 2025). The absence of a beam expander in SOPi maintains low illumination NA and extends the Rayleigh length, facilitating volumetric coverage.

The quadratic dependence of 2P excitation ($P_2(x,y,z) \propto [I(x,y,z)]^2$) ensures strong axial confinement, reducing out-of-plane excitation. Adjusting illumination NA enables trading off sheet thickness (and thus axial resolution) for reduced peak intensity, yielding substantial reductions (7–19×) in nonlinear photodamage (Keomanee-Dizon et al., 12 Nov 2025).

3. Imaging Performance and Resolution

2P-OPM achieves volumetric acquisition rates surpassing 3.2 million voxels per second, $>5\times$ faster than typical 2P point scanning, while maintaining subcellular resolution and single-molecule sensitivity (Keomanee-Dizon et al., 12 Nov 2025). For raw point spread function (PSF) measurements using 50 nm beads, 2P-OPM reports:

• Lateral resolution: $x = 292 \pm 40$ nm, $y = 331 \pm 40$ nm
• Axial resolution: $z = 653 \pm 84$ nm

For SOPi:

• Lateral FWHM (0.5 μm beads): 2P = $1.16 \pm 0.06$ μm
• Axial FWHM: ∼4 μm

The theoretically predicted sheet waist and Rayleigh length for 2P at $\lambda = 910$ nm and $\text{NA}_{\text{sheet}} = 0.06$ are $w_0 \approx 4.8$ μm and $z_R \approx 80$ μm (Kumar et al., 2018).

Depth penetration and contrast are enhanced due to the use of NIR wavelengths and nonlinear excitation. Measured SBR at 300 μm depth in mouse hippocampal slices for 2P SOPi is $\approx 0.37$ (compared to $\approx 0.05$ for 1P), reflecting an increased scattering length ($\ell_s \approx 300$ μm for 2P vs. 100 μm for 1P) (Kumar et al., 2018).

In molecular imaging, single-molecule FISH demonstrates 3D resolving power improvements of $\sim2.15\times$ in 2P versus 1P, with SNR per transcript ranging from 100–200 photons. In vivo measurements in Drosophila achieve $\sigma_x=32.9$ nm localization with ensemble diffusion measured via mean squared displacement (Keomanee-Dizon et al., 12 Nov 2025).

4. Volumetric Acquisition and Data Handling

Acquisition strategies utilize affine transformations to reconstruct isotropic Cartesian volumes from oblique image stacks. For $\theta = 45^\circ$, the transform involves:

$$S = \begin{pmatrix} 1 & 0 & 0 & 0\ 0 & 1 & 0 & 0\ 0 & 0 & 1/\sin\theta & 0\ 0 & 0 & 0 & 1 \end{pmatrix}, \quad H = \begin{pmatrix} 1 & 0 & 0 & 0\ 0 & 1 & 0 & 0\ 0 & -\sin\theta & 1 & 0\ 0 & 0 & 0 & 1 \end{pmatrix}$$

with $T = H S$ applied to transform the data stack (Kumar et al., 2018). This enables recovery of distortion-free 3D volumes at high temporal resolution, sufficient for functional imaging applications (e.g., 10 volumes/s for Ca$^{2+}$ imaging over $850\times300\times50$ μm$^3$ in zebrafish larvae with $\Delta F/F$ up to 20%) (Kumar et al., 2018).

5. Biological Applications

2P-OPM and 2P SOPi have been demonstrated in a breadth of structural and functional contexts:

• Mouse brain (Thy1-GFP slices): 2P SOPi resolves neuronal and dendritic structure over $750\times270\times500$ μm$^3$ in 30 s, with spine-level resolution and minimal shadowing at $\sim 500$ μm depth (Kumar et al., 2018).
• Zebrafish larvae: Pan-cellular volumetric captures ($450\times300\times200$ μm$^3$ in 1 s) with 1P, and rapid functional Ca$^{2+}$ imaging over large fields at high volume rates (e.g., $10$ VPS) (Kumar et al., 2018).
• Epithelial cell clusters (OptoEGFR): 2P-OPM enables optogenetic actuation and super-resolution imaging of protein clustering, with a 45% increase in heterogeneity at lower integrated dose relative to previous approaches (Keomanee-Dizon et al., 12 Nov 2025).
• Murine and Drosophila development: nuclear and cytoskeletal imaging in gastruloids and embryos, as well as live single-molecule mRNA tracking (achieving 20 Hz volumetric rates and robust SNR for fluorescent puncta) (Keomanee-Dizon et al., 12 Nov 2025).

A key feature is compatibility with standard glass-based mounting (e.g., #0 coverslips, glass-bottom dishes) without requiring custom sample holders or side-viewing objectives, streamlining multiparametric and multimodal integrations (Keomanee-Dizon et al., 12 Nov 2025, Kumar et al., 2018).

6. Photodamage, Contrast, and Depth Advantages

2P plane illumination reduces peak intensity per voxel ($\propto$ NA$^2$), shifting dose from damaging pulses to longer average dwell times afforded by faster scanning. This decreases higher-order nonlinear photodamage by $7$–$19\times$ (for $\alpha = 2.6$–$4.3$ reduction in NA) while maintaining SNR at elevated volumetric frame rates (Keomanee-Dizon et al., 12 Nov 2025). NIR wavelengths (750–1030 nm) also scatter $2$–$3\times$ less than visible, and the quadratic nonlinearity ensures fluorescence is confined to the evolutionary focal plane, further reducing background and maximizing contrast in scattering or index-mismatched samples (Keomanee-Dizon et al., 12 Nov 2025, Kumar et al., 2018).

Compared to 2P point scanning, 2P-OPM achieves $>5\times$ higher volumetric throughput at lower peak intensities and reduced photobleaching risk, supporting sensitive, high-contrast imaging in multicellular and in vivo contexts.

7. Current Limitations and Emerging Directions

The triply relayed detection architecture (O1→O2→O3→camera) imposes throughput losses (e.g., $\sim$60% at 488 nm), chromatic and pulse broadening, and exacerbates side-lobe attenuation in Bessel mode (Keomanee-Dizon et al., 12 Nov 2025). PSF anisotropy (300:330:650 nm, $x:y:z$) remains unresolved; real-time adaptive optics (AO) and field-dependent aberration correction may alleviate this but require further integration (Keomanee-Dizon et al., 12 Nov 2025). Thermal drifts and mechanical instabilities necessitate active stabilization.

Opportunities for further improvement include adopting resonant galvos for spatially averaged dose, optimizing dispersion compensation (e.g., grisms), advanced coatings for higher throughput, and the use of structured illumination or lattice-light-sheet variants for super-resolution (Keomanee-Dizon et al., 12 Nov 2025). The development of brighter, red/NIR fluorophores and two-step excitation chemistries may extend capabilities for ultra-deep, low-phototoxicity imaging.

A plausible implication is that the continued convergence of high-NA detection, novel beam engineering, adaptive optics, and advanced fluorescent probes will enable robust 4D molecular and functional imaging in increasingly complex and optically challenging biological contexts.

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Key references:

• "Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging" (Kumar et al., 2018)
• "Depth-enhanced molecular imaging with two-photon oblique plane microscopy" (Keomanee-Dizon et al., 12 Nov 2025)