Depth-enhanced molecular imaging with two-photon oblique plane microscopy

Abstract

High-numerical-aperture (NA) oblique plane microscopy enables noninvasive fluorescence imaging of subcellular dynamics without requiring radical sample modification. However, performance degrades at depth in multicellular specimens as scattering and refractive-index heterogeneity raise out-of-focus background. We report a two-photon oblique plane microscope that improves resolution at depth by combining high-NA single-objective detection with multiphoton plane illumination. The microscope achieves $\sim\!300$ nm lateral and $\sim\!650$ nm axial resolution, with single-molecule sensitivity in vivo. Compared with two-photon point scanning, the lower illumination NA delivers an order of magnitude lower peak intensity, enabling $>\!5\times$ faster volumetric acquisition (up to $3.25 \times 10^6$ voxels s$^{-1}$) with reduced photodamage. In multicellular contexts, near-infrared nonlinear excitation enhances contrast throughout the illumination depth by $\sim\!2\times$ and restores volumetric resolving power by $>\!2\times$ relative to linear excitation. We demonstrate these capabilities through molecular imaging of epithelial tissue, stem-cell-derived gastruloids, and living fruit fly embryos, including multicolor transcription-factor dynamics, optogenetic subcellular control, and single-mRNA tracking, all using standard glass-based mounting.

Figures (24)

• Figure 1: Two-photon oblique plane microscopy (2P-OPM). (Caption on next page)
• Figure 2: 2P-OPM performance. (a)$xy$ MIP of a beads field captured with 2P-OPM. Scale bar, 10 µm. Insets show $xy$ (top), $xz$ (middle), and $yz$ (bottom) MIPs of a representative bead from the region indicated by the white box. Zoomed-in regions of beads across an entire volume are shown in Fig. \ref{['fig:2P-OPM_beads']}. Scale bar, 1 µm. (b)$x$, $y$, and $z$ resolution, as measured by the FWHM, across the $x$-field of view for 2P-OPM and 1P-OPM ($N>90$ beads for each mode). The mean $x$, $y$, and $z$ FWHM $\pm$ SD values are 2P-OPM, 292 $\pm$ 40 nm, 331 $\pm$ 40 nm, 653 $\pm$ 84 nm, respectively; and 1P-OPM, 283 $\pm$ 27 nm, 324 $\pm$ 28 nm, 613 $\pm$ 60 nm, respectively.
• Figure 3: 2P-OPM improves the contrast and resolution in volumetric imaging of multicellular systems. (a)$xy$ MIPs of a 120-hr DAPI-stained gastruloid, captured with 2P-OPM (left) and 1P-OPM (right). Inset: transmitted light image showing the $100 \times 105 \times 30$ µm$\textsuperscript{3}$ volume imaged. See also Video \ref{['vid:oids_movie']}. Scale bar, 20 µm. (b) Intensity profiles along the dashed green line in (a), showing that 2P-OPM provides higher SNR than 1P-OPM. (c) Zoomed-in $xy$ slice from the box in (a), highlighting cleaner images with 2P-OPM. Scale bar, 5 µm. (d) Fourier transforms of $xy$ MIPs in (a). Resolution bands (white circles at 1 µm$\textsuperscript{-1}$) highlight the increased spatial frequency content of 2P-OPM compared to 1P-OPM. The average amplitudes of the Fourier spectra (right) show that 2P-OPM reaches the noise floor more slowly, indicating superior effective resolution. (e)$xz$ MIPs of a 120-hr gastruloid stained by immunofluorescence for FOXC1, recorded with 2P-OPM (left) and 1P-OPM (right). Top inset: transmitted light image showing the $70 \times 100 \times 30$ µm$\textsuperscript{3}$ volume imaged. Yellow arrow indicates the light-sheet propagation direction ($y'$), corresponding to the $y'$-distance from the glass interface. Scale bar, 20 µm. (f) Quantification of image contrast as a function of $y'$-distance from the glass interface, showing improved contrast over the full illumination depth with 2P-OPM over 1P-OPM. Each $xz$' slice is normalized to the surface value. (g) Zoomed-in $xz$ orthoslice from the box in (e), revealing that 2P-OPM reduces background and better resolves individual cell nuclei deep in the gastruloid tissue than 1P-OPM. Scale bar, 2 µm. (h) Intensity profiles along the dashed green line in (e), demonstrating the improved SNR and optical sectioning of 2P-OPM.
• Figure 4: 2P-OPM enhances molecular imaging performance in low SNR fluorescence. (a)$xz$ MIPs of mRNA activity for the eve gene, labeled via smFISH, in a fixed Drosophila embryo at NC 14, comparing 2P-OPM (left) and 1P-OPM (right). Inset: $xy$ view, highlighting activity in individual nuclei. Scale bar, 5 µm. (b)$x$ (top), $y$ (middle), and $z$ (bottom) line intensity profiles through the transcript from the subregion indicated in (a), showing better resolution in all dimensions. Color points: raw data; solid lines: Gaussian fit. (c)$x$ (top), $y$ (middle), and $z$ (bottom) resolution, measured by the FWHM; for each modality, the same ($N = 20$) transcripts were chosen from the tissue represented in (a). All boxes denote mean $\pm$ standard error; center values are means; whiskers represent the spread of the data. The mean $x$, $y$, and $z$ FWHM $\pm$ SD values are 2-OPM, 299 $\pm$ 46 nm, 377 $\pm$ 50 nm, 692 $\pm$ 24 nm, respectively; and 1P-OPM, 373 $\pm$ 69 nm, 458 $\pm$ 75 nm, 975 $\pm$ 30 nm, respectively.
• Figure 5: 2P-OPM enables fast, multicolor, single-molecule imaging in living animals. (a) Left: Transmitted light (brightfield) image of a live fruit fly embryo at NC 14, expressing Bcd-eGFP (overlaid in green), marking the $100 \times 115 \times 15$ (xyz) µm$^{3}$ volume imaged with 2P-OPM. The white rectangle indicates the 3.5-µm-thick slab shown at right. Right: Zoomed $xy$ MIP of Bcd-eGFP (gray) and nascent hb transcripts, tagged with MS2-MCP-mCherry (orange hotspots). Scale bar, 10 µm. (b)$xy$ (top) and $xz$ (bottom) MIPs from a $100 \times 115 \times 15$ µm$^{3}$ volume in a developing fruit fly embryo, showing active hb transcription loci labeled with MS2-MCP-mNeonGreen at the end of NC 12. See also Video \ref{['vid:4D-transcription_movie']}. Scale bar, 20 µm. (c) Zoomed time-lapse of NC events from (b). Left: beginning of NC 13; middle: peak of NC 13; right: peak of NC 14. Scale bar, 10 µm. (d) Transcription traces of 148 hb spots sampled every $\sim\!\!10$ s from a $100 \times 15 \times 15$ µm$^{3}$ subvolume within a $100 \times 115 \times 15$ µm$^{3}$ imaged field of a fly embryo. This subvolume corresponds to mid-anterior along the AP axis, centered at $x/L$$\approx$ 0.45 (spanning $\sim$0.37-0.53). Mean activity is overlaid (white). (e) Temporal standard-deviation projection showing hb mRNAs as diffraction-limited puncta and transcription sites as larger, brighter hotspots. $100 \times 20$ µm$^{2}$ field of view captured at 20 Hz over 50 time points (Video \ref{['vid:SM_movie']}). Scale bar, 10 µm. (f,g) Zoomed views of the boxed regions from (e), showing the first time point with single-particle trajectories overlaid from the full sequence (tracks $\ge$2 steps; colors indicate track identity). Scale bar, 2 µm. (h) Mean squared displacement (MSD) of hb mRNA molecules as a function of time ($\Delta t=50$--$200$ ms). Points are mean $\pm$ standard error across step pairs; a linear fit to the first two $\Delta t$ points yields an apparent ensemble diffusion coefficient of $D\approx1.04~µm^{2}\per s$.