Lauri A et al. (2019), Whole-Cell Photoacoustic Sensor Based on Pigmen...

XB-ART-55646

ACS Sens 2019 Mar 22;43:603-612. doi: 10.1021/acssensors.8b01319.

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Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Lauri A , Soliman D , Omar M , Stelzl A , Ntziachristos V , Westmeyer GG .

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Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

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Species referenced: Xenopus laevis
Genes referenced: cps1 gprc6a mip pmch

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	Graphical Abstract
	Figure 1. RSOM imaging and photoacoustic signal frequency analysis of melanophores after background adaptation of a zebrafish larva in vivo. (a,b) Top-view maximum intensity projections (MIP) of a 5-day-old zebrafish larva imaged with RSOM after being adapted to a dark (a) or a light (b) environment. Dorsal view, anterior is left. The insets show a magnification of the trunk region delineated by the white dashed boxes. White arrows highlight positions in which background-dependent melanosome aggregation was observed. (c,d) Corresponding brightfield (BF) microscopy images of the same fish as shown in (a) and (b) obtained after optoacoustic imaging. The insets show a magnification of the regions indicated by the red dashed outline, corresponding to the insets in (a) and (b). Red arrows mark the same cells containing aggregated melanosomes as labeled in (a) and (b). (e) Quantification of the melanosome aggregation effect for the two conditions and imaging modalities shown in (a)–(d) (mean pigmented area normalized to the maximum of each modality ± SEM from 19 cells (BF) or 9 clusters of cells (RSOM)). A significantly reduced pigmented area was observed after light background adaptation with both RSOM and BF (paired t test, *** indicates p < 0.001). (f) Difference in photoacoustic signal cumulative power spectra (CPS) normalized to the CPS peak value of the control measurement. The light blue curve shows the difference between the CPS obtained from the light background adapted fish and the dark-adapted fish. The gray curve represents the CPS obtained from the dark-adapted fish.
	Figure 2. RSOM imaging of a zebrafish larva before and after exposure to MCH. (a,b) Top-view MIPs of a zebrafish larva imaged with RSOM before (a) and after (b) 45 min of exposure to 100 μM of MCH dissolved in fish water. Dorsal view, anterior is left. The insets show a magnification of the trunk region delineated by the white dashed boxes. White arrows highlight regions of prominent melanosome aggregation. (c) Differences in photoacoustic cumulative power spectra (CPS) normalized to the control CPS peak value. The dark blue curve shows the difference between the CPS obtained after 20 min of treatment with MCH and the CPS of the control condition. The light blue curve shows the same analysis for 45 min of treatment with MCH. The CPS of the control condition is shown in gray color as a reference.
	Figure 3. Cultured frog melanophores imaged with brightfield microscopy and RSOM before and after exposure to MCH. (a–d) Brightfield microscopy images showing cultured frog melanophores before (a) and after (b) 15 min of treatment with 30 μM of MCH. (c,d) Close-ups of a subset of cells shown in (a) and (b). The cell identities are indicated with colored dots, whereas the cell membranes are delineated by dashed lines. A reduced pigmented area by 58% was observed as compared to the control image (Wilcoxon matched-paired signed rank test, p-value <0.0001, n = 25). (e,f) RSOM imaging of cultured frog melanophores before (e) and after (f) exposure to 30 μM of MCH, which triggered melanosome aggregation. The images show top-view MIPs of the RSOM measurement of the cell layer, while the insets represent magnifications of the regions delineated by the dashed boxes. Upon MCH stimulation, the overall pigmented area was found to be reduced by 22% as compared to the control measurement (paired t test, p-value: 0.006, n = 10).
	Figure 4. Optoacoustic imaging of melatonin-induced melanosome aggregation in xenotransplanted frog melanophores in zebrafish in vivo. (a) Xenografted frog melanophores (orange color map) in rag2E450 fs mutant fish. Shown is a top-view MIP of the RSOM measurement of the fish before stimulation (“Control”). Background signals from endogenous pigmentation are displayed with a blue color map, whereas the eyes are additionally marked with red asterisks. The inset shows a corresponding brightfield image of the head region, which was recorded before RSOM imaging. Scale bar: 200 μm. (b,c) Zoom into the region indicated by the white dashed box in (a). Four melanophores are labeled for reference and shown before (b) and after (c) 30 min of stimulation with 0.1 μM of melatonin (“Stimul.”). (d,e) Side-view MIPs of the RSOM reconstructions in a direction indicated by the white arrow in (a). The gray dashed lines in (e) indicate the part of the reconstructed volumes that was subjected to the photoacoustic frequency analysis. (f) Difference of cumulative power spectra (CPS) (Stimul. minus control; blue curve) normalized to the maximum of the control CPS (gray curve). The red curve represents a local regression fit to the blue spectrum to highlight the global trend. (g) MORSOM imaging of the same specimen ex vivo after the stimulation experiment showing a side-view MIP of the 3D reconstruction. Cyan color: native cells and eyes. Orange color: region around the xenotransplanted frog cells.
	Figure 5. RSOM imaging of Xenopus melanophores expressing a designer receptor exclusively activated by designer drugs (DREADD) hMD4i xenografted into rag2E450 fs mutant zebrafish. (a–c) Schematic showing the experimental design comprising coexpression of the DREADD hMD4i and the enhanced GFP (hMD4i-2A-eGFP) to enable fluorescence-activated cell sorting (FACS) of the most strongly expressing cells. (b) Brightfield imaging of melanosome aggregation in response to clozapine-N-oxide (CNO) stimulation of the transplanted melanophores expressing the hMD4i-2A-eGFP. (c) Dot blot showing the cell population selected by FACS cell sorting via eGFP fluorescence. (d,e) Photoacoustic top-view MIPs of a rag2E450 fs mutant zebrafish with xenografted genetically modified frog melanophores (hMD4–2A-eGFP, orange color map) before (d) and after (e) stimulation with CNO. Cyan color map: endogenous structures and fish eyes. Orange color map: region around the xenotransplanted frog melanophores.

References [+] :

Amano, Melanin-concentrating hormone: A neuropeptide hormone affecting the relationship between photic environment and fish with special reference to background color and food intake regulation. 2009, Pubmed