Visualising the Emerging Platform of Using Microalgae as a Sustainable Bio-Factory for Healthy Lipid Production through Biocompatible AIE Probes

Abstract

Nowadays, a particular focus is using microalgae to get high-valued health beneficiary lipids. The precise localisation of the lipid droplets (LDs) and biochemical changes are crucial to portray the lipid production strategy in algae, but it requires an in vivo tool to rapidly visualise LD distribution. As a novel strategy, this study focuses on detecting lipid bioaccumulation in a green microalga, Chlamydomonas reinhardtii using the aggregation-induced emission (AIE) based probe, 2-DPAN (C₂₄H₁₈N₂O). As the messenger molecule and stress biomarker, hydrogen peroxide (H₂O₂) activity was detected in lipid synthesis with the AIE probe, TPE-BO (C₃₈H₄₂B₂O₄). Distinctive LDs labelled with 2-DPAN have elucidated the lipid inducing conditions, where more health beneficiary α-linolenic acid has been produced. TPE-BO labelled H₂O₂ have clarified the involvement of H₂O₂ during lipid biogenesis. The co-staining procedure with traditional green BODIPY dye and red chlorophyll indicates that 2-DPAN is suitable for multicolour LD imaging. Compared with BODIPY, 2-DPAN was an efficient sample preparation technique without the washing procedure. Thus, 2-DPAN could improve traditional fluorescent probes currently used for lipid imaging. In addition, the rapid, wash-free, multicolour AIE-based in vivo probe in the study of LDs with 2-DPAN could advance the research of lipid production in microalgae.

Keywords: aggregation-induced emission; green microalgae; health beneficiaries; healthy lipid; visualisation.

Figure 1

Effects of nutrient and light manipulation on the growth of Chlamydomonas reinhardtii at different time intervals. Treatment 1: MBL medium; Treatment 2: MBL, (−) N₂; Treatment 3: MBL, (−) N₂, (−) Ca²⁺; Treatment 4: MBL, (−) N₂, (−) Ca^{2 +}, (+) Sodium acetate (2.0 g/L) (24 h light) and Treatment 5: MBL, (−) N₂, (−) Ca²⁺, (+) Sodium acetate (2.0 g/L) (24 h dark) conditions.

Figure 2

Fluorescence properties of 2-DPAN. Absorption spectra (a) and fluorescence spectra (b,c) of 2-DPAN (10 µM) in 90% water fraction and DMSO-water mixtures, respectively.

Figure 3

Confocal images of lipid drops in Chlamydomonas reinhardtii cells of Treatment 4 (MBL medium, (−) N₂, (+) Sodium Acetate (2.0 g/L), (−) Ca²⁺, (24 h light condition)). Cells were labelled with traditional BODIPY™ 505/515 (5 μM; incubation-5 min) (c) and AIE probe, 2-DPAN (20 μM; incubation-30 min) (d). (Bright-field images: (a); Fluorescence images—Chlorophyll: (b) (λ_ex: 488 nm, λ_em: 685–758 nm); BODIPY: (c) (λ_ex: 488 nm, λ_em: 490–517 nm); 2-DPAN: (d) (λ_ex: 488 nm, λ_em: 570–650 nm); Merged image: (e–i) and (j) are the enlarged regions of a–d respectively); (k) intensity profile of BODIPY and 2-DPAN in green and yellow channels, respectively; (l) intensity scatter plot for the colocalised channels; Pearson correlation coefficient and Mander’s overlap coefficient were calculated as 0.91 and 0.92, respectively; (m) Relative fluorescence intensity/cell for lipid-specific probes. Values are relative to the control BODIPY dye. Averages shown as mean ±SE; * p < 0.05. Images were taken with Zeiss LSM 880 Airyscan confocal microscope.

Figure 4

Flow cytometry measurements for lipid in Chlamydomonas reinhardtii cells in different treatments labelled with BODIPY™ 505/515 and AIE probe, 2-DPAN. (a–e) Flow cytogram of FITC-A vs. SSC-A for BODIPY fluorescence in different treatments; (g–k) Flow cytogram of KO525-A vs. SSC-A for 2-DPAN fluorescence. Cells were cultured in Treatment 1: modified Woods Hole (MBL) medium; Treatment 2: MBL, (−) N₂; Treatment 3: MBL, (−) N₂, (−) Ca²⁺; Treatment 4: MBL, (−) N₂, (−) Ca²⁺, (+) sodium acetate (2.0 g/L) (24 h light); and Treatment 5: MCM, (−) N₂, (−) Ca²⁺, (+) sodium acetate (2.0 g/L) (24 h Dark) conditions. Histogram of BODIPY™ 505/515 (f) and 2-DPAN (l) fluorescence for cells. (m) Relative fluorescence of BODIPY™ 505/515 and 2-DPAN / cell for different treatments. Values are relative to the control condition of Treatment 1. Averages shown as mean ± SE; * p < 0.05; ** p < 0.01. All plots are in the logarithmic scale for both axes.

Figure 5

Labelling lipid drops with lipid-specific AIE nanoprobe, 2-DPAN (20 μM; incubation-30 min) in Chlamydomonas reinhardtii. Cells were cultured in (A) Treatment 1: modified Woods Hole (MBL) medium; (B) Treatment 2: MBL, (−) N₂; (C) Treatment 3: MBL, (−) N₂, (−) Ca²⁺; (D) Treatment 4: MBL, (−) N₂, (−) Ca²⁺, (+) sodium acetate (2.0 g/L) (all the treatments were in 24 h light condition). Bright-field images: Aa,Be,Ci,Dm Fluorescence images-2-DPAN: Ab,Bf,Cj,Dn (λ_ex: 488 nm, λ_em: 570–650 nm),and Chlorophyll: Ac,Bg,Ck,Do (λ_ex: 488 nm, λ_em: 685–758 nm); Merged images: Ad,Bn,Cr,Dp. (E) Relative fluorescence intensity/cell for different treatments. Values are relative to the control condition (Treatment 1: modified Cramer–Myers medium (MCM)). Averages shown as mean ±SE; * p < 0.05; Images were taken with Zeiss LSM 880 Airyscan confocal microscope.

Figure 6

Hydrogen peroxide content in the Chlamydomonas reinhardtii cells of different treatments. Treatment 1: modified Woods Hole (MBL) medium; Treatment 2: MBL, (−) N₂; Treatment 3: MBL, (−) N₂, (−) Ca²⁺; Treatment 4: MBL, (−) N₂, (−) Ca^{2 +}, (+) sodium acetate (2.0 g/L) (all the treatments were in 24 h light condition). Data represented as mean ± SE, n = 3, * p < 0.05; ** p < 0.01.

Figure 7

Confocal images of H₂O₂ activities in Chlamydomonas reinhardtii cells during lipid induction. H₂O₂ and lipid drops were labelled with AIE-probes, TPE-BO (100 μM; incubation-15 min) and 2-DPAN (20 μM; incubation-30 min), respectively. (a–e) Treatment 1: modified Woods Hole (MBL) medium; (f–j) Treatment 2: MBL, (−) N₂; (k–o) Treatment 3: MBL, (−) N₂, (−) Ca²⁺; (p–t) Treatment 4: MBL, (−) N₂, (−) Ca^{2 +}, (+) sodium acetate (2.0 g/L) (all the treatments were in 24 hr light condition). Bright-field images: a, f, k and p; Fluorescence images—Chlorophyll: b, g I and q (λ_ex: 488 nm, λ_em: 685−758 nm); 2-DPAN: (c,h,m,r) (λ_ex: 488 nm, λ_em: 570–650 nm); TPE-BO: (d,i,n,s) (λ_ex: 405 nm, λ_em: 428–499 nm); Merged image: (e,j,o,) and (t,u) Relative fluorescence intensity/cell for different treatments. The red arrow indicates H₂O₂ activity in normal cells and during cell division; the orange arrow indicates H₂O₂ activity in lipid accumulated cells; The white arrow indicates H₂O₂ activity in autophagic cells. Averages are shown as mean ±SE; * p < 0.05; Images were taken with Zeiss LSM 880 Airyscan confocal microscope.

Figure 8

Imaging of lipid drops in H₂O₂ treated Chlamydomonas reinhardtii. Lipids were labelled with lipid-specific AIE nanoprobe, 2-DPAN (20 μM; incubation-30 min). Cells were cultured in 0.0 mM (a–d), 0.4 mM (e–h) and 0.6 mM (i–l) H₂O₂ supplemented MBL medium. Bright-field images: (a,e,i); Fluorescence images—2-DPAN: (b,f,j) (λ_ex: 488 nm, λ_em: 570–650 nm) and Chlorophyll: (c,g,k) (λ_ex: 488 nm, λ_em: 685–758 nm); Merged images: (d,h,l). (m) Relative fluorescence intensity/cell for different treatments. Values are relative to the control condition (0.0 mM H₂O₂). Averages shown as mean ± SE; ** p < 0.01; Images were taken with Zeiss LSM 880 Airyscan confocal microscope.