This protocol outlines the visualization and quantification of a particular protein within cells at the cellular level for the phycoerythrin-containing cyanobacterium, Crocosphaera watsonii.
Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.
The physiological variation from cell to cell (commonly referred to as "heterogeneity") in metabolic activities within microorganisms, including cyanobacteria, has been documented through studies on clone cultures1,2,3,4. This heterogeneity encompasses diverse metabolic activities such as cell division5, carbon assimilation6,7,8, and nitrogen assimilation9,10. For instance, recent investigations have indicated that N2 fixation activity in colonial cyanobacteria C. watsonii and C. subtropica (Cyanothece) exhibits single-cell level variability, being present in subpopulations of cells while absent in others within the community. Notably, the nitrogen uptake or N2 fixation activities also exhibit variability among cells in situ11,12,13. These findings have been substantiated by stable 15N isotope analyses conducted with isotope ratio mass spectrometers (NanoSIMS)14,15. However, despite NanoSIMS offering a novel avenue for analyzing isotopic composition at the individual cell level, its use remains constrained due to its technical complexity and cost.
An alternative approach to observe intracellular heterogeneity in metabolic activities is through immunodetection. Earlier reports have demonstrated the immunodetection of nitrogenase in individual cells, but this poses challenges due to the autofluorescence emitted by their photosynthetic pigments16,17,18. Marine cyanobacteria, particularly those adapted to oceanic waters such as the major oceanic diazotrophs C. watsonii and Trichodesmium, contain substantial amounts of phycobilins that emit autofluorescence in shorter wavelengths: phycoerythrin and phycourobilin19. To circumvent this autofluorescence, blue-emitting fluorochromes with UV excitation have been favored for cyanobacteria studies16,20,21. However, this strategy hasn't consistently yielded success, as cells treated solely with primary antibodies emitted strong blue to bluish-yellow autofluorescence under UV excitation20,21. Efforts have been made to mitigate this issue by subjecting cells to blue or UV light exposure prior to observation and by employing semiconducting nanocrystals22. The present study employs a different strategy that enhances protein fluorescence signals using the tyramide signal amplification system (TSA) to visualize proteins with low cellular content.
TSA, also known as catalyzed reporter deposition (CARD), is a highly sensitive enzymatic method enabling the detection of low-abundance targets in immunocytochemistry. This technique leverages peroxidase's catalytic activity to covalently deposit labeled tyramide in proximity to target proteins in situ23,24. In the presence of hydrogen peroxide, peroxidase catalyzes the oxidative condensation of tyramide into reactive tyramide radicals, which then bind to electron-rich moieties such as tyrosine, phenylalanine, and tryptophan25. This enhances signals by up to 10 to 200-fold compared to standard methods, making the signal detectable via standard chromogenic or fluorescent techniques. Consequently, this technique facilitates the rapid and simultaneous assessment of multiple proteins alongside phenotypic markers in heterogeneous populations and rare cell subsets. Notably, as of now, the amalgamation of immunolabeling and TSA systems for cyanobacteria has been limited to a single study that visualized saxitoxin in Cylindrospermopsis raciborskii26.
The method outlined herein permits the investigation of protein production under varying environmental conditions at the single-cell level, enabling the assessment of metabolic activities in target cyanobacteria. The availability of whole-cell immunofluorescence protein detection allows for swift and semi-quantitative visualization of proteins in C. watsonii at the single-cell level. Moreover, this method can be easily adapted for use with other cyanobacterial cells containing phycourobilin and phycoerythrin.
1. Cyanobacteria cultivation
2. Preparation of reagents
3. Harvesting cells
4. Fixation and preservation of cells
5. Permeabilization and blocking
6. Preparation of samples for imaging
7. Detection of fluorescence signal using a fluorescence microscope
8. Detection under a confocal microscope
9. Quantify the intensity of the signal using Fiji
The fluorescence signal was observed from extracellular substances in the negative control, where the 1st antibody was not used (Figure 1A–C). The fluorescence signal of the tyramide-boosted reagent, conjugated to the large subunit of the Rubisco protein (RbcL), was successfully detected in C. watsonii under a fluorescence microscope using a DAPI filter with UV excitation (Figure 1D–F). Additionally, fluorescent signals were visible from the extracellular polymeric substance (EPS) (Figure 1C,F).
Moving forward, the protocol was applied to the enzyme responsible for nitrogenase Fe protein (NifH). To confirm the specificity of the fluorescence signal resulting from the immunochemical reaction, the fluorescence intensity was quantified using the Fiji software. The intensity of signals from the cells treated with the complete protocol was significantly higher than from cells lacking the 1st and/or 2nd antibodies (Figure 2) (Mann-Whitney test30, p < 0.001 and Steel-Dwass post-hoc test31, p < 0.001). The signals from the cells lacking the first and/or second antibodies were slightly higher than those from the control with no antibody or tyramide reagent, using the maximum excitation wavelength at 350 nm (Tyramide 350) (Mann-Whitney test, p < 0.001 and Steel-Dwass post-hoc test, p < 0.001). However, a small fraction of cells from the control and cells lacking the first and/or second antibodies exhibited high-intensity signals (~200). Nonetheless, these cells constituted less than 1% of all cells within a given treatment. In contrast, over 83% of cells treated with the full procedure displayed a signal intensity of 200 or higher. Furthermore, less than 3% of cells in the control group and less than 5% of cells lacking the first and/or second antibodies exhibited higher-intensity signals (>133) than the lowest signal measured for cells treated with the complete procedure (Figure 2).
The positive impact of the tyramide signal amplification system was verified by the relationship between the incubation time of the tyramide working solution (step 6.3) (Figure 3). The proportion of cells with positive signals increased with longer incubation times, up to 7.5 min. The positivity threshold for the signal was defined as being greater than 25% of the maximum value within the same image. The error bars represent the standard deviation32.
The fluorescence spectra and images obtained via a confocal microscope are presented as the final step. Due to the confocal microscope's minimum excitation wavelength of 405 nm, the Tyramide 405 reagent was employed instead of Tyramide 350. When the second antibody was conjugated with the Tyramide 405 reagent, fluorescence emission spectra peaking around 460 nm could be distinguished from fluorescence peaks emitted by phycourobilin at 503 nm and phycoerythrin at 566 nm (Figure 4). The error bars denote the standard deviation32.
The procedure is summarized as a flowchart (Figure 5).
Figure 1: Bright-field and fluorescence image of C. watsonii PS0609 culture under the fluorescence microscope. (A–C) Negative control, treated without primary antibody. (D–F) Positive control treated as described in the protocol (Figure 5). (A,D) Bright-field image. (B,E) Phycoerythrin autofluorescence detection with FITC filter set (Excitation 460-495 nm/Emission 510 nm). (C, F) Immunofluorescence detection of RbcL with DAPI filter set (Excitation 360-370 nm/Emission 420-460 nm). The blue signal shows the fluorescence from the Tyramide 350. The white arrow shows the Tyramide 350 attached to the extracellular polymeric substance. Scale bars: 10 µm. Please click here to view a larger version of this figure.
Figure 2: The confirmation of fluorescence signals being from the target protein. (A) Each dot represents fluorescence from a single cell. The parenthesis shows the number of cells analyzed. (B–E) Bright-field and fluorescence image of C. watsonii PS0609 culture under the fluorescence microscope. (F–I) Immunofluorescence detection of nitrogenase Fe protein (NifH)28 with DAPI filter set (Excitation 360-370 nm/Emission 420-460 nm). Cont.; no amendment, Tyramide; tyramide reagent. Please click here to view a larger version of this figure.
Figure 3: Optimization of booster incubation time. The average percentage of cells with the positive signal for nitrogenase Fe protein (NifH). Each incubation time of the tyramide reagent with standard deviation. The threshold of the positivity of the signal was defined as >25% of the maximum value in the same image. Parentheses show the number of cells observed under a given condition. Error bar shows standard deviation. Please click here to view a larger version of this figure.
Figure 4: The fluorescence emission spectra. Immunostaining against RbcL (A) and NifH (B). The average single-cell fluorescence emission spectra of C. watsonii PS0609 with (RbcL or NifH: black line) or without 1st nor 2nd antibodies (Cont.: grey line) incubation with RbcL (A) or NifH (B) as 1st antibody followed by Tyramide 405. Data are normalized at 503 nm. The parentheses show the number of cells analyzed. A typical image of C. watsonii PS0609 cells without 1st antibody (C) and with NifH antibody (D) obtained by Tyramide 405 channel (detected at 410-436 nm) confocal imaging. r. u.; relative unit. PUB: phycourobilin, PE: phycoerythrin, Tyramide: tyramide reagent. Error bar shows standard deviation. Please click here to view a larger version of this figure.
Figure 5: Flowchart of the present protocol. MtOH: Methanol, PFA: paraformaldehyde, PBS: phosphate-buffered saline, PBG: 0.2% gelatin + 0.5% BSA, HRP: horseradish peroxidase. Please click here to view a larger version of this figure.
For cyanobacteria, the TSA system has found widespread use in TSA-fluorescence in situ hybridization (TSA-FISH, CARD-FISH), targeting specific rRNA. However, its application for proteins remains limited26. In this study, a TSA procedure was applied to enable whole-cell immunodetection of the N2-fixing cyanobacterium C. watsonii, incorporating modifications based on a previous reference20. Notable revisions encompassed permeabilization, accomplished through a combination of lysozyme and achromopeptidase22, and signal enhancement using TSA systems23. Furthermore, leveraging the TSA technique and RGB image separation through Fiji software enabled the visualization and quantification of antibody-conjugated fluorescence signals amidst the inherent auto-fluorescence of phycourobilin and phycoerythrin.
The visualization of proteins in unicellular cyanobacteria offers insight into cell-to-cell variations in protein content. Previously, analyses of cell-to-cell metabolic heterogeneity in cyanobacteria relied on isotope ratio mass spectrometers (NanoSIMS)11,12,13,14,15. Nonetheless, utility of NanoSIMS has been curtailed by instrument scarcity and high costs. In contrast, immunocytochemistry necessitates standard equipment present in most labs. The provided protocol can be readily adapted for other cyanobacterial cells. For instance, it has successfully been employed for nitrogenase (NifH) in C. watsonii WH8501 and Rubisco (RbcL) in Synechocystis sp. PCC 6803, yielding anticipated outcomes.
Critical protocol stages include cell envelope permeabilization. In the case of C. watsonii, achromopeptidase use was pivotal, a step introduced following the methodology of reference22. The same protocol yielded positive results with Synechocystis sp. PCC 6803. Nevertheless, since cell envelope structure varies among cyanobacteria species, optimization of incubation times and permeabilization agents might be required for other species.
While the signals obtained through the full protocol significantly exceeded those from control cells or cells lacking 1st and/or 2nd antibodies, it's noteworthy that high signals were recorded in certain cells missing antibodies or control cells. This phenomenon could relate to challenges in segregating EPS from cells, possibly due to fluorescence dye binding to EPS (Figure 1C,F). Hence, the rinsing step assumes significance in mitigating such issues. This method holds potential for quantifying protein content33,34, akin to quantification achieved through signal detection via flow cytometry35. The current protocol effectively enables the assessment of target protein presence in C. watsonii. This technique promises swifter and more cost-effective monitoring of intracellular metabolic heterogeneity compared to NanoSIMS11,14,15. This approach could be employed in forthcoming studies to identify organisms exhibiting target metabolic functions, such as in situ N2 fixation.
The authors have nothing to disclose.
We appreciate Dr. Radek Kana and Barbora Šedivá for assistance with confocal microscopic analysis and Dr. Roman Sobotka and Dr. Kateřina Bišová for advice in immunodetection and fluorescence microscopy analysis. This research was financially supported by Czech Research Foundation GAČR (project 20-17627S to OP and TM), the Mobility plus project between JSPS and Czech Academy of Sciences (JPJSBP 120222502), and JSPS KAKENHI (project 23H02301).
Achromopeptidase | FUJIFILM | 014-09661 | |
Alexa Fluor350 | Thermo Scientific | B40952 | Tyramide-350 |
Alexa Fluor405 | Thermo Scientific | B48254 | Tyramide-405 |
Alexa Fluor488 Tyramide SuperBoost Kit | Thermo Scientific | B40922 | Goat anti-rabbit IgG |
Bovine serum albumin | Sigma-Aldrich | A2153 | |
Centrifuge | Eppendorf | 5804 R | |
Centrifuge tubes (15 mL) | VWR | 525-1085 | For harvesting cells |
Confocal microscope | Zeiss | LSM880 | Equipped with Airyscan |
Fluorescence microscope | Olympus | BX51 | DAPI filter: Ex.360-370 nm, Em. 420-460 nm |
Gelatine | Merk | 4070 | |
High precision microscope cover glasses for confocal microscope | Deckgläser | No. 1.5H | |
Liquid Blocker Regular/Mini | Daido Sangyo Co., Ltd. | Part 6505 | For keeping the cells on the slide glass |
Lysozyme | ITW Reagents | A4972 | |
Methanol | Carl Roth | 67-56-1 | |
Monopotassium Phosphate | Penta | 12290 | |
Monunting medium | Sigma-Aldrich | 345789-20ML | FluorSave Reagent |
Mounting medium | Vectashild | H-1300 | |
Objective lens used in the confocal microscope | Zeiss | Plan-Apochromat 63x/1.4 Oil DIC M27 | |
Paraformaldehyde | Sigma-Aldrich | 158127 | |
Poly-lysine coated slide glass | Sigma-Aldrich | P0425-72EA | |
Potassium chloride | Lach-Ner | ||
Safe lock tube (1.5 mL) | Eppendorf | 0030 120.086 | For treating cells and storing chemicals |
Sodium chloride | Penta | 16610 | |
Sodium hydrogen phosphate | Penta | 15130 | |
Triton X-100 | Sigma-Aldrich | X100 |
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