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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2487:15–26. doi: 10.1007/978-1-0716-2269-8_2

Multi-dimensional Fluorescence Live-cell Imaging for Glucosome Dynamics in Living Human Cells

Songon An 1,2,*, Prakash Parajuli 1, Erin L Kennedy 1, Minjoung Kyoung 1,2,*
PMCID: PMC9191769  NIHMSID: NIHMS1747605  PMID: 35687227

Abstract

Fluorescence live-cell imaging that has contributed to our understanding of cell biology is now at the frontline of studying quantitative biochemistry in a cell. Particularly, the technological advancement of fluorescence live-cell imaging and associated strategies in recent years have allowed us to discover various subcellular macromolecular assemblies in living human cells. Here we describe how the real-time dynamics of a multienzyme metabolic assembly, the “glucosome,” that is responsible for regulating glucose flux at subcellular levels, has been investigated in both 2- and 3-dimensional space of single human cells. We envision that such multi-dimensional fluorescence live-cell imaging will continue to revolutionize our understanding of how intracellular metabolic pathways and their network are functionally orchestrated at single-cell levels.

Keywords: Live-cell Imaging, Fluorescence Microscopy, Lattice Light-sheet Microscopy, Glucosome, Metabolic Condensate, Metabolic Complex, Macromolecular Assembly, Glycolysis, Glucose Metabolism, Cancer Metabolism

1. Introduction

Fluorescence microscopy has been one of the popular techniques in the field of cell biology and has contributed significantly to our current understanding of cell anatomy [1,2]. However, its application had been technically limited when studying the dynamic nature of intracellular biomolecules in living cells due to the lack of subcellular specificity necessary to visualize specific biomolecules at single-cell levels [3,4]. Recent scientific and technical developments in biophysics and biochemistry have allured us to explore previously untouchable territories of life sciences [3–6]. Multi-dimensional fluorescence live-cell imaging has now opened a foreseeable avenue of cellular biochemistry by revealing the existence of multienzyme metabolic complexes in living cells and thus their real-time functional contributions to the cell [7–9]. Here we demonstrate how we have investigated subcellular dynamics of a multienzyme metabolic assembly, namely the “glucosome,” that regulates glucose flux in single human cells under fluorescence live-cell imaging [10–12]. Our strategy has allowed us to advance our knowledgebase of not only how glucose metabolism is regulated in space and time as part of a functional metabolic network in human cells, but also its functional association with other cellular processes at the level of single human cells.

2. Materials

2.1. Human Cancer Cell Lines

Glucosomes have been successfully visualized in various human cancer cell lines, including HeLa (cervical adenocarcinoma) [12], Hs578T (breast carcinoma) [12], BT-549 (breast ductal carcinoma), MCF-7 (breast adenocarcinoma) and MDA-MB-436 (breast adenocarcinoma).

2.2. Cell Culture and Imaging

  1. General disposable plasticware and small equipment for mammalian cell culture.

  2. Glass-bottomed 35-mm cell culture petri dishes.

  3. DFG medium: 450 mL of Dulbecco’s modified eagle medium with high glucose (DMEM) is supplemented with 50 mL of fetal bovine serum (FBS), 55 mg of sodium pyruvate, 25 uL of 1 g/mL gentamycin sulfate.

  4. RFG medium: 450 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) is supplemented with 50 mL of dialyzed FBS (see Note 1) and 25 uL of 1 g/mL gentamycin sulfate.

  5. Gibco Opti-MEM-I reduced serum medium (Opti-MEM-I).

  6. Lipofectamine 2000 transfection reagent.

  7. A plasmid encoding human phosphofructokinase liver type that is fused with monomeric enhanced green fluorescent protein (PFKL-mEGFP) [12].

  8. Buffered-saline solution (BSS) [7,12]: Add about 700 mL autoclaved water to a 1 L graduate cylinder. Add 20 mL of 1 M HEPES, pH 7.4, 33.8 mL of 4 M NaCl, 5 mL of 1 M KCl, 2 mL of 0.5 M MgCl2, 3.6 mL of 0.5 M CaCl2, and 1.9 mL of 3 M D-glucose. Mix and make up to 1 L with autoclaved water. Perform sterile filtration and store at 4 °C.

  9. Nikon Eclipse Ti inverted C2 confocal microscope equipped with a 60x objective lens (Nikon CFI Plan Apo TIRF, 1.45 NA), a monochrome CCD camera (Photometrics CoolSnap EZ), and a filter set appropriate for mEGFP detection.

  10. Imaging software for wide-field and confocal microscopy: Nikon NIS-Elements.

  11. Lattice light-sheet microscope (LLSM) and imaging software [10,13].

  12. Image analysis software: ImageJ [14,15] and MatLab (version 9.7.0 (R2019b), The MathWorks Inc).

3. Methods

All media and reagents described here were warmed up to room temperature for a few hours or 37 °C for an hour before use. Please note that all procedures were carried out at room temperature unless otherwise specified.

3.1. Human Cell Culture

  1. A frozen stock vial of each cell line (HeLa, Hs578T, BT-549, MCF-7 and MDA-MB-436) that contains ~ 2–3 × 106 cells, is thawed and cultured in a T25 tissue culture flask containing the DFG medium.

  2. After the cells are maintained in the DFG medium for about 2 weeks by sub-culturing them in every 3–4 days, they are passed into the DFG medium or the RFG medium following 3–4 days sub-culture for another two weeks before preparing samples for live-cell imaging (see Note 2).

  3. The cells are now ready for transient transfection and imaging in every passage cycle for another 2 months.

3.2. Transient Transfection

  1. Cells are seeded into a glass bottom 35 mm petri dish in a total of 2 mL volume of the desired growth medium without antibiotics (i.e. DF or RF media) for wide-field and confocal microscopy. Alternatively, cells are seeded into 5 mm round coverslips that are placed on a glass bottom 35 mm petri dish for lattice light-sheet microscopy (LLSM) (see Note 3). Cells should be uniformly distributed on the glass-bottom imaging area of the dish or on the 5 mm round coverslips by rocking it slowly and incubating overnight at 37 °C under 5% CO2 and 95% humidity.

  2. The next day, transient transfection is performed using Lipofectamine 2000 when cells are approximately 70–80 % confluent at the time of transfection (see Note 4).

  3. A plasmid expressing PFKL-mEGFP as a glucosome marker (0.8μg) [11,12] is added into 50 μL of Opti-MEM-I. In a separate tube, 1.5 μL of Lipofectamine 2000 is added into 50 μL of Opti-MEM-I. Each mixture is gently mixed and incubated individually for 5 min at room temperature (see Note 5).

  4. Combine both mixtures, mix gently and incubate for 30 min at room temperature.

  5. While incubating the plasmid-transfection reagent mixture, take out cell dishes from the CO2 incubator and rinse the cells twice with 2 mL of Opti-MEM-I. Then, add 1 mL of Opti-MEM-I to each dish (see Note 6).

  6. After 30 min incubation of Step 4, add 900 μL of Opti-MEM-I into the plasmid-transfection reagent mixture.

  7. Add the entire plasmid-transfection reagent mixture (~ 1 mL) dropwise to the dish containing cells (see Note 7).

  8. Incubate the cells at 37°C in a CO2 incubator (95% humidity) for 5–6 hours.

  9. Exchange the medium with fresh growth medium without antibiotics and continue incubation for 18–24 hours at 37°C in a CO2 incubator (95% humidity).

3.3. 2-Dimentional (2D) Visualization of Glucosome Using Wide-field or Confocal Microscopy

  1. Remove the cell dishes from the CO2 incubator and visualize for adherence under a cell culture microscope (see Note 8).

  2. Rinse the cells with 2 mL of 1xBSS solution at room temperature.

  3. Add 2 mL of 1xBSS solution and incubate for 10 min at room temperature. Repeat this step twice.

  4. Add 2 mL 1xBSS solution again to the cells and incubate for 1–2 hours at room temperature prior to imaging (see Note 9).

  5. Imaging is now performed at ambient temperature (~25°C) with the Nikon Eclipse Ti inverted C2 confocal microscope. PFKL-mEGFP is shown to form spatially resolved glucosomes at various sizes in living human cells (Figure 1). We have then categorized them into 3 sub-classes [12,16]; namely, small (< 0.1 μm2) (see Note 10), medium (< 3 μm2) (see Note 11) and large sizes (< 8 μm2) of glucosome assemblies (see Note 12).

  6. Colocalization Analysis: If cells are transfected with two plasmids, dually transfected cells are imaged in each color channel. Digitally merged images are analyzed using the ImageJ software for colocalization between PFKL-mEGFP and another protein that is fused with either mCherry or mOrange fluorescent protein [12].

  7. Addition of temporal resolution: Since glucosomes are being visualized in live cells, real-time dynamics of glucosomes can be monitored as a function of time in the presence and/or absence of additives to investigate the importance of glucosome dynamics in biological processes of interest [11,12].

Figure 1. Glucosomes in Human Breast Cancer Cells.

Figure 1.

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Representative images of PFKL-mEGFP as a glucosome marker are from BT-549 (A), Hs578T (B), MCF-7 (C) and MDA-MB-436 (D) cells. Scale bars, 10 μm.

3.4. 3D Visualization of Glucosome using Lattice Light-sheet Microscopy (LLSM)

  1. After rinsing the cells as described in Steps 1–3 in Section 3.3 above, add 2 mL 1xBSS solution again to the cells along with a diluted solution of TetraSpeck™ fluorescent beads and allow the beads to settle on the glass surface for at least 10 minutes (see Note 13).

  2. Prior to LLSM imaging, it is important to align illumination pathways daily as described below in Steps 3–8 in Section 3.4.

  3. Using a 30 mm cage alignment plate with Ø0.9 mm hole prior to the spatial light modulator (SLM) while projecting a single beam pattern in the ‘Continuous’ illumination mode, ensure that all laser beams are centered on the pattern. This can be done by carefully adjusting the knobs on the mounted filters that are close to the individual lasers.

  4. Align the single beams through the annular mask by matching the illuminated single beam pattern to the position of the annular mask. This can be observed in the back focal plane guppy camera and adjusted with the knobs on the second mirror before the annular mask [13].

  5. Use the flip mirror to direct the lasers into the illumination objective and examine that the exiting beam of illumination is symmetrical. Add diluted fluorescein and sulfarhodamine B solutions in the sample chamber to visualize Bessel beams. Using the Orca Flash 4.0 camera, adjust the focus on the detection objective using the travel stage knob until the beam is in focus (see Note 14).

  6. Using the ‘Continuous Z-stack’ mode without moving the detection objective piezo stage, ensure that the plane of illumination is orthogonal to the plane of detection. Adjust the travel stage on the flip mirror behind the illumination objective until both ends of the Bessel beam focus at the same time (see Notes 15 & 16).

  7. Ensure that the entire plane of Bessel beams is in focus by switching the SLM to a multibeam pattern. Knobs on the mirrors prior to the flip-mirror can be adjusted until perfect focus of all Bessel beams in the multibeam patterns are in the same plane.

  8. If any of the later adjustments have changed, revisit earlier adjustment steps (i.e. Steps 2–7 in Section 3.4) to ensure that no additional alignment is needed (see Note 17).

  9. Subsequently, the point spread function (PSF) should be obtained daily as follows (i.e. Steps 10–14 in Section 3.4).

  10. Dilutions of TetraSpeck™ fluorescent microspheres are sonicated at room temperature for 5–10 min. Afterwards, a single droplet is added to clean a 5mm coverslip. The fluorescent beads adhere to the coverslip surface after 10 min of undisturbed incubation at room temperature.

  11. Securely load the coverslip into the LLSM sample holder and rinse the sample chamber with fresh 1xBSS. Bring the beads into the detection region using the ‘Motion Console’ mode that controls the sample stage piezo stages.

  12. Using a single isolated bead with the ‘Autofocus Bead’ mode, determine the z-galvo offsets that are associated with each laser to ensure optimal illumination for all laser channels. This step allows to overlap the different laser illumination in the same sample plane to each other (see Notes 18 & 19).

  13. Switch acquisition to the ‘single Z-stack’ mode and obtain 3D images of a bead for each color channel to determine the PSF daily (see Note 20).

  14. Change acquisition to the ‘XZ PSF’ mode and capture a cross-section of the pattern. If any variance in illumination is observed, repeat from Steps 2–7 in Section 3.4 to ensure that all regions of the experimental data are valid for image analysis (see Note 21).

  15. LLSM Imaging with live cells is now performed at ambient temperature (~25°C).

  16. Clean the sample chamber thoroughly with fresh 1xBSS.

  17. Load the 5 mm coverslip that contains transfected cells into the sample holder and position in the sample chamber (see Note 22).

  18. 3D sample scans are taken of each sample for each illumination channel by moving the sample stage in the ‘SI Scan’ mode. The step size of the sample piezo is approximately half of the z-dimension of the PSF. All sample specific factors including the PSF are used in the image deconvolution process using the 3D deconvolution algorithm in MATLAB [13,17] (see Notes 23 & 24).

  19. TetraSpeck fluorescent beads are isolated from the captured deconvolved images in all color channels. To register two color channels, the centroids of the beads obtained from the ‘3D Objects Counter’ plugin [18] in the ImageJ software are calibrated using the imtranslate syntax of Matlab. After alignment of the color channels, beads and non-target cells are cleared from the processed images for analysis (see Note 25).

  20. PFKL-mEGFP as a glucosome marker [11,12] is now visualized at various sizes in living human cells. The ‘3D Objects Counter’ plugin [18] is used to identify glucosome assemblies and to create an 8-bit binary 3D objects mask of the identified glucosome assemblies (see Note 26).

  21. Colocalization Analysis (Figure 2): If cells are transfected with two plasmids, dually transfected cells are imaged in each color channel. The ‘Coloc 2’ plugin [14] is used to assign mCherry-fusion protein to Channel 1, mEGFP-fusion protein to Channel 2, and the objects mask as the Region-of-Interest (ROI) Mask [10]. Note that the colocalized TetraSpeck beads are served as an internal colocalization control.

  22. Addition of temporal resolution: Since glucosomes are being visualized in live cells, real-time dynamics of glucosomes in 3D space can be monitored as a function of time in the presence and/or absence of additives to investigate the importance of glucosome dynamics in biological processes of interest under LLSM imaging [10].

Figure 2. 3D Colocalization Analysis.

Figure 2.

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Colocalized TetraSpeck™ beads overlaid in the red and green channels as a 2D projection of the image (A) or as the 3D image (B) compared against a dataset showing mCherry-fusion protein (in red) colocalized with mEGFP-fusion protein (in green) as a 2D projection (C) or as the 3D image volume (D). The Pearson’s coefficients are calculated by the ‘Coloc 2’ plugin in the ImageJ software (E). Note that the representative cell image here (C and D) shows PFKL-mCherry in red and mEGFP-PKM2 in green. Yellow color indicates their colocalization. Scale bars, 1 μm (A) or 10 μm (C).

4. Notes

  1. We have dialyzed FBS against 0.9% NaCl solution at 4 °C for ~2 days using 12–14 kDa MWCO dialysis membrane [12,16].

  2. This means that it will take about 1 month for cells to be ready for glucosome imaging from a frozen stock. This protocol appears to be one of the indispensable factors that influence on the reproducibility of our imaging data.

  3. The 5 mm round coverslips are cleaned in 1M KOH solution, rinsed with autoclaved Millipore water, and subsequently sterilized under UV light prior to placing them on glass bottom 35mm petri dishes.

  4. For HeLa, Hs578T and MCF-7 cells, we seed ~2 × 105 cells per petri dish in 2 mL of a growth medium without antibiotics [10–12]. However, for BT-549 and MDA-MB-436 cells, ~ 3 × 105 cells per petri dish appear to work better.

  5. The amount of the plasmid for glucosome imaging should be determined for each cell line for the best results by evaluating various experimental factors, specifically considering the ratio (ug/uL) of the plasmid to Lipofectamine 2000, and the dilution ratio of the plasmid-transfection reagent mixture to Opti-MEM-I. At the same time, transfection efficiency of the protein also varies with the type of cell lines. It is necessary to optimize at least the described parameters above to ensure the optimal transfection efficiency for reproducible best results. Note that transfection efficiency should be determined for each cell line by counting fluorescence cells over the total number of cells. If dual-color imaging and colocalization experiment are desired, a second plasmid expressing a protein of interest with mCherry or mOrange fluorescence protein is added into the plasmid mixture together with PFKL-mEGFP in a total of 50 uL of Opti-MEM-I. In this case, the amount of the second plasmid and the ratio of two plasmids should be optimized for best co-transfection efficiency in addition to the parameters mentioned for single transfection above.

  6. We usually take out cell dishes and perform this step ~20–25 min after Step 4 in Section 3.2.

  7. This step usually takes ~30–60 sec per plasmid-transfection reagent mixture per dish.

  8. Reduction in cell population is anticipated because of the cytotoxicity of the transfection reagent. In addition, degree of such reduction in cell population depends on the cell line of interest.

  9. HeLa cells have been experimentally shown to require at least 1 or 2 hours to be equilibrated at a desired temperature prior to imaging [19]. This step is particularly important if one would like to evaluate the effect of additives on glucosome assemblies from live cells.

  10. Due to the optical diffraction limit of light, the sizes of fluorescent assemblies in single cells cannot be determined if they are smaller than 0.1 μm2. We have thus established 0.1 μm2 as a first threshold to distinguish small sizes of glucosomes from others.

  11. 3 μm2 is then set as a second threshold because, unlike cancer cells, in human non-cancerous breast tissue cells (i.e. Hs578Bst) we have barely observed glucosome assemblies that are larger than 3 μm2 [12]. To emphasize glucosomes that are larger than 3 μm2 as biologically independent entities, the medium size of glucosomes is defined to range from 0.1 to 3 μm2.

  12. We also observe apparent fluorescent aggregates, which are larger than 8 μm2, inside ~5 % of transfected cells. The aggregate formation is determined to be independent of the identity of transfected proteins [7,12,16]. We also confirmed using a biophysical technique, so-called fluorescent recovery after photobleaching, that these aggregates contain only immobile fractions of transfected proteins [12,16]. Therefore, ~5 % of transfected cells in our cell culture conditions appear to suffer from ectopic overexpression.

  13. Although TetraSpeck™ fluorescent microspheres are shown to be not cell-permeable nor cause distress inside cells, we still recommend diluting the fluorescent beads so that at least one bead can be observed within a field of view.

  14. Multiple modes of acquisition are used throughout the alignment and experimental process in the LLSM. The ‘Continuous’ mode is used to quickly observe the plane of illumination and detection and the z-galvo and objective piezo stage are not moving in this mode. When bringing the single Bessel beam into focus or observing the total plane focus of the multibeam pattern it is important not to dither the x-galvo.

  15. When using the ‘Continuous Z-stack’ mode to make the illumination and detection planes orthogonal, the z-galvo mirror is mobile but the detection objective piezo and the x-galvo are stationary.

  16. When using the ‘Continuous Z-stack’ mode to capture the PSF daily, the detection objective piezo and the z-galvo mirror are moving in unison while the x-galvo dithers.

  17. It is recommended that after a thorough alignment, permanent adjustable iris diaphragms are added throughout the laser pathways to reduce time needed for total realignment later. The iris diaphragms are located after all lasers are combined, after the acousto-optic tunable filter, prior to the cylindrical lenses, and after the annular mask.

  18. Additional adjustment using the ‘Autofocus Bead’ mode from the LLSM imaging software is desired throughout the experiment to keep the illumination plane aligned with the detection plane.

  19. For the ‘Autofocus Bead’ and the ‘XZ PSF’ modes, the z-galvo and objective piezo are moving independently and the x-galvo is dithering. The purpose of ‘Autofocus Bead’ is to determine the best offset position for the z-galvo to ensure that the illumination and detection planes are perfectly matched for each other.

  20. The PSF observed at any given day of experiment should be symmetrical. The size of the PSF varies depending on wavelengths, choices of SLM patterns, media conditions, and specific alignments, which are why it must be imaged every day at the time of experiment.

  21. The purpose of the ‘XZ PSF’ mode is to calculate the cross-section of illumination at the sample plane which provides insight into alignment before imaging.

  22. We usually allow cells to equilibrate with 1xBSS imaging solution for ~ 60 min prior to LLSM imaging.

  23. In order to avoid cross-channel bleed-through or crosstalk, LLSM is equipped with a filter wheel station containing laser-specific emission filters prior to the Orca Flash 4.0 camera, and the laser channels were manually selected for illumination.

  24. The ‘SI Scan’ mode holds the z-galvo and objective piezos stationary, dithers the x-galvo, and moves the sample piezo stage through the illumination plane.

  25. Using the beads as a colocalization standard is important because spectrally distinct PSFs present different sizes due to the diffraction limit and chromatic aberration. Such PSF differences result in imperfect colocalization factors, such as the Pearson’s R values obtained with the ‘Coloc 2’ plugin analysis [14].

  26. Glucosomes vary in size and intensity, so intensity thresholds and size filters in the ‘3D Objects Counter’ plugin [18] of the ImageJ software are used to determine the sizes of glucosome assemblies in LLSM imaging.

Acknowledgement

We wish to thank all former and current members who have contributed to the described protocol. We would also like to thank Dr. Gerald M. Wilson and Dr. Jiayuh Lin for sharing MCF-7 and MDA-MB-436 cell lines, respectively. This work is financially supported by the National Institutes of Health: R01GM134086 (M.K.), R01GM125981 (S.A.), R03CA219609 (S.A.), T32GM066706 (E.L.K.), and R25GM55036 (E.L.K.).

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