Fluorescence Lifetime-Assisted Probing of Protein Aggregation with sub-Organellar Resolution

Materials and reagents

Biological materials

1. Chinese hamster ovary cells (CHO-K1, ATCC CCL-61) with stable expression of HT-aggr^ER (HaloTag with M21K F86L mutations)

   
   

1. Nutrient mixture F12 Ham [Sodium bicarbonate (+), L-Glutamine (-)] (Sigma Merck, catalog number: N4888-500ML)

2. Fetal bovine serum (FBS) (Sigma, catalog number: F9665-500ML)

3. Penicillin-streptomycin (Pen/strep) (Thermo Scientific, catalog number: 15140122)

4. L-Glutamine (Thermo Scientific, catalog number: 25030024)

5. P1 solvatochromic fluorophore (prepared in-house—protocol described in General Notes and Supplementary File 1)

6. 1× Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190169)

7. 0.05% Trypsin-EDTA (1×) (Thermo Fisher, catalog number: 25200056)

8. Janelia Fluor 646 HaloTag ligand (Promega, catalog number: GA1120) (see General Notes for further discussion of the use of commercially available HaloTag ligands to label HT-aggr)

   
   

1. Complete nutrient mixture F12 Ham (see Recipes)

2. P1 staining solution (see Recipes)

3. Janelia Fluor 646 HaloTag staining solution (see Recipes)

   
   

1. Complete nutrient mixture F12 Ham

   Reagent	Stock concentration	Final concentration	Amount
   Nutrient mixture F12 Ham	100×	90%	500 mL
   FBS	100×	10%	50 mL
   Pen/strep	10,000 U/mL	1%	5.5 mL
   L-Glutamine	200 mM	1%	5.5 mL

   
   

2. P1 staining solution

   Reagent	Final concentration	Amount
   Complete nutrient mixture F12 Ham	NA	500 μL
   P1 (2 mM)	4 μM	1 μL

   
   

3. Janelia Fluor 646 HaloTag staining solution

   Reagent	Final concentration	Amount
   Complete nutrient mixture F12 Ham	NA	500 μL
   Janelia Fluor 646 HaloTag (200 mM)	200 μM	0.5 μL

   
   

Laboratory supplies

1. Coverslip bottomed dishes, IBIDI (IBL Labor GmbH, catalog number: D35C4-20-1-N)

2. 10 cm dish (Falcon, catalog number: 353003)

3. Centrifuge tube, 15 mL (Appleton Woods Ltd, catalog number: AB031)

Equipment

1. Leica Stellaris 8 FALCON FLIM Microscope (Leica, Wetzlar, Germany, model: STELLARIS8)

2. Benchtop centrifuge (Eppendorf, model: 5810 R, product code: 12813252)

Software and datasets

1. Leica LAS X (v5.2.2, 01.02.2024)

2. Icy (v2.5.2.0, 08.02.2024)

Procedure

1. Preparation of CHO HT-aggr^ER cells for 3D-FLIM imaging

   1. Grow cells in complete nutrient mixture F12 media (see Recipe 1) in a cell culture incubator at 37 °C and 5% CO₂ until cells reach 70%–80% confluency in a 10 cm dish.

   2. Aspirate complete nutrient mixture F12 from cells, wash once with 1× DPBS, add 1 mL of Trypsin-EDTA, and return cells to incubator for ~3 min.

   3. Once detached, add 3 mL of complete nutrient mixture F12 media to cells and pipette up and down 10 times to make a single-cell suspension.

   4. Count cells using a hemacytometer or automated cell counter.

   5. Transfer cells to a 15 mL falcon tube and centrifuge at 200× g for 5 min.

   6. Aspirate supernatant and resuspend cells with complete media at 1×10⁶ cells/mL.

   7. Seed cells at 3,000 cells/cm² in a coverslip glass-bottomed cell culture vessel.

   8. Return to incubator and grow to ~70% confluency.

      
      

2. Pulse labeling of HT-aggr^ER with P1 solvatochromic fluorophore

   1. Dilute P1 stock to 4 μM final concentration in prewarmed complete nutrient mixture F12 media (see Recipe 2).

   2. Take cells grown from Section A and replace media with P1 staining solution.

   3. Return cells to the incubator for 45 min.

   4. Remove P1 staining solution and wash with prewarmed complete media.

   5. Replace with prewarmed complete nutrient mixture F12 media.

   6. Now that HT-agg^ER has been pulse-labeled, the cells can be subjected to a treatment of choice prior to imaging to observe the effect of the treatment on protein aggregates over time. For example, we have observed that mild ER stress induced by tunicamycin or thapsigargin results in a decrease in long-lifetime aggregates [8].

      
      

3. Counter-labeling with 646 HaloTag ligand to visualize ER

   1. Directly before imaging, newly synthesized HT-aggr^ER can be pulse-labeled with Janelia Fluor 646 HaloTag ligand (see Recipe 3) (this only labels non-aggregated HT and thus serves as a counterstain for the endoplasmic reticulum).

   2. Stain cells with Janelia Fluor 646 HaloTag staining solution for 45 min prior to imaging.

   3. Remove Janelia Fluor 646 HaloTag staining solution and wash twice with prewarmed 1× DPBS.

   4. Add 500 μL of fresh complete nutrient mixture F12 media to the well and subject to imaging. We have not observed any difference in fluorescence lifetime of our probe in media with or without phenol red or serum.

      
      

4. FLIM live cell imaging of P1-label

   1. Warm the microscope stage to 37 °C and set CO₂ level to 5%.

   2. Place the cell sample on the microscope stage.

   3. Locate and focus on cells through eyepieces using brightfield illumination to minimize bleaching of P1 fluorophore.

   4. Set microscope parameters in LAS X software as follows:

      1. FLIM mode

      2. Laser 1: Excitation/emission 440/450–500 nm, pulsed laser (see notes; for P1 imaging, faster laser repetition rate may result in apparent shortening of fluorescence lifetime)

      3. Laser 2: Excitation/emission 646/655–750 nm (for 646 HaloTag ligand imaging, follow steps C1–4)

      4. HyDx detector in counting mode

      5. Frame size 512 × 512 pixels

      6. Scan speed 400 Hz

      7. Count frame average of 15–20

      8. Set Z-stack for the cell (begin and end)

   5. Start image acquisition (Start Experiment button).

      
      

5. Confocal live imaging in the LIGHTNING mode of 646 HaloTag ligand

   1. Immediately after FLIM imaging of cells, deselect FLIM mode and select LIGHTNING mode of LAS X software.

   2. Do not change the Z-stack or frame-size settings acquired in FLIM mode.

   3. Start image acquisition (Start experiment button).

      
      

6. Image rendering

   1. Raw FLIM image rendering (Figure 1) produces a map of mean arrival time for each pixel of the acquired Fast FLIM image. The values are color-coded to distinguish protein aggregates with longer lifetime.

      1. Select FLIM mode.

      2. Set the lifetime range (2.5–6 ns). In this range, pixels with a lifetime of >4.5 ns will appear in yellow/red, allowing aggregates to be distinguished as red puncta from green-folded probe species. We also observe concentrated areas of shorter lifetime (blue) signal. Further work is required to understand the biological relevance of these regions. Since their lifetime does not correspond to that of aggregated HT-aggr such as that induced by heat shock [8], we do not consider these as aggregates when we perform image analysis.

      3. Export image in TIFF or PNG format (ensure “Save Palette Image as RGB” is selected to maintain the defined FLIM lookup table in the saved image).

      4. Export in the format of 3D-movie.

         
         

      
      Figure 1. Illustration of Raw 3D FLIM image. A. Settings for acquiring FLIM image using Leica Stellaris 8 confocal. B. Micrograph of 2D plane of CHO M21K F86L cells acquired using settings as in A. C. Micrograph of 3D FLIM image of cell as in B. White arrows point toward aggregates. Images are at 63× magnification and were acquired using Leica Stellaris 8 FALCON. Scale bar: 20 µm. Lifetime range: 2.5–6.0.

      
      

   2. Aggregate size analysis of FLIM data in Icy Software (Figure 2)

      1.  Open the exported TIFF or PNG FLIM image files in Icy Software (v2.5.2) [12].

      2.  Select Build RGB Image.

      3. Deselect Ch1 and Ch2 (green and blue) channels to leave only red pixels, reflecting regions of fluorescence lifetime >4.5 ns.

      4. Open the Aggregates Detector protocol.

      5. Adjust the Threshold and Min volume settings to identify aggregates of the desired intensity and size. We typically use threshold = 125 and min volume = 5 to avoid quantification of diffuse signal.

      6. Run Aggregates Detector and then navigate to the ROI tab on the right-hand side of the viewer. There will be a list of ROIs corresponding to aggregates with corresponding shape descriptors such as area. This table can be exported to Excel for subsequent analysis of shape descriptor metrics (Figure 2D).

         
         

      
      Figure 2. Schematic representation of calculating frequency distribution of aggregates’ sizes extracted from FLIM using Icy software. A. 2D FLIM image is imported into Icy and converted to RGB image. B. Green and blue channels are deselected to leave only red pixels corresponding to longer lifetime areas. C. Aggregates Detector plugin is utilized to identify and extract shape descriptors from long-lifetime aggregates. D. Representative graph of aggregate size in correspondence to its relative frequency.

      
      

   3. 3D FLIM image rendering (Figure 3). This produces a 3D image of the FLIM map overlayed with the high-resolution ER counter image. This should be used for visual purposes only and not for quantitative analysis of aggregates.

      1. Select 3D tool on LASX software.

      2. Open 3D FLIM and corresponding LIGHTNING confocal images.

      3. Under the Processing tab, select the concatenate function.

      4. Select both 3D FLIM and LIGHTNING images to concatenate by channel.

      5. Apply concatenation.

      6. Deselect blue and green channels.

      7. Set minimum intensity threshold to 40.

      8. Using movie editor, export image in movie mode (Video 1).

         
         
   
   Figure 3. Processed high-resolution 3D FLIM image. A. Micrographs of FLIM and ER acquired using FLIM mode and LIGHTNING confocal mode, respectively. B. 3D overlay of micrograph of FLIM (red/green) and ER (grey) image. Red spots represent aggregated probes. Images are at 63× magnification and were acquired using Leica Stellaris 8 FALCON. Scale bar: 20 μm. Lifetime range: 2.5–6.0 ns.

   
   

   
   Video 1. 3D fast-FLIM image of P1-labeled HT-aggr^ER aggregates acquired by FLIM (red), overlayed with HaloTag ligand 646-labeled newly synthesized HT-aggr^ER acquired by confocal (grey). Red spots represent longer lifetime aggregates.

Validation of protocol

This protocol has been used and validated in the following research article [8]:

1. Melo et al. (2022). Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP. Nat Commun (Figure 2, panel e).

General notes and troubleshooting

1. Here, the protocol has been demonstrated on the CHO-K1 cell line harboring stable integration of the HT-aggr^ER construct with M21K F86L mutations. This cell line and other mutant cell lines used in Melo et al. [8] are available upon request.

2. Alternatively, the plasmids used in Melo et al. [8] and this protocol are available via Addgene. We recommend using stable cell lines harboring the plasmids, as the formation of protein aggregates occurs several days after expression and thus cannot be visualized transiently.

3. P1 solvatochromic HaloTag ligand was synthesized by Dr. Ana Costa at the University of Algarve, Portugal, and is available upon request to Dr. Edward Avezov. A full methodology for P1 synthesis can be found in Supplementary File 1.

4. Other HaloTag ligands can be used to label HT-aggr^ER but behave differently from P1 in terms of binding propensity and fluorogenicity to aggregates. For example, Janelia Fluor HaloTag TMR and 646 do not label the aggregates we observe with long fluorescence lifetime as measured by P1 labeling. However, diffuse HT-aggr and other short-lifetime puncta are labeled by these ligands.

5. Overnight P1 labeling can also be performed.

6. Before staining cells with HaloTag ligand 646, wash away P1 stain and allow cells to grow in fresh nutrient mixture F12 medium for a minimum of 4 h. This is to allow sufficient time for new HT-aggr^ER to be synthesized after washing excess P1 label away, which will be available to covalently bind to HaloTag ligand 646 and thus act as a counterstain for the ER.

7. Fluorescence lifetime is dependent on the frequency of the laser used. For the original research article in which the protocol was validated, Zeiss LSM710 with 20 MHz laser was used for 2D FLIM. For this Bio-protocol, Leica Stellaris 8 FALCON FLIM microscope with 80 MHz laser was used to acquire 3D FLIM images. The differing laser frequencies have an impact on the observed fluorescence lifetime of the P1-labeled probe.

8. 3D FLIM is enabled by improved imaging speeds of Fast-FLIM, made possible by reduced detector dead time, which allows for ~80 mega counts per second photon detection speeds. Other microscope systems capable of TCSPC FLIM with comparable detection speed, such as Becker & Hickl FASTAC FLIM or picoQuant MultiHarp 150, should thus be able to reproduce our approach when combined with a sub-diffraction limit resolution confocal system such as Airyscan or stimulated emission depletion (STED).

Acknowledgments

E.A. in this work is supported by the UK Dementia Research Institute through UK DRI Ltd, principally funded by the Medical Research Council, The Evelyn Trust and Alzheimer’s Society. K.G. is supported by Cambridge Indian Ramanujan Scholarship. E.P.M. received Portuguese national funds from FCT - Foundation for Science and Technology through projects UIDB/04326/2020 (DOI:10.54499/UIDB/04326/2020), UIDP/04326/2020 (DOI:10.54499/UIDP/04326/2020) and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020). This protocol is adapted from Melo et. al [8].

Competing interests

The authors declare no competing interests.

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Supplementary information

The following supporting information can be downloaded here:

1. Supplementary File 1