Hyperspectral CARS imaging reveals FA-accumulated organelles in stressed cells
To image organelle dynamics in real-time, we designed an integrated multimodal CARS-MPEF microscope as depicted in Fig. 1A. A dual-output laser system was used as the light source for both CARS and MPEF. The fixed-wavelength 1040 nm output was used as the Stokes beam while the wavelength-tunable output was used as the pump beam for CARS. To image lipids in the cells, we tuned the pump beam to 800 nm, corresponding to the Raman shift centered at 2884 cm− 1. For CARS imaging, we first spatially and temporally combined the pump and Stokes beams before chirping the beams using two SF-10 glass rods (each 150 mm long). The combined and chirped beams were guided to a 2D galvo scanning system. A 40X water immersion objective lens (Olympus, LUMPlanFl, NA = 0.8) was used to focus the laser beams onto live cells cultured on glass-bottom dishes. A dish heater was installed to control the temperature at the sample. CARS signals were acquired in the forward direction using a photo-multiplier tube (PMT, Hamamatsu S3994), while MPEF signals were collected in the epi direction using separate PMTs. For MPEF imaging of autofluorescent molecules, femtosecond laser pulses were directly used for two-photon excitation and no glass rods were implemented. Hyperspectral CARS spectra were derived using a spectral-focusing method by tuning the optical delay between the two chirped beams while collecting a single-color CARS image at each delay step25–27.
To better visualize mitochondrial structure and dynamics, we implemented a denoising method (CANDLE-J, ImageJ-based denoising plugin) to improve the quality of the CARS images28. To perform denoising, a stack of 100 time-lapse images was used (see Fig. 1B). The denoised images give better contrast for selecting the best regions of interest for hyperspectral CARS analysis.
MIAPaCa-2 cells were removed from an incubator and exposured to room temperature (24 °C) for about 1 hr prior to imaging the cells with the CARS microscope. We discovered the formation of rod-like structures in the cells, having strong signals in the lipid CH2 vibration (see Fig. 1C, inside the yellow square). A denoised image from the same region gives better contrast to highlight the lipid-accumulated rod-like structures (Fig. 1D). Figures 1E and 1F are magnified original and denoised images from the yellow squares in Figs. 1C and 1D, respectively, in which the bright rod-like organelles are visible (see arrows). The time-lapse images of this region shown in Fig. 1G reveal that these rod-like structures were transported as whole units over time, which differentiates them from aggregates of LDs, which appear as round ‘dots’ in cells and usually undergo rapid contact and dissociation over time. We first fixed the cells and acquired hyperspectral CARS images, after which we compared the CARS spectra in the C-H region for different organelles in the cells. We found that a lipid-rich rod-like organelle (see ROI 1) presents a weaker CH2 stretching CARS signal at 2850 cm− 1 than the LDs, but a stronger CH2 signal than the structure having very similar shape and size (see ROI 2) next to it (see Fig. 1H-J). However, if we compare the CH3 CARS signal at 2940 cm− 1, the two rod-like structures have very similar intensities (Fig. 1J). Since lipids have long acyl chains with abundant CH2 moieties, while proteins have much less CH2 groups but a much higher portion of CH3 groups, our observation indicates the bright rod-like structures accumulated much more lipid molecules compared to other darker rod-like organelles, but still had less lipid composition than the LDs.
Mitochondrion-specific fluorescent-labeling verifies mitochondria structures
To verify the structures of these organelles, we used standard fluorescence labeling. We added a mitochondria-targeted dye solution (MitoTracker® Green, Cell Signaling) into the culture medium of living MIAPaCa-2 cells and maintained them for 1 hr at room temperature (24 °C) before imaging. As shown in Fig. 2A-C, a rod-like organelle having stronger CH2 signals in the CARS channel also showed a strong fluorescence signal from the MitoTracker® Green in the MPEF channel 1 (571±72 nm). This is distinctly different from the LDs, which are dot-like organelles that exhibit a very strong signal in the CARS channel but no signal in the MPEF channel. Figure 2D shows magnified images from the highlighted square areas in the CARS and the CARS/MPEF channels.
We also used immunofluorescence to further confirm the mitochondria structures. We applied the anti-adenosine triphosphate synthase subunit beta (ATPB) antibody to target the ATP synthase in mitochondria. We then applied an Alex Fluor 488 secondary antibody (ab150113, Abcam) to visualize the mitochondria in our MPEF channel 1 (571 ± 72 nm). We compared CARS and MPEF images and selected three locations to compare the CARS spectra. The spectral signatures of LDs, mitochondria with high CH2 signals, and mitochondria with low CH2 signals (see Fig. 2H) show similar relationships as compared to our previously shown results (see Fig. 1J). Here, the CH2 signal differences from the mitochondria are reduced, likely due to the use of detergents which may have washed away some lipid molecules during the sample preparation.
Therefore, we hypothesize that the rod-like structures with strong CH2 signals are mitochondria. Mitochondria are typically protein-rich and generate weaker signals in the CH2 vibrations. The stronger CH2 CARS signals generated by some of these mitochondria indicate the accumulation of FA molecules in these organelles, which is observed for the first time. For MIAPaCa-2 cells, these structures were not observed in the physiological temperature (37 °C) but only detected after the 24 °C hypothermia exposure.
Multiphoton autofluorescence reveals altered NADH metabolism
Mitochondria are key regulators of cellular NADH molecules. We wanted to explore if the fatty-acid-accumulated mitochondria also have abnormal NADH metabolism. NADH molecules are autofluorescent and can be excited by the 800 nm pulses through two-photon absorption and by 1040 nm pulses through three-photon absorption29,30. To ensure sufficient excitation efficiency for the NADH autofluorescence, we used femtosecond laser pulses directly for both MPEF and CARS. We removed the glass rods used for hyperspectral CARS imaging in previous experiments and directly used the laser output at 800 nm and 1040 nm for CARS and MPEF. We found that the lipid-accumulated mitochondria structures show strong signals in both the transmission CARS channel and the epi-direction MPEF channel 2 (451±103 nm) which detects the NADH autofluorescence emission band (Figs. 3A-C). Figures 3D-F show magnified areas in Figs. 3A-C, highlighting a target of interest (yellow arrow).
Figure 3G compares the CARS and NADH MPEF signal intensities of LDs, FA-accumulated mitochondria, and normal mitochondria. We found that FA-accumulated mitochondria have much higher lipid and NADH signals compared to normal mitochondria. Figure 3H plots the lipid (CARS) and NADH (MPEF) signals from the three types of organelles in a 2D space for better visualization and comparison. The lipid and NADH signal intensity differences allow one to distinguish these different types of organelles, especially the FA-accumulated mitochondria, which have very different optical signatures compared to the LDs and normal mitochondria. Figure 3I compares the intensity ratio of NADH MPEF signals and the CARS signals of the three organelles (the ratio has an arbitrary unit), from which we can further distinguish the FA-accumulated mitochondria from other organelles.
Figure 3J depicts FA and NADH metabolic pathways of mitochondria. It is reasonable to believe that hypothermia impacted the electron transport chain, which converts NADH to NAD+, and subsequently induces the accumulation of NADH in the organelles. The increased concentration of NADH possibly slows down the FA β-oxidation since NADH is the net product of this catabolic process, thus resulting in the accumulation of FA in the mitochondria.
We also compared the flavin adenine dinucleotide (FAD) level of the FA-accumulated mitochondria using the MPEF channel 1 (571±72 nm), and also found the accumulation of FAD molecules in these organelles (Figs. S1B and S1E). If we take the intensity ratio of NADH/(NADH + FAD), which is defined as the redox ratio, we find that these FA-accumulated mitochondria tend to have a higher value than other parts of the cell (Figs. S1C and S1F), indicating an abnormal redox metabolism of these organelles.
Degradation of FA-accumulated mitochondria at 37 °C
We found that hypothermia can induce the accumulation of FA and NADH in certain mitochondria of MIAPaCa-2 cells. These changes in organelle metabolism resulted in organelle dysfunction which might be toxic to cells. One of our recent studies showed that the LD dynamics of MIAPaCa-2 cells tend to recover quickly after a short term hypothermia exposure31, suggesting that the cells develop mechanisms to recover from metabolic changes induced by hypothermia exposure. Therefore, it is reasonable to believe that cells can remove and degrade these dysfunctional mitochondria after being heated back to 37 °C.
To observe the degradation process, we first created a hypothermia condition, allowing FA-accumulated mitochondria to form, as shown in Fig. 4A. In a MIAPaCa2 cell, we first identified 3 FA-accumulated mitochondria (see red, blue, and yellow arrows) and set this time as time 0. We found that these mitochondria underwent fission and fusion (156.2 s -200.2 s), which is a common behavior when mitochondria are under stress conditions32–34. Later, at 220 s, the fourth FA-accumulated mitochondrion (see green arrows) appeared. At time 340 s, we reheated the sample back to 37 °C. Starting from about 619.4 s, slightly less than 300 s after the reheating started, the rod-like organelles started to form into dot-like structures (Fig. 4B-C, the blue arrow first, the red arrow second, the yellow arrow next, and the green arrow last). Eventually, all the 4 identified FA-rich mitochondria were converted to bright dot-like structures in the CARS images (Fig. 4D). This process is likely the mitophagy process during which mitochondria are wrapped by autophagosomes and eventually degraded by the cells35–37. Videos of FA-rich mitochondria changes can be found in the Supplementary Information. These stress-induced organelles are rich in lipid and are likely recycled by the cell.