Live-cells Details and Dynamics with Structured Illumination Microscopy | SIMTRUM Photonics Store

To demonstrate the SIM Basic optimal performance in following fast biological  events, we performed live imaging on HeLa cells permanently expressing GFP-alpha  tubulin and stained for lysosomes with  Spirochrome SiR Lysosome probe.

In Figure 1 we compared Widefield (WF),  Confocal (CF) and SR images demonstrating  a significant improvement in terms of resolution over traditional microscopy.  Moreover, in Figure 2, a parallel comparison between deconvolved spinning disk and SR data allows appreciating how some details of lysosomal vesicles are detectable, sharper and certainly clearer in SR images respect to deconvolved CF images.

Thanks to the possibility of using the SpinDisk Advance spinning disk together with the SIM Basic SR module, it was possible to perform fast live imaging on a large field of view (FOV) (Figure 3A),  obtaining significant data from a large  number of cells, and then focus on  a  single cell to follow lysosomes dynamics at  subcellular level (Figure 3B).

The SIM Basic high-speed acquisition modality allows the capture of relevant data at high resolution, minimizing light exposure and the resulting risk of phototoxicity. This functionality, as nicely reported in Figure 4, allowed us to explore the dynamics of delicate specimens and trace lysosomal vesicles over time. In particular, a continuous 30 second SIM Basic SR time-lapse was carried out (Figure 4A), monitoring fast events without worrying about bleaching. Image quality and lysosomes motility were preserved over time and this made it possible to extract important data from the tracking of lysosomes such as the distance travelled  (um) and the speed (um/s) of vesicles  (Figure 4B).

Altogether these data demonstrate that the SIM Basic combines high-speed imaging with light efficiency and sensitivity. Fast biological events can be monitored over time at a subcellular level without bleaching problems giving the opportunity to capture relevant data at high resolution also on live specimens.

Figure 2: Parallel comparison between deconvolved CF and SR data showing lysosomal vesicles details; deconvolution of  CF images was performed by 3D Richardson-Lucy algorithm provided by NIS Elements software. These images were acquired with SIMTRUM SpinDisk Advance CF spinning disk system coupled with SIM Basic SR add-on and equipped with CFI Plan Apo Lambda 60X oil objective.

Taking advantage of using the SIMTRUM SpinDisk Advance spinning disk together with the SIM Basic SR module, we had the chance to perform a CF acquisition to have a general overview of the sample (Figure 5A). Once the area of interest was identified, thanks to the fast and easy switch between different imaging modalities, it was possible to spot a dividing cell and follow its entire mitotic  process through SR imaging. As a matter of fact, 33 minutes of live SR time-lapse is reported in Figure 5B showing different mitotic phases with very crisp details.

One of the main drawbacks of the conventional structured illumination microscopy (SIM) is the photon dosage and its  compatibility with delicate biological events;  a lasting exposure to light can compromise a  biological phenomenon in progress and this  can be a problem especially if we consider  the multiple images necessary for SIM image reconstruction.

In this regard, to prove SIM Basic’s ability not to trade-off sample vitality and physiology, we monitored cells for more than 12 hours and, as reported in Figure 6, the health of the cells was not compromised at all, as evidenced by cell divisions and absence of cell death. Notably, image quality is preserved over time, allowing for longer time-lapse experiments without phototoxicity and photobleaching issues.

In conclusion, altogether these data demonstrate that with a temporal resolution greater than 10fps and a low photon burden, the SIM  Basic SR module enables the effortless study of live- cell dynamics and provides structured  localization imaging using routine sample  preparation protocols. Both long-term and fast biological events can be monitored over time without worrying about bleaching, giving the opportunity to trace biological dynamics at the subcellular level and capture meaningful data with  SIM also on live samples.

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Figure 5: Live imaging on HeLa cells stained with Spirochrome SPY 505-DNA (green) and SPY 555-Tubulin (red) probes. (A) CF acquisition on large FOV; (B) single-cell focus showing 33 minutes of live SR time-lapse. These images were acquired with SIMTRUM SpinDisk Advance CF spinning disk system coupled with SIM Basic SR add-on and equipped with CFI Plan Apo Lambda 60X oil objective.

Cells staining was performed with Spirochrome(https://spirochrome.com/) fluorescent probes for live bioimaging (Spirochrome). In particular, we labelled cells with SPY 505-DNA, SPY 555-Tubulin and SiR Lysosome probe.

Movies for lysosomes tracking were recorded for 30 seconds with no delay; movies forcellular division imaging were recorded for 33 minutes (3 minutes delay) (Figure 5B) and for 12 hours and 15 minutes (15 minutes  delay) (Figure 6A).

HeLa cells shown in this application note  were kindly provided by Dr. Lia Asteriti  and Dr. Giulia Guarguaglini, Institute of  Molecular Biology and Pathology, Consiglio  Nazionale delle Ricerche c/o Sapienza  Università di Roma.