structured illumination microscopy

Breaking the diffraction limit: discovering cellular organelles with structured illumination microscopy

An organelle is a subcellular structure that contributes to a variety of cellular functions through its molecular composition and environmental interactions.
Standard fluorescence microscopy techniques were traditionally used for organelle studies and focused on identifying the unique characteristics of individual compartments. However, the finer structures of organelles, as well as many key sub-cellular details, are smaller than 200 nm and therefore cannot be sufficiently characterized by diffraction-limited conventional microscopy.

Advances in imaging technologies have enabled simultaneous studies of multiple organelles’ structure and dynamics, as well as their movements, interaction, and reshaping. In this context, the development of super-resolution (SR) microscopy techniques represents a key driver to exceeding the diffraction limit of light and enables the investigation of cellular structures and organelles at the nanometer scale.

Through the use of a multi-spot structured illumination system, DeepSIM provides reliable, easy-to-use, and affordable solutions to study sub-cellular structures with an XY resolution of 100 nm without requiring any special sample preparation protocol. In this Application Note, we demonstrate that with the DeepSIM it is possible to follow live organelles dynamics at a cellular and sub-cellular level. In particular, we focused on different intracellular structures ranging from endosomes, actin, mitochondria, tubulin, endoplasmic reticulum (ER), and lysosomes discovering subcellular details under the diffraction limit of light.

SR live imaging follows cytoskeletal and endosomal dynamics

Endosomes are subcellular organelles where internalized cargoes are organized and sorted toward different trafficking pathways; their content recycling is orchestrated by multimolecular complexes promoting new actin filaments polymerization.
The study of actin dynamics in the regulation of endosomal recycling, and the interaction between these two cellular components, can undergo considerable improvement thanks to SR techniques that allow studying the intracellular structures at the nanometer scale.

To demonstrate the DeepSIM optimal performance in following these fast biological events, we performed live imaging of cells permanently expressing cytoskeletal (actin) and endosomal markers.

In Figure 1 we compared Widefield (WF), Confocal (CF) and SR images demonstrating a significant resolution increment between the three imaging modalities.

Figure 1: Comparison of WF, CF spinning disk and DeepSIM structured illumination microscopy SR images of cells permanently expressing actin (green) and endosomal (cyan) markers. These images were acquired with CrestOptics X-Light V3 CF spinning disk system coupled with DeepSIM X-Light SR system and equipped with CFI SR Plan Apo IR 60X water immersion objective.

Moreover, thanks to the DeepSIM high-speed acquisition modality, we captured relevant data in a continuous 30-second SR time-lapse. As represented in Figure 2, we were able to monitor fast events in SR without worrying about bleaching and demonstrating how image quality and biological dynamics were preserved over time.

Figure 2: Movie showing 30 seconds fast live imaging of cells stably expressing actin (green) and endosomal (cyan) markers. This time-lapse was acquired with a DeepSIM X-Light SR system equipped with CFI SR Plan Apo IR 60X water immersion objective.

SR live imaging reveals mitochondrial meshwork details.

The mitochondria are the cellular powerhouses of most eukaryotes. They are composed of an intricated network of tubules that are tightly integrated with other cellular compartments.

The size of most mitochondrial structures is just at the resolution limit of optical microscopy making their analysis always challenging using diffraction-limited optical microscopes. In this context, SR microscopy provides additional insight into mitochondrial morphology, distribution, and inter-mitochondrial contacts.

As evidence of that, in Figure 3, we compared WF, CF, and SR images demonstrating a significant resolution increment between the three imaging modalities, especially if we focus on an intricately interconnected network of tubules (Figure 3B).

Figure 3: Comparison between WF, CF spinning disk, and DeepSIM SR images of cells permanently expressing a mitochondrial marker. The entire network of a cell (A) and the zoom of mitochondrial details (B) are shown. These images were acquired with CrestOptics X-Light V3 CF spinning disk system coupled with DeepSIM X-Light SR system and equipped with a CFI Plan Apo Lambda 100X oil immersion objective.

By removing out-of-focus and scattered light, CF imaging can provide excellent quality images respect to WF modality; however, to observe biological details below the refractive index, a SR technique is required. The improvement in image quality provided by DeepSIM was compared to the one achieved when applying deconvolution to CF images, as it has been shown to improve mitochondrial structure detection (Figure 4).

Although deconvolution on CF acquisition increases image contrast, the improvement in resolution is limited compared to that achievable using structured illumination microscopy (SIM) (Figure 4A). As demonstrated by the analysis of the intensity profile (Figure 4B), subtle inter-mitochondrial contacts (160 nm) are only effectively distinguishable through SR microscopy (green line profile), which allows viewing of mitochondrial meshwork structural details.

B

Figure 4: Parallel comparison between deconvolved CF and SR data showing mitochondrial structures. The inter-mitochondrial contacts (A) and the related intensity profiles (B) are shown (the red line and the green line represent the intensity profiles of the deconvolved CF and DeepSIM respectively). Deconvolution of CF images was performed by 3D Richardson-Lucy algorithm provided by NIS Elements software. These images were acquired with CrestOptics X-Light V3 CF spinning disk system coupled with DeepSIM X-Light  SR system and equipped with a CFI Plan Apo Lambda 100X oil immersion objective.

Mitochondria are highly dynamic organelles undergoing fission and fusion cycles in order to maintain their shape, distribution, and size. After demonstrating the need for a gain in terms of resolution for the study of inter- and intra-mitochondrial structures, we leveraged the DeepSIM structured illumination microscopy high-speed acquisition modality for the study of these mitochondrial dynamics.

A

B

Figure 5: 2D (A) and 3D (B) movies showing 30 seconds fast live imaging of cells stably expressing a mitochondrial marker. These time-lapses were acquired with the DeepSIM stand-alone SR system equipped with CFI SR Plan Apo IR 60X water immersion objective.

For this purpose, in Figure 5 we reported 30 seconds of continuous 2D (Figure 5A) and 3D (Figure 5B) time-lapse to follow mitochondrial movements at the subcellular level. The use of structured illumination microscopy (SIM) enhances contrast and improves the resolution of mitochondrial structures thus enabling the correct visualization of biological details otherwise invisible with standard microscopy methodologies even after applying deconvolution algorithms.

SR live imaging enhances details and follows the main intracellular organelles’ dynamics.

The ER is the biggest organelle and it extends throughout the cytoplasm coming into contact with the other types of intracellular structures. On the other hand, lysosomes are small subcellular vesicles containing hydrolytic enzymes responsible for the digestion of macromolecules such as nucleic acids, proteins, and polysaccharides. Both organelles are among the most dynamic within the cell and many scientists are interested in tracking these structures while maintaining a high resolution to distinguish individual vesicles thus increasing the accuracy of their results.

A

B

Figure 6: Fast live imaging of cells stained for lysosomes (A) and ER (B). These time-lapses were acquired with DeepSIM X-Light SR system equipped with CFI SR Plan Apo IR 60X water immersion objective.

Thanks to its temporal resolution of 12 fps, the DeepSIM allows for the capture of meaningful data at high resolution while minimizing light exposure and photo-toxicity risk. Delicate and live specimens can be explored in real-time monitoring cellular and subcellular changes. This functionality, as nicely reported in Figure 6, allowed exploring lysosomal vesicles (Figure 6A) and ER movements (Figure 6B) demonstrating how fast biological events can be monitored over time with an impressive resolution.

Conclusions

Altogether these data demonstrate that DeepSIM combines SR with high-speed imaging, light efficiency, and sensitivity. Imaging membranes in live cells with SR techniques reveals structural dynamics which represent the crucial point for understanding cellular functions. However, many of these intracellular movements occur very rapidly and it is not easy to follow such dynamic events, especially if we consider that SR techniques require the acquisition of a set of raw images necessary for the creation of one super-resolved image. Therefore, it is very important to have an instrument capable of acquiring with a high frame rate, without damaging the sample or blocking the ongoing biological event.

In this context, because of its imaging speed and resolution power, the DeepSIM enables the effortless study of live-cell dynamics giving the opportunity to trace fast biological events at the subcellular level.

Acknowledgments

The data shown in figures 1, 2, 4, and 5 were acquired during the Imaging Bootcamp 2023 held at Hokkaido University. Thanks to Dr. Fujioka (Hokkaido University) for the invitation and Yasutomo Kubota (Givetechs) for installation support.

Beautiful dynamic lysosomes shown in figure 6A have stained with a Spirochrome SiR Lysosome probe; thanks to Spirochrome for this excellent fluorescent probe.

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