Structured Illumination Microscopy with Low Magnification Objectives | SIMTRUM Photonics Store

In Figure 1, different visualizations of a cleared mouse intestine section (0.55 mm thickness) with blood vessels in green and nuclei in red are shown. In particular, thanks to the fast switch between three microscopy methods, we report a global comparison of WF, CF spinning disk and SR acquisitions of a volume of 125 um (Figure 1A) and a 3D movie of the SIM Basic SR acquisition showing fine details of intestinal villi (Figure 1B).

In Figure 2, a cleared mouse brain section (0.55 mm thickness) with GFP-expressing neurons is shown. Notably, we acquired a 150 um Z stack of an intricate neuronal network shown as maximum intensity projection  (MIP) (Figure 2A) and 3D volume view (Figure  2B).

As nicely appreciated from these images, switching from WF, CF and SR modalities allows reaching an increasing resolution  gain that enhances the fine details of the  neuronal network even at high thickness.

Moreover, we decide to compare the 20X SR  acquisition with a 60X CF acquisition and,  as demonstrated in figure 2C and 2D, the  acquisitions with the SIM Basic not only allow  to have an evident resolution improvement  compared to the  20X CF  acquisitions,  but it enables to reach an image quality comparable to those obtained with the CF  spinning disk equipped with a CFI Plan Apo  Lambda 60X oil objective (NA 1.4, WD 0.13).  In addition to a noticeable enhancement in neuronal details quality with respect to a 20X CF (Figure 2D), the use of the SIM Basic with 20X objective allows going even deeper into the sample than what is permitted by the maximum working distance of a common  60X oil objective (130 um) (Figure 2C).

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Figure 2: Cleared mouse brain section with GFP-expressing neurons. (A) Comparison of WF, CF spinning disk and SIM Basic SR acquisition acquired with 20X air objective and shown as maximum intensity projection from Z stack; (B) 3D volume views acquired with 20X air objective, 150 um thickness. (C) Comparison between 3D volume views acquired with SIM Basic SR module equipped with 20X air objective (left) and CF spinning disk equipped with 60X oil objective (right). (D) Focus on neuronal details and comparison between three different acquisition modalities: CF spinning disk equipped with 20X air objective (left), CF spinning disk equipped with 60X oil objective (middle) and SIM Basic SR module with 20X air objective (right).

Super-Resolution imaging of conventionally prepared samples at low magnification

Therefore, we have tested some conventionally prepared samples, such as brain organoids and mouse brain sections, making acquisitions that are usually done with a confocal microscope.

Human whole-brain organoids are able to recapitulate the very first neurodevelopmental events in vitro, both in terms of cellular interactions and tissue architecture, mimicking the real 3D organization of the developing human brain.  In Figure 3, a human brain organoid section (50 um thickness) showing CTIP2-positive deep layer cortical neurons (green) and pan-neuronal MAP2 marker (red) is represented.  In particular, we focused on a cortical plate performing a comparison between WF,  CF spinning disk and SIM Basic SR images  acquired with 20X air objective and shown as  MIP from Z-stack (15 um). Again, switching between the three acquisition modalities, we obtained a notable increase in resolution and image quality with the SIM Basic, enhancing  fine cellular structures of cortical deep-layer  neurons that, differentiating towards the  periphery, shape the typical laminar cortical  structure.

In Figure 4, we report images of another conventionally prepared sample: a hippocampal coronal slice from Thy1- GFP mouse brain (50um thickness) and, analyzing the acquisitions made with 20X air objective (Figure 4A), the resolution gain obtained with the SIM Basic is evident, also  in this very scattering sample. Furthermore, focusing on neuronal details inside the intricate organization of the hippocampal neuronal network, we compared the 20X SR acquisition with a 60X CF image. As nicely reported in figure 4B, the SIM Basic equipped with the 20x air lens allows reaching an image quality comparable to  those obtained with the CF spinning disk  equipped with the 60X oil objective.

In conclusion, all these data taken together demonstrate the capability of SIM Basic to bring out fine details in extra-large objects using routine preparation protocols.  Affordable easy-to-use low magnification dry objectives can also be used to acquire both conventional and non-conventional specimens, doubling the objective performance and obtaining an image quality very similar to that reached with  immersion lenses.

The use of the SIM Basic with low magnification objectives allows benefiting all the advantages of an air lens, like the absence of immersion media or a greater working distance, without compromising the quality  of the image. This type of configuration greatly increases the plasticity of the system and gives our customers more flexibility in applications, applying the SIM Basic as  a powerful platform for SR imaging and  laying the foundations for the use of SR in  high-content screening.

Figure 3: Human brain organoids showing CTIP2-positive deep layer cortical neurons (green) and pan-neuronal MAP2 marker (red). Comparison of WF, CF spinning disk and SIM Basic SR images acquired with 20X air objective and shown as maximum intensity projection from Z stack.

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Figure 4: Hippocampal coronal slice from Thy1-GFP mouse brain. (A) Comparison of WF, CF spinning disk and SIM Basic SR images acquired with 20X air objective and shown as maximum intensity projection from Z stack. (B) Focus on neuronal details and comparison between three different acquisition modalities: CF spinning disk equipped with 20X air objective (left), CF spinning disk equipped with 60X oil objective (middle) and SIM Basic SR module with 20X air objective (right).

All the acquisitions of this Application Note had performed through a Nikon Eclipse Ti2 microscope equipped with SIMTRUM SpinDisk Advance spinning disk system coupled with SIM Basic super-resolution add on, LDI laser illumination (89 North) and Prime BSI Scientific CMOS (sCMOS) camera with 6.5 um pixels (Photometrics). We used a CFI Plan Apo Lambda 20X air objective (NA 0.75, WD 1) and a CFI Plan Apo Lambda 60X oil objective (NA 1.4, WD 0.13).

Human cerebral organoids shown in Figure 3 and mouse brain sections represented in Figure 4 were kindly provided by Prof.  Silvia Di Angelantonio, Dr Maria Rosito,  Dr Federica Cordella and Dr Caterina  Sanchini, Center for Life Nano- & Neuro-  Science (CLN2S@Sapienza Università di  Roma – Istituto Italiano di Tecnologia).