Advancing Microscopy with Specialized Objectives
- Mouse brain hemisection embedded for Expansion Microscopy (pre-expansion), labeled with secondary antibodies against GFP (Alexa Fluor 488, green), SV2 (Alexa Fluor 565, red) Homer (Alexa Fluor 647, blue). (Sample courtesy of Dr. Ed Boyden and Dr. Fei Chen, MIT)
- Fig. 1: Capturing light from deep inside a tissue sample is challenging due to scattering and absorption.
- Fig. 2: (a) Silicone oil closely matches the refractive index of living cells, leading to better deep-tissue imaging. (Images courtesy of Motokazu Uchigashima M.D., Ph.D., Department of Anatomy, Hokkaido University Graduate School of Medicine).
- Fig. 2: (b) XZ image captured using a silicone oil immersion objective (Olympus UPLSAPO 60XS).
- Fig. 3: Ovule culture device for visualizing plant embryogenesis.
- Fig. 4: Time lapse images of a seed in the ovule culture device. Images captured on Olympus IX microscopy platform using the UPLSAPO 30XS silicone objective (NA = 1.05, WD = 0.8 mm).
- Fig. 5: Methylation in focus. (a) Transgenic mice with and without the methylation label (see online article).
- Fig. 5: Methylation in focus. (b) Olympus’ UPLSAPO 60XS2 silicone objective enabled researchers to study the degree of DNA methylation in 3D with sharp fluorescent images throughout the embryo during the first days of embryonic development.
- Fig. 6: Confocal images of mouse cells inside the placenta captured using a 40x silicone objective (Olympus UPLSAPO 40XS) (Images courtesy of: Asako Sakaue-Sawano and Atsushi Miyawaki, Brain Science Institute.) (a).
- (b) Overlay of Fucci and DAPI images
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In life science microscopy applications – such as 3D imaging of living specimens or long-term live-cell imaging – brightness, resolution and focus stability are key challenges. Silicone oil is an inert, stable immersion medium that helps to maintain focus in long-term imaging applications. It also has a refractive index close to living cells, which minimizes spherical aberrations in 3D – making silicone oil immersion objectives well suited for deep-tissue imaging of live cells.
In deep-tissue imaging, scattering and absorption can lead to low light efficiency at the objectives. In order to produce clear images from deep inside a sample it is essential to use objectives that capture the available light in the most efficient manner.
Researchers at the University of Hokkaido demonstrate this problem in a study on the effect of depth on light capture, where they used an immersion oil to produce XZ images of mouse brain slices. Their results clearly show the drop-off in light intensity when imaging at depths above 30 µm (fig. 1).
A key reason behind low-efficiency light capture is a mismatch in the refractive indices of the media the light needs to travel through. The use of an immersion medium that matches the refractive index of the sample improves efficiency and reduces spherical aberrations leading to brighter, more detailed images at high depth.
In life science imaging, where cells are often the target, silicone oil immersion objectives are well suited for high-depth imaging. Silicone oil reduces the refractive index mismatch with living cells by 60–85% compared to water or conventional immersion oils (fig. 2). Using silicone oil also improves the focus along the Z axis, which results in reduced blurring and minimal spatial distortions in 3D imaging.
In addition to their improved accuracy in capturing the true shape of 3D objects, silicone objectives also provide benefits in long-term imaging applications, such as time lapse studies. In these applications, microscopes need focal stability and should not be affected by temperature changes. Due to its stable physical characteristics and low rate of evaporation, silicone oil enables reliable observation of living cells for days or even weeks without losing focus.
A further benefit over water immersion objectives is their higher numerical aperture.
Several super-resolution and multiphoton applications can also be improved using silicone objectives. Compared to other immersion objectives, these objectives produce an improved point spread function.
The following application examples give an overview of recent, cutting-edge imaging studies that used silicone objectives to reduce aberrations, improve light capture and ensure stable imaging.
Thanks to the combination of stable, high-depth imaging capabilities and a wide field of view, researchers at Nagoya University were able to use a silicone objective to carry out the world’s first long-term observation of plant zygote embryogenesis . Seeing this process in action has been challenging, because the microscope’s light has to travel through multiple layers of cells covering the ovule to reach the objective.
The researchers achieved the imaging of a developing embryo by creating a specially designed ovule culture system (fig. 3). They removed the outer protective cell layers from the seeds and fixed them between the pillars of the culture device. By using the highly stable Olympus IX microscope frame and a silicone objective with a high numerical aperture and working distance and a wide field of view, they were able to follow the process of embryogenesis in real time for 67 hours (fig. 4).
Maintaining focus is an important factor in obtaining high-quality data in time lapse studies. The setup should be able to keep a sample in focus for periods of up to several weeks. In these applications, a steady microscope frame – together with an inert, stable immersion medium – is essential.
At the Department of Genetic Engineering of Kindai University, researchers visualized DNA methylation in embryos by using transgenic mice with a red fluorescent methylation label (fig. 5a) . To study these embryos in 3D, the researchers previously used a combination of an oil lens and a water immersion objective for deep observations. By switching to a silicone objective, they were able to study fluorescently labeled methylated DNA on the surface and in the center of a developing embryo in great detail – from the one-cell stage to the blastocyst stage (fig. 5b).
Meant to Be Together
Another application where the use of silicone objectives can provide a significant improvement is in fluorescence colocalization experiments. In these studies, chromatic aberration – resulting in the shifting of features depending on their wavelength – can have a detrimental effect on results. Silicone objectives have minimal chromatic aberration, enabling precise visualization of fluorophore colocalization.
A study at the Laboratory for Cell Function Dynamics of the Riken Brain Science Institute demonstrated the low chromatic aberration of silicone objectives . In this study, researchers wanted to visualize the different stages of the cell cycle. To achieve this, they labeled placental cells with the Fucci cell cycle indicator, which produces different colors at different stages of the cell cycle.
Figure 6a shows images at different depths of the Fucci indicators as well as the DAPI nuclear stain. Fucci–DAPI colocalization was clearly demonstrated thanks to the low chromatic aberration of silicone objectives, which prevents Z shifts between different wavelengths (fig. 6b).
Silicone oil immersion objectives are designed to suit a wide variety of applications in life science imaging. Due to a refractive index that closely matches living cells, silicone oil is the ideal medium for deep-tissue and live-cell imaging in high resolution without spherical aberrations.
In addition to this, the stability of silicone oil and the low chromatic aberration and wide field of view of silicone objectives also benefit applications such as time lapse studies, and several multiphoton and super-resolution techniques – making silicone objectives a valuable, versatile addition to the life science microscopy toolkit.
 Gooh, K. et al.: Live-Cell Imaging and Optical Manipulation of Arabidopsis Early Embryogenesis, Developmental Cell 34(2): 242-51 (2015)
 Ueda, J. et al.: Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO, Stem cell reports 2(6): 910-924 (2014)
 Sakaue-Sawano, A. et al.: Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle, Molecular Cell 68(3): 626–640 (2017)