CLEM for Life Science

What is CLEM

CLEM（Correlative Light and Electron Microscopy）is a method for effective observation and analysis by integrating information of the same specimen obtained by a light microscope(LM) and transmission electron microscope(TEM) or scanning electron microscope(SEM). The advantages of both can be utilized by doing this for observation and analysis.

Please click here and check each method of CLEM for Life Science.

polish CLEM / milling CLEM

This is a method to observe the same field of view as an LM image (optical image) by using a scanning electron microscope by exposing it to the surface by polishing or ion milling, etc. It is possible to observe the entire image or fluorescent staining image of cells by using LM, and then observe the detailed structure inside the cell using SEM. The capability of simple and extensive observation is the feature. The use of a specialized linkage holder makes it easy to align the position.

Polish CLEM / milling CLEM workflow

Cells are cultured on a dish with a marker and pre-fixed and stained . After an LM observation, the preparation sample is attached to the holder and the coordinates are confirmed. The observation surface is exposed by polishing and ion milling, and observed by using an SEM.

on chip CLEM

This is a method to observe an ultra-thin specimen on the same specimen mount (SiN window chip) by using both a light microscope (LM) and transmission electron microscope (TEM). Because there is no process to cause a deformation of a specimen between observations by LM and TEM, highly accurate overlaying is possible. This method can be also used for the material field.

on chip CLEM workflow

The on chip CLEM is to put the ultra-thin section specimen prepared for TEM on a SiN window chip. The SiN window chip can be attached to the holders for the light microscope, and TEM by using a specialized retainer. By switching the use of a specialized retainer to the LM holder and TEM holder, LM・TEM observation can be easily performed.
Since the same sections can be observed, highly accurate correlations can be obtained.

LLP-CLEM

Limitless Panorama (LLP) has a function to import images taken externally, such as with a light microscope. LLP can match the area of observation of LLP with the specimen coordinates of a transmission electron microscope. After matching the coordinates of each observation image, a montage pictures of the area that was observed by LM, are taken by using the TEM. A wide range can be observed with high resolution.

LLP-CLEM Workflow

Cells cultured on a dish with markers are pre-fixed and stained. After the LM imaging, the target area of the specimen after post-fixation and resin-embedding is cut out using a marker as a landmark. An ultra-thin section of the specimen is prepared by using ultramicrotome and observed by using the TEM.
Alignment is performed by specifying three or more feature points in common between the LM image that was taken and the TEM image of the low magnification image. Specify an area and perform LLP imaging.

In resin CLEM

In resin CLEM is a technique to prepare an ultra-thin section after fluorescent staining and resin-embedding of the specimen, revive the fluorescence, and observe the same section by using a light microscope and electron microscopes (TEM/SEM). To revive the fluorescence, a fluorescence reviver (TUK solution) is used. The same section can be observed with both light and electron microscopes, allowing highly accurate superposition of optical and electron microscopic images.

In resin CLEM workflow

An ultra-thin section is mounted on the SiN window chip (for TEM) or Si wafer or slide glass (for SEM) that has a smooth surface. Fluorescent reviver (TUK solution) is applied and observed by a light microscope, followed by an electron microscope.

Multi-color CLEM

Various modifications are possible with organic silica nano particles, such as mixing fluorescent dyes or adding heavy metals. Organic silica nano particles with fluorescent dyes and heavy metal are taken into cells. The organic silica nano particles with several kinds of heavy metals can be distinguished by the heavy metal added through EDS mapping. Thus, it is possible to observe it in multiple colors not only by light microscopes, but also by electron microscopes. It is also possible to use this as a marking when the area observed by a fluorescent microscope is observed by an electron microscope in detail.

Multi-color CLEM workflow

Organic silica nano particles are modified such as "green fluorescence + gold particle", and "red fluorescence + iron particle". This is introduced to the cultured cell and then confirmation of the same area is possible by using fluorescence observation and EDS mapping.

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