Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

Wouters, C. H.

doi:10.1111/j.1365-2818.1987.tb02814.x

Cited by 8 publications

(3 citation statements)

References 33 publications

(56 reference statements)

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“…In a system built by Wouters et al . in the 1980s (Wouters & Koerten, ; Wouters et al ., ; Wouters, ), the light microscope was integrated in a scanning electron microscope (SEM) under an angle of 45° with respect to the SEM optical axis. An illumination lens for transmission LM was added at the opposite side of the SEM objective lens.…”

Section: Introductionmentioning

confidence: 99%

“…This combination is called correlative light and electron microscopy (CLEM). CLEM thus closes the gap between LM and EM by giving complementary functional and structural information from the same region of interest (ROI; Webster et al, 1978;Wouters, 1987;Giepmans, 2008;Caplan et al, 2011;Lucas et al, 2012). In recent years, CLEM has received increasing interest from the scientific community (Agronskaia et al, 2008;Kanemaru et al, 2009;Pluk et al, 2009;Spiegelhalter et al, 2010;Kukulski et al, 2011;Murphy et al, 2011;Watanabe et al, 2011;Jahn et al, 2012;Kopek et al, 2012;Maruyama et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Integration of a high‐NA light microscope in a scanning electron microscope

Zonnevylle

Tol

Liv

et al. 2013

Journal of Microscopy

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Summary We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Integration of a high‐NA light microscope in a scanning electron microscope

Zonnevylle

Tol

Liv

et al. 2013

Journal of Microscopy

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“…To settle this problem nearly perfectly, the VM has to be successfully incorporated into the SEM specimen chamber. Techniques for combining light microscopy and SEM have been studied (see review by Wouters 1987). Two projects progress in our laboratory to acquire the identical view image.…”

Section: Experiments For Confirming Usefulness Of the Frequency Characmentioning

confidence: 99%

Natural color scanning electron microscopy based on the frequency characteristics of the human visual system

Oho

Watanabe

2001

Scanning

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Summary:The principles of image formation in natural color scanning electron microscopy (NC-SEM) are discussed in detail. This method is based on the frequency characteristic of the human visual system. It is shown that the Mach effect and masking effect are important in the characteristics. The former, which can enhance structural details, is visually similar to the edge effect in secondary electron (SE) images, and the latter is required for proper representation of very degraded color information obtained from a light microscope. When using these effects suitably, an NC-SEM image with the resolution equivalent to that of an SEM image can be acquired, though it is composed of an SEM image and a special video microscopy (VM) image with a resolution much lower than the SEM image of the identical view. The NC-SEM is more effective than the SEM in observation, interpretation, and analysis of various samples with important color information.

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A natural color scanning electron microscopy image

Oho

Ogashiwa

1996

Scanning

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Summary:A method for obtaining a scanning electron microscopy (SEM) image with natural color information (NCSEM image) has been developed by using an SEM operated under low vacuum or low voltage, a video microscope (VM), and a high-performance personal computer. In order to produce an NCSEM image with both the fine structures of the SEM image and the color information included in the VM image, an improvement of the depth of focus in VM image is required. This can be successfully performed by some digital image processing techniques. A difference in resolution between the SEM image and the VM image does not disturb our study as shown in the text. In addition, the well-known hue, saturation, intensity (HSI) color model, which is frequently utilized for color image manipulation, was originally altered for the present purpose.

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Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

Cited by 8 publications

References 33 publications

Integration of a high‐NA light microscope in a scanning electron microscope

Integration of a high‐NA light microscope in a scanning electron microscope

Natural color scanning electron microscopy based on the frequency characteristics of the human visual system

A natural color scanning electron microscopy image

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