3D live cell imaging analysis of epidermal keratinocytes

Published on February 13, 2025

The Laboratory for Evolutionary Cell Biology of Skin, Cosmetics Course, School of Bioscience and Biotechnology, Tokyo University of Technology, is researching epidermal barrier formation mechanisms. To elucidate the mechanisms of epidermal barrier formation, it is necessary to image the epidermis in three dimensions. For this purpose, they have introduced our MAICO^® MEMS confocal Unit.

We interviewed Professor. Takeshi Matsui from the same laboratory about the background of introducing the MAICO MEMS confocal unit, his impressions of its use, and the prospects for future research.

Current research
Challenges in 3D live-cell imaging of the epidermis
Decisive factor for introducing the MAICO MEMS confocal unit
Imaging examples
Usability of the MAICO MEMS confocal unit
Prospects for research
Researcher profile

Current research

Could you tell us about your research?

Our laboratory studies the mechanisms of adaptive evolution in the skin, particularly focusing on how the epidermal barrier is formed. The skin consists of three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis is a stratified squamous epithelium made up of multiple layers, with its outermost layer being the stratum corneum (cornified layer) composed of dead cells. This stratum corneum evolved when ancient amphibians made the transition to land, and it serves as a crucial barrier that enables terrestrial vertebrates to survive on land. The stratum corneum is formed through a specialized type of cell death called corneoptosis, which occurs in the SG1 cells located in the uppermost part of the granular layer. Our laboratory has developed a live-cell imaging system using optical microscopy to observe and understand the process of corneoptosis.

Professor Takeshi Matsui

Cross-sectional view of skin epidermal tissue

Challenges in 3D live-cell imaging of the epidermis

Live-cell imaging system using MAICO

Professor Matsui also uses our ORCA^®-Fusion BT for regular epifluorescence imaging.

What are the challenges of live-cell imaging of the epidermis?

Since the epidermis we studied has a multilayered structure, we needed to image its structure in three dimensions. Regular fluorescence microscopes and cameras couldn't capture this 3D structure clearly, so we had to use confocal microscopy. However, confocal microscopes are expensive, making it difficult for our laboratory to purchase one. Previously, we had to rely on shared equipment available at our research institute and university.

Our experiments required time-lapse imaging to observe the process of corneoptosis in granular layer cells, sometimes necessitating continuous imaging over several days. However, the shared equipment operated on a reservation system with limited time slots. Additionally, each imaging session required considerable setup time – we needed an incubator for cell culture and had to configure settings for multi-point time-lapse imaging.

Given these circumstances, we wanted to install our confocal microscope in our laboratory so we could conduct experiments without time constraints or the hassle of repeated setup procedures.

Decisive factor for introducing the MAICO MEMS confocal unit

What made you decide to introduce MAICO?

We had been using Hamamatsu Photonics' sCMOS cameras for fluorescence imaging. While we were looking for a confocal microscope, a sales representative informed us about the newly released MAICO MEMS confocal unit, which could transform existing microscopes into confocal microscopes simply by adding it on. We first requested a demonstration.

Although I was initially concerned about the image quality and sensitivity due to its very reasonable price, I remember being surprised during the demonstration - it could capture high-quality images comparable to other companies' confocal microscopes. We ultimately decided to implement it based on several factors: its reasonable price, image quality comparable to other confocal microscopes, the ability to start with just one wavelength and add more later through its modular subunit structure, and the compact size of the equipment.

Another deciding factor was MAICO's unique feature of simultaneous multi-wavelength observation without crosstalk. Since our laboratory conducts live cell imaging, we wanted to capture multiple wavelengths simultaneously. Usually, this causes wavelength bleeding problems, but MAICO solved this issue, making it extremely valuable for our work.

Recently, we have completed the installation of an XY stage for multi-point time-lapse imaging and a Z-drift compensator to maintain focal position, in addition to the MAICO MEMS confocal unit. Now that we finally have a complete setup for confocal time-lapse imaging, we're about to begin actual imaging.

Professor Takeshi Matsui

Imaging examples

Z-stack image of mouse skin epidermal granular layer cells

After isolation of granular layer cell sheets from EGFP-expressing mouse skin, they were stained using Hoechst 33342 and Z-stack images were acquired.

Data provided by: Prof. Takeshi Matsui, Laboratory for Evolutionary Cell Biology of the Skin School of Bioscience and Biotechnology Tokyo University of Technology

Usability of the MAICO MEMS confocal unit

Professor Takeshi Matsui

How would you feel about the usability of MAICO?

I find it very user-friendly with a simple operation. For example, while the pinhole settings don't allow for detailed adjustments, they can be set in three levels - S, M, and L - which is simple and easy to understand. This makes it smooth to explain to new students joining the laboratory each year. Additionally, for optimal imaging conditions with each lens, Hamamatsu Photonics provides a resolution simulator, which I find helpful as it allows us to adjust the Z-slice thickness according to the sample structure and thickness while referring to the simulation.

Prospects for research

Could you tell us about future research prospects?

Currently, we primarily conduct experiments using human and amphibian epidermis samples. In the future, we hope to observe and study the epidermis of amphibians and reptiles to investigate species-specific differences and evolutionary mechanisms. We plan to conduct more detailed research by combining confocal microscopy images with electron microscopy analysis and genetic information.

Since our laboratory is in the community of dermatological research, we sometimes collaborate with dermatologists and cosmetics manufacturers on various research projects, including studies on atopic dermatitis (where the skin barrier is compromised) and the evaluation of cosmetic ingredients. The live cell imaging system I'm currently developing to elucidate epidermal barrier formation mechanisms could potentially be applied to these studies as well. Therefore, we're also focusing on developing new evaluation systems.

Case study: 3D live cell imaging analysis of epidermal keratinocytes

Researcher profile

Professor Takeshi Matsui

Takeshi Matsui
Professor, Laboratory for Evolutionary Cell Biology of Skin, Cosmetics Course, School of Bioscience and Biotechnology, Tokyo University of Technology

Mar. 2000

Ph. D., Graduate School of Medicine and Faculty of Medicine, Kyoto University

Apr. 2000

Group Leader, Research Group of Dermatology, KAN Research Institute, Inc.

Oct. 2006

Industry-Academia-Government collaboration Researcher, Kyoto University

Nov. 2006

Project Researcher, Graduate School of Medicine, Osaka University

Apr. 2007

Assistant Professor, Laboratory of Biological Science, Graduate School of Frontier Biosciences, Osaka University

Nov. 2007

Lecturer, Medical Top Track (MTT) Program, Medical Research Institute, Tokyo Medical and Dental University

Apr. 2011

Assistant Professor, Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University

Apr. 2013

Senior Researcher, Laboratory for Skin Homeostasis, RIKEN Center for Integrative Medical Sciences

Apr. 2016

Deputy Team Leader, Laboratory for Skin Homeostasis, RIKEN Center for Integrative Medical Sciences

Apr. 2021

Professor, Laboratory for Evolutionary Cell Biology of Skin, Cosmetics Course, School of Bioscience and Biotechnology, Tokyo University of Technology

Click here for the website of the Laboratory for Evolutionary Cell Biology of Skin, Cosmetics Course, School of Bioscience and Biotechnology, Tokyo University of Technology

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3D live cell imaging analysis of epidermal keratinocytes

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