Single-molecule fluorescence observation of cells using ORCA^®-Quest

The Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University, aims to elucidate the molecular mechanisms inside cells and on cell membranes. In 2022, this laboratory introduced the ORCA-Quest camera for single-molecule fluorescence observation.

We interviewed Professor Kenichi Suzuki and Researcher Koichiro Hirosawa of this laboratory, as well as Rinshi Kasai, a former member of the laboratory until May 2023 and currently the Unit Head of the Division of Advanced Bioimaging at the National Cancer Center Research Institute, regarding the motivations for introducing the ORCA-Quest, their experiences using it, and the perspectives for future research directions.

Current research
Problems in single-molecule fluorescence observation
Deciding factor in introducing ORCA-Quest
Imaging example
Prospects for future research
Division of Advanced Bioimaging, National Cancer Center Research Institute
Researcher profiles

Current research

The Cell Biophysics Laboratory at the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University, aims to elucidate the molecular mechanisms by observing individual molecules of proteins, lipids, and other cellular components, obtaining statistic data on event time and frequency.

To elucidate molecular mechanisms, it is essential to observe their behaviors by microscopy. We use ultra-sensitive cameras to capture molecular images, adjusting between high-speed and high-resolution settings as dictated by the experimental requirements, to gain insights into molecular dynamics and interactions. In addition, we are also developing optimal optical systems and imaging techniques to enhance such observations.

Dr. Rinshi Kasai (left), Dr. Kenichi Suzuki (center), Dr. Koichiro Hirosawa (right)

Problems in single-molecule fluorescence observation

The biggest problem in single-molecule fluorescence observation is that the signal emitted from the molecule is very weak. In addition, several factors complicate this process, such as the necessity for high-speed imaging to capture molecular behaviors, which reduces the incident signal per frame. Weak signals can also become obscure by background noise in conventional epi-illumination observations, further complicating signal detection. To reduce background interference, total internal reflection fluorescence (TIRF) microscopy is commonly used in single-molecule fluorescence studies. However, it is also important to reduce the readout noise of the camera, which ultimately outputs the signal as data.

The common approach to address this issue is to use an EM-CCD camera, which detects weak signals by increasing their magnification, or to combine an I.I. (image intensifier) with an sCMOS (scientific CMOS) camera for imaging. However, both EM-CCD cameras and I.I. cameras have the disadvantage that the signal fluctuation is generated during signal multiplication, resulting in low signal quantitativity and resolution. Our laboratory has recently been performing single-molecule super-resolution imaging. Because the reduced resolution due to fluctuations leads to a reduction in the quality of super-resolution images, we required a camera that can detect signals without reliance on I.I. or other methods.

Deciding factor in introducing ORCA-Quest

The reason why we decided to implement the ORCA-Quest was the significant improvement in image quality due to reduced noise, which became immediately evident upon reviewing the demonstration images.

Although we knew from the spec sheet that the noise levels were reduced, observing the actual output images and instantly perceiving the remarkable improvement in performance underscored the significant advancements in camera technology.

In our laboratory, we also use the ORCA-Flash4.0, ORCA-Fusion, and other cameras before the ORCA-Quest. While these cameras possess high sensitivity, at the frame rates we require, the signal from a single molecule is too weak to yield satisfactory images with the camera alone. Therefore, we had to combine these sCMOS cameras with I.I. to capture adequate images. However, with ORCA-Quest, we achieved satisfactory single-molecule fluorescence images solely with the camera. This led us to adopt the ORCA-Quest, as we anticipate it would enhance the quality of single-molecule tracking and super-resolution imaging.

Currently, we use both the ORCA-Quest and the I.I.+ previous sCMOS camera setup, selected based on the experimental requirements. For imaging that demands very high speed, the I.I.+sCMOS camera combination may offer greater sensitivity, but in most cases, we have been able to obtain satisfactory images with the ORCA-Quest.

Imaging example

The chemokine receptor CXCR4, labeled with the fluorescent protein mStayGold, was expressed in CHO cells derived from Chinese hamster ovaries, and single-molecule fluorescence observation was performed using the ORCA-Quest.

Data provided by: Dr. Rinshi Kasai, Division of Advanced Bioimaging, National Cancer Center Research Institute

Scan mode：Standard scan

Frame rate：40 frames/sec（1024 × 1024）

Scan mode：Ultra-quiet scan

Frame rate：40 frames/sec（512 × 512）

Prospects for future research

As for prospects for future research, we would like to develop our research mainly by using the following two imaging techniques.

1. Multi-wavelength imaging

2. Three-dimensional super-resolution observation

Regarding multi-wavelength imaging, we currently use two wavelengths to observe single-molecule fluorescence. However, we aspire to expand this capability to three or even four wavelengths. For example, if simultaneous three-wavelength imaging becomes feasible, we could observe molecular behaviors at two wavelengths while capturing super-resolution images at the third. This advancement would enable the analysis of molecular interactions and behaviors in even more complex systems than is presently achievable.

Regarding three-dimensional super-resolution observation, our focus has primarily been on monitoring molecular behavior on the cell membrane surface. However, by simultaneously imaging the interior of the membrane with three-dimensional super-resolution observation, we can observe molecular interactions with the endoplasmic reticulum and Golgi apparatus located within. This approach has the potential to elucidate how the endoplasmic reticulum and Golgi apparatus are involved in phenomena occurring on the membrane surface and to clarify how information from events occurring on the membrane surface is transmitted to the inside of the membrane.

There are several methods to capture three-dimensional super-resolution images, but our laboratory adopts a method that uses a cylindrical lens. To acquire a three-dimensional super-resolution image using this method, it is necessary to back-calculate the Z-axis positional information of molecules from their shapes in the two-dimensional image. While the use of an I.I. improves sensitivity, the resolution is reduced, blurring molecular shapes and degrading image quality in super-resolution imaging. The ORCA-Quest addresses this issue by eliminating the need for I.I., thereby preserving image clarity and resolution.

We believe that the ORCA-Quest is a camera poised to significantly advance our research development.

Division of Advanced Bioimaging, National Cancer Center Research Institute

In June 2023, the National Cancer Center Research Institute established Division of Advanced Bioimaging with Dr. Kenichi Suzuki as Chief and Dr. Rinshi Kasai as Laboratory Head.

In this laboratory, the microscopy system identical to that of the Cell Biophysics Laboratory at the Institute for Glyco-core Research (iGCORE) will be used to elucidate signal regulation and signal transduction mechanisms in cancer cells, mainly by single-molecule fluorescence observation.

Researcher profiles

Kenichi G. N. Suzuki
Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University: Professor. 　Division of Advanced Bioimaging, National Cancer Center Research Institute: Chief.

Jan. 1997

Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University, Ph. D.

Nov. 1996

Research Associate in Michael P. Sheetz Laboratory, Duke University Medical Center, Department of Cell Biology

Feb. 1999

Researcher in ERATO Kusumi Membrane Organizer Project

Apr. 2005

Research Assistant Professor, Kyoto University, Institute for Frontier Medical Sciences

Oct. 2008

PRESTO Researcher, Japan Science and Technology Agency (JST)

Apr. 2011

Associate Professor, Kyoto University, Institute for Integrated Cell-Material Sciences (iCeMS); Visiting Associate Professor, The National Centre for Biological Sciences (NCBS) / Institute for Stem Cell Biology and Regenerative Medicine (inStem)

Apr. 2017

Professor, Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University

Jan. 2021

Professor, Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University

Apr. 2023

Chief, Division of Advanced Bioimaging, National Cancer Center Research Institute (Concurrent)

Rinshi Kasai
Division of Advanced Bioimaging, National Cancer Center Research Institute: Laboratory Head. Hoshi University: Visiting Senior Lecturer.

Mar. 2005

Department of Biological Science, School of Science, Nagoya University, Ph. D.

Apr. 2005

Researcher, International Cooperative Research Project (ICORP), Japan Science and Technology Agency

Apr. 2010

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

Apr. 2011

Assistant Professor, Institute for Frontier Medical Sciences, Kyoto University

Oct. 2016

Assistant Professor, Institute for Frontier Life and Medical Sciences, Kyoto University

Apr. 2021

Program-Specific Associate Professor, Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University

Jun. 2023

Laboratory Head, Division of Advanced Bioimaging, National Cancer Center Research Institute

Apr. 2024

Visiting Senior Lecturer, Hoshi University

Koichiro Hirosawa
Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University: Researcher

Mar. 2010

Department of Micro Engineering, Kyoto University, Ph.D.

Apr. 2010

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

Apr. 2017

Researcher, Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University

Jan. 2021

Researcher, Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University

The website of the Cell Biophysics Laboratory of the Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System, Gifu University, is here.

The website of the Division of Advanced Bioimaging, National Cancer Center Research Institute is here.

Product used in this case study

ORCA-Quest
C15550-20UP

The C15550-20UP is the world's first camera to incorporate the qCMOS^Ⓡ image sensor. The camera achieves the ultimate in quantitative imaging.

*The successor model, C15550-22UP, is currently available.

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