Challenges in 3D Live Cell Imaging

A short overview on 3D live cell imaging is given. Relevant samples are described and various problems and challenges—including 3D imaging by optical sectioning, light scattering and phototoxicity—are addressed. Furthermore, enhanced methods of wide-field or laser scanning microscopy together with some relevant examples and applications are summarized. In the future one may profit from a continuous increase in microscopic resolution, but also from molecular sensing techniques in the nanometer range using e.g., non-radiative energy transfer (FRET).

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Detection and Tracking of Dual-Labeled HIV Particles Using Wide-Field Live Cell Imaging to Follow Viral Core Integrity

Methods in Molecular Biology - HIV Protocols ◽

10.1007/978-1-4939-3046-3_4 ◽

2016 ◽

pp. 49-59 ◽

Cited By ~ 9

Author(s):

João I. Mamede ◽

Thomas J. Hope

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Wide Field ◽

Viral Core ◽

Detection And Tracking

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Characterization and Use of Wide-Field Fluorescence Microscopy and Image Restoration in Quantitative Live Cell Imaging

Microscopy and Microanalysis ◽

10.1017/s1431927602100377 ◽

2002 ◽

Vol 8 (S02) ◽

pp. 266-267

Author(s):

Melpomeni Platani ◽

Angus I Lamond ◽

Jason R. Swedlow

Keyword(s):

Fluorescence Microscopy ◽

Image Restoration ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Wide Field

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High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)

Microscopy and Microanalysis ◽

10.1017/s1431927600026180 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 22-23

Author(s):

P. W. Wiseman ◽

J. C. Bouwer ◽

S. Peltier ◽

M. H. Ellisman

Keyword(s):

Live Cell Imaging ◽

High Speed ◽

Cell Imaging ◽

Live Cell ◽

Image Correlation ◽

Scanning System ◽

Optical Sectioning ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

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Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1287 ◽

2015 ◽

Vol 26 (9) ◽

pp. 1743-1751 ◽

Cited By ~ 41

Author(s):

Shinichi Hayashi ◽

Yasushi Okada

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Rotating Disk ◽

Confocal Microscope ◽

Diffraction Limit ◽

Live Cell ◽

Wide Field ◽

Structured Illumination ◽

Stripe Pattern ◽

Spinning Disk

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of microtubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.

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