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Principles of Light Microscopy: From Basic to Advanced pp 139–172Cite as

Live-Cell Imaging: A Balancing Act Between Speed, Sensitivity, and Resolution

What You Will Learn in This Chapter

Live-cell imaging is perhaps one of the most exciting and challenging activities in the field of microscopy. It is exciting as recent developments in microscope technology have enabled scientists to visualize cellular and subcellular processes in real time down to the molecular level. With this comes the prospect of studying the mechanisms of diseases in greater detail and finding possible therapeutic solutions. Nevertheless, live-cell imaging is equally challenging because cells themselves and in fact—all cellular processes—are extremely sensitive to the very impact of using light for their visualization. The aim of this chapter is to provide a practical overview for early PhD students as well as more experienced post-docs, who will spend considerable time mastering the most important challenges and prerequisites in the very rapidly evolving field of live-cell microscopy.

Keywords

  • Live cells
  • Microscopy
  • Phototoxicity
  • Controlled environment
  • Focus drift
  • Fluorescent probes
  • Automation

“In honour of Professor Anirban Banerjee, who taught us the wonders of observing living cells…”

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Change history

  • 29 January 2023

    The original version of this chapter was inadvertently published with errors. The following corrections have been made after publication.

Abbreviations

AOTF:

Acousto-optical tunable filter

BF:

Brightfield

CCD:

Charge-coupled device

CIN:

Chromosome instability

CMOS:

Complementary metal-oxide-semiconductor

CON:

Confocal microscopy

CRC:

Colorectal cancer

CRISPR:

Clustered regularly interspaced short palindromic repeats

CRISPR-HOT:

CRISPR-Cas9-mediated homology-independent organoid transgenesis

CSLM:

Confocal scanning laser microscopy

DAPI:

4′,6-diamidino-2-fenylindool

DIC:

Differential interference contrast

EM-CCD:

Electron multiplying charged-coupled device

EPI:

Epifluorescence microscopy

FL:

Fluorescence

FOV:

Field of view

FRAP:

Fluorescence recovery after photobleaching

FRET:

Fluorescence resonance energy transfer

GFP:

Green fluorescent protein

HCA:

High-content analysis

HCS:

High-content screening

HEPA:

High-efficiency particulate air

HUVEC:

Human umbilical vein endothelial cells

IR:

Infrared

LED:

Light-emitting diode

NA:

Numerical aperture

NIR:

Near-infrared

PFS:

Perfect focus system

PMT:

Photomultiplier tube

PSF:

Point spread function

PUM-HD:

Pumilio-homology domain

PZF:

Polydactyl zinc finger

RCM:

Re-scanning confocal microscopy

RFP:

Red fluorescent protein

ROI:

Region of interest

SIM:

Structured illumination microscopy

SNR:

Signal-to-noise ratio

STED:

Stimulated emission depletion microscopy

STORM:

Stochastic optical reconstruction microscopy

TALE:

Transcription activator-like effector

TIRF:

Total internal reflection fluorescence

TTL:

Transistor-transistor logic

UV:

Ultraviolet

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Author information

Authors and Affiliations

  1. Confocal.nl, Amsterdam, Noord-Holland, The Netherlands

    Jeroen Kole

  2. UCB Pharma, Slough, Berkshire, UK

    Haysam Ahmed

  3. Nikon India Private Limited, Bangalore, Karnataka, India

    Nabanita Chatterjee

  4. Chromatin Labeling and Imaging Group, Department of Nanobiophotonics, Max Planck Institute for Biophysical Multidisciplinary Sciences, Göttingen, Germany

    Gražvydas Lukinavičius

  5. M4N, Amsterdam University Medical Centers - VUmc, Amsterdam, The Netherlands

    René Musters

Authors
  1. Jeroen Kole
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  2. Haysam Ahmed
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  3. Nabanita Chatterjee
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  4. Gražvydas Lukinavičius
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  5. René Musters
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Editor information

Editors and Affiliations

  1. Nikon Europe B.V., Leiden, The Netherlands

    Volodymyr Nechyporuk-Zloy

Appendix: Microscope Company and Resources List with Internet-Links

Appendix: Microscope Company and Resources List with Internet-Links

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Kole, J., Ahmed, H., Chatterjee, N., Lukinavičius, G., Musters, R. (2022). Live-Cell Imaging: A Balancing Act Between Speed, Sensitivity, and Resolution. In: Nechyporuk-Zloy, V. (eds) Principles of Light Microscopy: From Basic to Advanced . Springer, Cham. https://doi.org/10.1007/978-3-031-04477-9_6

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