3 min. Newsletter from Diagnocine, 03222023

Excellence in Science on March, 2023

Andreas Ettinger and Torsten Wittmann

Fluorescence Live Cell Imaging

Abstract

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods
to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main
experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio,
and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on
microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental
control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein
constructs by spinning disk confocal microscopy.


1. Fluorescence Microscopy Basics

Fluorescence imaging relies on illumination of fluorescently labeled proteins or other intracellular molecules with a defined wavelength of light ideally
near the peak of the fluorophor excitation spectrum, and detection of light emitted at a longer wavelength. An important question is how much
excitation light is actually needed to obtain a useful image. At the objective front lens, the light power of our spinning disk confocal microscope with a
100 mW 488 nm solid state laser at 100% illumination is approximately 6 mW (measured with an X-Cite XR2100 light power meter, EXFO Photonic
Solutions; Chapter 4). Divided by the area of the spinning disk aperture of ~6000 µm2 at 100× magnification this results in an irradiance of ~100 W
cm−2. At lower magnification, the excitation light is spread over a larger area, thus the irradiance decreases proportional to the square of the
magnification ratio (i.e. ~36 W cm−2 for 60×). For comparison, the direct solar irradiance at ground level on a bright sunny day at noon is ~1000 W
m−2 (i.e. 0.1 W cm−2) across all wavelengths or ~1–1.5 W m−2 nm−1 for specific wavelengths within the visible part of the spectrum1. Although this
should be considered a rough estimate, it shows that the maximum light intensity in a spinning disk confocal is ~1000 times higher compared with
the total irradiance of direct sunlight, and one million times higher at a specific excitation wavelength. Similar calculations can be made for widefield
epifluorescence illumination and result in similar values depending on the light source. Because laser scanning confocal microscopes utilize a
focused beam to illuminate a very small area at a time, typical irradiance values can be several orders of magnitude higher.

This difference in specimen irradiance between spinning disk and laser scanning confocal microscopes explains partly why spinning disk confocal
microscopes are the better choice for live cell imaging. Fluorescence emission is linearly related to the excitation light intensity as long as the
majority of fluorescent molecules in a population are not in the excited state. At higher rates of photon flux, however, that are quite easily reached in
laser scanning confocal microscopes, a large proportion of fluorophors populates the excited state and thus can no longer absorb additional photons
(Wang, Babbey, & Dunn, 2005). This is referred to as ground-state depletion, and additional excitation light will only yield sub-proportional increases
in fluorescence signal, but still contribute to photodamage. Because in spinning disk confocal microscopes, the excitation laser light is spread over
thousands of pinholes that scan across the specimen rapidly (Chapter 9), ground-state depletion is not reached even with excitation lasers with
hundreds of mW power output. It is interesting to note that ground state depletion can be used to achieve PALM/STORM-type superresolution
(Lalkens, Testa, Willig, & Hell, 2012).

~ ~ more info. click the below link, 

The original research paper link is here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198327/
 

Keywords: Environmental control; Fluorescence microscopy; Fluorescent proteins; Live cell microscopy; Photobleaching; Spinning disk confocal microscopy.



Figure 3
Influence of tagging strategy on the apparent localization of FP-tagged MT1-MMP

HeLa cells were co-transfected with the constructs indicated. The two constructs localize to mostly distinct intracellular compartments.
 

The original research paper link is here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198327/

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