Insights in Cell Science – Towards a Quantitative Approach of Fluorescence Microscopy that Unravels Cellular Function

Ben Loos*

Department of Physiological Sciences, Stellenbosch University, South Africa

*Corresponding Author:
Ben Loos
Distinguished University Professor, Department of Physiological Sciences
Stellenbosch University
South Africa
Tel: +27218089196
Fax: +27218083145
E-mail: [email protected]

Received date: October 01, 2015 Accepted date: December 31, 2015 Published date: January 02, 2016

 
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Mini Review

It is undoubtedly so, the time for fluorescence microscopy in cellular sciences has come, allowing the unraveling of the ‘inside’ of cells and their molecular itinererary in a novel and most precise manner. This is supported by the in 2014 awarded Noble price in chemistry, received for the development of the superresolution fluorescence microscopy techniques photo-activation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED). These techniques provide for the first time a platform for subdiffraction molecular imaging complementing the growing arsenal of fluorescence based techniques such as live cell imaging, photo-activation, fluorescence resonance energy transfer (FRET) or fluorescence recovery after photo-bleaching (FRAP). The challenge however remains, not to be drowning in a sea of imagedata, that may be building up to terabytes of data, but to be able to extract meaningful and statistically sound data points, that are suitable to inform on cellular function and dysfunction [1]. More so, it is envisaged to expand our fluorescent toolbox in a manner that not only allows the utilization of fluorescence based image data in a most powerful manner, but also that may contribute towards enabling predictive and preventative medicine [2]. Here we comment on some of the recently developed and employed applications and trends, that enable to powerfully provide “Insights in cell science”, both visually and quantitatively. In doing so, it is hoped to offer a perspective on current applications, trends and challenges that define our present research landscape of cellular imaging. This may assist in better and more carefully designed experimental settings that are most informative and well aligned with the powers of fluorescence microscopy technology.

Cellular decision making centers around maintaining homeostasis and cell viability. When the cell is unable to meet its metabolic demands, or is exposed to some form of cellular injury, cell death will be induced [3]. Although it is highly important to assess the overall contribution of cell death modalities, life cell imaging in combination with FRET-based caspase-sensors [4] allow for the quantification of the exact onset of cell death induction (Figure 1A) [5]. This enables to inform dynamically about cellular decision making, to trace the fate of individual cells and to assess the time span particular cells were able to maintain viability. Such an approach, based on long series of time lapses may reveal a more accurate understanding of cellular pathology and cell disease pathogenesis [6] compared to a static approach that merely quantifies parameters at few selected time points.

Integral part in this context is the availability of ATP, and its presence in regions of ATP consuming processes. The tubulin network (Figure 1B) and ATP dependent molecular motors such as kinesin and dynein [7] ensure the transport of mitochondria and autophagosomes to regions where either ATP is required (such as synaptic processes) or where fusion processes with other organelles (such as lysosomes in the perinuclear region) take place [8]. In the past, it has been a major challenge to visualize ATP, and usually luciferase-based methods have been employed to quantify overall ATP availability [9]. However, recently, the development of sensitive sensors, that are based on fluorescence resonance energy transfer (FRET) have been developed [10] and successfully employed to visualize and localize intracellular ATP hotspots (Figure 2a). Such an approach may not only allow to assess where intracellular ATP demand is highest or lowest and how such localization may change in disease or intervention, but it may also, through live cell imaging, enable the quantification of the duration a cell is able to maintain favorable ATP availability. This enhances greatly our understanding of metabolic processes and cellular decision making during metabolic adaptations or perturbations and cellular injury, such as in cancer or neurodegeneration. The precise labeling of selective proteins through either a transfection- or immuno-fluorescence based approach enables to visualize specific organelles of interest. The employment of super-resolution techniques, such as superresolution structured illumination microscopy (SR-SIM) allows to do implement such approach at enhanced resolving power, in a three-dimensional space. This in turn enables to not only visualize organelles of interest, but, through optical z-stacking, to acquire complete cellular organelle pool sizes with their subsequent quantification. This is powerful, since, although higher resolution may be achieved through transmission electron microscopy (TEM), complete cell volume and organelle pool size quantification is enabled using a subdiffraction limit resolving power. In that manner, for example lysosomes (Figure 2B) can be visualized and their distribution expressed as complete lysosomal pool size nA [11]. SR-SIM also enables to assess the mitochondrial network at much greater detail (Figure 2C), allowing its quantification more precisely than using confocal-based images, by employing form factor analyses or aspect ratio approaches [12]. Together with techniques such as photoactivation-based mitochondrial fission and fusion dynamics [13], network properties can be assessed in their dynamic nature, which is more sensitive and by far more rapid in response compared to a change in protein expression levels. Here, fluorescence microscopy techniques compile a powerful tool box to understand mitochondria at their nexus in cell death and survival. If combined with pool size analyses of associated organelles (such as autophagosomes, (Figure 2D), the dynamic behavior of interactions such as mitophagy or protein degradation through macroautophagy can be better understood and quantitatively described.

stemcells-Apoptosis-onset

Figure 1: Apoptosis onset (1A) and the microtubule network (1B). Cells were transfected with a CFP-DEVD-YFP construct and loaded with tetramethylrhodamineethylester (TMRE) to monitor the onset of caspase-3 activation and mitochondrial depolarization. B: Acetylated tubulin allows for the assessment of tubulin network structural integrity. Image was acquired from z-planes and projected in maximum intensity. (From Loos et al., 2011).

stemcells-neuronal-cells

Figure 2: GT 1-7 neuronal cells indicating ATP (A) targeted to the mitochondria, using a FRET construct. Superresolution structured illumination microscopy (SRSIM) enables the visualization of labeled organelles down to 80 nm spatial x-y resolution. Fluorescent micrographs indicate lysosomes labeled for the lysosomal associated membrane protein-1 (B), mitochondria (C) and the interaction between GFPLC3 labeled autophagosomes and mitochondria (D).

Advances in organic chemistry and molecular biology as well as material sciences have enabled the research community to choose from a wide range of classes of fluorescent probes for imaging in cell biology. Here, a combination of inherently binding fluorochromes, fluorescent proteins and an immunofluorescence approach allows the concomitant assessment of a number of parameters of interest (Figure 3). Such multi-colour imaging in turn enables the assessment of organelle and/or protein-protein interactions through colocalization analysis in a powerful manner, reporting on cell function and dysfunction. Multi-parameter imaging enables a better understanding of the relationship between the parts of interest in the cellular system. If performed in combination with super-resolution techniques, such intermolecular relationships can report on function and localization in a most precise manner [14].

stemcells-targeting-methods

Figure 3: A combination of targeting methods and fluorescent probes. Mouse embryonic fibroblasts transfected with GFP-LC3 (green). After fixation cells were immune-labeled for mitochondrial DNA with a mouse monoclonal antibody (red) and counterstained using Alexa fluor 647 phalloidin (magenta) and Hoechst 33342 (blue). Images were acquired from z-planes and projected in maximum intensity. Scale bars: 20 μm.

The large amount of data that is being generated through fluorescence microscopy based techniques demands an integrative approach, in order to interpret distribution and intensities of fluorescent signal in a meaningful manner. This will become of even greater importance as we venture into PALM- 3D and super-resolution time lapse acquisition techniques. Here, systems biology and computational modeling may become an even more crucial part in the future to integrate the vast amount of biological information, as quantification of molecular elements of a biological system is at the heart of this discipline [2]. This becomes particularly clear in current trends using fluorescence-based image data at a multi-scale level [15], i.e. from molecular density mapping based on PALM [16] to in vivo whole organ imaging based on light sheet microscopy [17]. With no doubt such an approach will enable us to map proteins in cells and tissues spatiotemporally like never before, building dynamic and most comprehensive organ systems based on real fluorescence microscopy derived data. When doing so, and when doing so quantitatively, we are likely one step closer towards enabling predictive or preventative medicine. Taken together, we can look forward to an exciting future, where insights in cell science will matter greatly and where the use of fluorescence microscopy techniques will play a fundamental and integral part.

Acknowledgements

This research work was supported by the National Research Foundation (NRF) and the Medical Research Council (MRC), South Africa. I wish to thank Dr Tavaré (University of Bristol, UK) for providing the CFP-DEVD-YFP construct, Prof Hiromi Imamura, Kyoto University, Japan for the provision of the ATP-FRET construct and Prof Noboru Mizushima, Tokyo University, for the provision of GFP-LC3 expressing mouse embryonic fibroblasts.

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