Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells

Abstract

Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2-3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×10(6) while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage.

Noninvasive Continuous Monitoring of Adipocyte Differentiation: From Macro to Micro Scales

“…Error bars are the standard error, statistical significance between time points (using analysis of variance): p < 0.0001. which is different but adopts concepts from other commonly used techniques applied in quantitative biology, e.g. a hemocytometer to determine the number of living cells in a culture (Strober, 1997) and pre-engraved gridded plates or gridded stickers (Weinmeister et al, 2008;Finlayson & Freeman, 2009;Neubrand et al, 2010;Doerner et al, 2015;Kraus et al, 2016;Macias-Romero et al, 2016;Thompson et al, 2016;Toneff et al, 2016) for locating, identifying, counting, and tracking movement of cells, cell clusters, or of other organisms. These devices are not used or provide the option to map or estimate the adipogenic propagation.…”

Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level

“…We used the interfacial water response to construct maps of the electric surface potential by relating the interfacial electrostatic field ( E DC ) and corresponding surface potential ( Φ 0 ) via 34 – 36 : where is the surface second-order susceptibility and an effective third-order susceptibility of the aqueous phase that primarily depends on oriented water in the electric double layer, and f 3 is an interference term that takes the value 1 for a transmission experiment 36 , 37 . Given the low photo toxicity of the high throughput microscope 38 , it is possible to use the same approach, namely the reorientation of water in the electric double layer of a membrane to map membrane potentials in living neurons optically and label-free.…”

Mapping of real-time morphological changes in the neuronal cytoskeleton with label-free wide-field second-harmonic imaging: a case study of nocodazole

“…After 20 min following the washout, the SH intensity has recovered back to ∼80% of its original value, in agreement with previous studies. 26,37 As the wide-field SH imaging system can be employed to record 0.25-s integration time images, without damaging the cells, 43 in what follows we demonstrate two possible applications of SH imaging: to probe morphological drug-induced changes in neurons of different age and to dynamically image spatiotemporal changes in real time.…”

“…The method combines an increase in throughput of 3 orders of magnitude of the SH imaging process 41,42 with low photodamage. 43 We use the well-known effects of nocodazole on microtubules as an example of a system where spatiotemporal changes are expected. We determine the orientational distribution of microtubules and map the relative intensity of different morphological structures and perform in-vitro imaging of the effects of nocodazole on the axon of a corpus of neurons at different stages of maturity.…”