High throughput second harmonic imaging for label-free biological applications

Macias-Romero, Carlos; Didier, Marie; Jourdain, Patrick; Marquet, Pierre; Magistretti, Pierre J.; Tarun, Orly B.; Zubkovs, Vitālijs; Radenović, Aleksandra; Roke, Sylvie

doi:10.1364/oe.22.031102

Cited by 47 publications

(56 citation statements)

References 65 publications

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“…In order for the wide-field and scanning systems to display the same temperature rise after an illumination time of 5 ms, the imaging volume in the wide-field system would have to be increased by 255 times. With such an increase in the volume and the scanning parameters given above, a 270-fold improvement in the imaging throughput is expected with the wide-field configuration, which is indeed observed ( [13], Eq. 2, Fig.…”

Section: Photodamage Mechanismsmentioning

confidence: 71%

“…Figure 1(b) shows the rise in temperature calculated for two cases keeping the illuminated volume and deposited energy constant: First, the effect of tightly focused beam (1.1 NA, 1035 nm wavelength, 80 MHz repetition rate, red curve), exemplary of a point scanning multiphoton microscope is considered. Second the effect of a weakly focused beam (0.02 NA, 1035 nm wavelength, 200 kHz repetition rate, blue curve), exemplary of a wide-field imaging system is computed [13]. For the scanning system, we considered a dwell time of 10 µs and a scanning resolution equal to a.…”

Section: Photodamage Mechanismsmentioning

confidence: 99%

“…2 Ref. [13]); a higher number indicates a better imaging throughput. It can be seen that for a medium repetition rate (100 kHz) and a tight focus (500 nm diameter), very high peak intensities (900 -2000 GW/cm 2 ) can be used [24].…”

Section: Proliferation Assay On Hek Cellsmentioning

confidence: 99%

“…), and using short dwell times of 10 -80 µs per pixel [3]. Recent approaches, which are capable of imaging with higher throughput [11][12][13], use the same pulse duration but with lower repetition rates (100-200 kHz), wide-field illumination (100 µm diam. ), and higher pulse energies (0.1 µJ).…”

Section: Introductionmentioning

confidence: 99%

“…Here, we consider the effects of these three pathways to optical damage and model photodamage using the heat transport equation, paying particular attention to the effects induced by changing from scanning to wide-field imaging and lowering the repetition rate [13]. We combine this analysis with two-photon fluorescence (2PF) experiments of solutions and endogenous in living cells.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Macias-Romero

Zubkovs

Roke

2016

Biomed. Opt. Express

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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.

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Section: Photodamage Mechanismsmentioning

confidence: 71%

Section: Photodamage Mechanismsmentioning

confidence: 99%

Section: Proliferation Assay On Hek Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Macias-Romero

Zubkovs

Roke

2016

Biomed. Opt. Express

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Wide‐field Stokes polarimetric microscopy for second harmonic generation imaging

Castaño

Mirsanaye

Kontenis

et al. 2023

Journal of Biophotonics

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We employ wide-field second harmonic generation (SHG) microscopy together with nonlinear Stokes polarimetry for quick ultrastructural investigation of large sample areas (700 μm Â 700 μm) in thin histology sections. The Stokes vector components for SHG are obtained from the polarimetric measurements with incident and outgoing linear and circular polarization states. The Stokes components are used to construct the images of polarimetric parameters and deduce the maps of ultrastructural parameters of achiral and chiral nonlinear susceptibility tensor components ratios and cylindrical axis orientation in fibrillar materials. The large area imaging was employed for lung tumor margin investigations. The imaging shows reduced SHG intensity, increased achiral susceptibility ratio values, and preferential orientation of collagen strands along the boarder of tumor margin. The wide-field Stokes polarimetric SHG microscopy

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Roadmap on Label‐Free Super‐Resolution Imaging

Astratov,

Sahel,

Eldar

et al. 2023

Laser & Photonics Reviews

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Label‐free super‐resolution (LFSR) imaging relies on light‐scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super‐resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state‐of‐the‐art in this field, and to discuss the resolution boundaries and hurdles that need to be overcome to break the classical diffraction limit of the label‐free imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction‐limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super‐resolution capability that are based on understanding resolution as an information science problem, on using novel structured illumination, near‐field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere‐assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

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High throughput second harmonic imaging for label-free biological applications

Cited by 47 publications

References 65 publications

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

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

Wide‐field Stokes polarimetric microscopy for second harmonic generation imaging

Roadmap on Label‐Free Super‐Resolution Imaging

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