RESUMO
Tendon stem/progenitor cells (TSPC) are potential targets for regenerative medicine and the treatment of tendon injuries. The frequency of such injuries increases in elderly patients while the proportion of functional TSPCs in tendon tissue decreases, protracting tendon repair. Using atomic force microscopy (AFM), we show that cell stiffness and size increase in TSPCs isolated from elderly patients (A-TSPC) compared to TSPCs from younger patients (Y-TSPC). Additionally, two-photon excited fluorescence (TPEF) microscopy revealed a denser, well-structured actin cytoskeleton in A-TSPC, which correlates with the augmented cell stiffness. Treating A-TSPC with ROCK-inhibitor, reverses these age-related changes, and has rejuvenating effect on cell morphology and stiffness. We assume that cellular stiffness is a suitable marker for cell aging and ROCK a potential target for therapeutic applications of cell rejuvenation.
Assuntos
Amidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Células-Tronco/citologia , Tendões/citologia , Quinases Associadas a rho/antagonistas & inibidores , Adulto , Idoso , Fenômenos Biomecânicos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Módulo de Elasticidade/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Rejuvenescimento , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tendões/efeitos dos fármacos , Tendões/metabolismo , Adulto Jovem , Quinases Associadas a rho/metabolismoRESUMO
In this manuscript, we present a detailed investigation of the impact of dispersion on the spectral resolution achievable by the application of spectral focusing in coherent Raman imaging. Our results reveal the detrimental effect of third order dispersion that limits the resolution for group delay dispersion of 100 000 fs2 and more. Experimental examples for the exact determination of the described effects are given as well as a condensed presentation of the known equations. We introduce useful approximations to the latter, which serve to facilitate the straightforward integration of spectral focusing into any multimodal microscope.
RESUMO
We present a two-color two-photon stimulated emission depletion microscopy technique (2C2P-STED) that correlates a confocal image with a super-resolved image employing the inherent self-referencing mechanism of nonlinear excitation. The novel approach overcomes the substantial challenge posed by two different imaging modalities in laser-scanning fluorescence microscopy for colocalization on the nanometer scale. Demonstrating the principle of 2C2P-STED, we show for the first time super-resolved images of the gram-positive bacteria Streptococcus pneumoniae TIGR4 pilus type-1. A signal-to-noise ratio (SNR) greater than 10 was achieved in 2C2P excitation mode and approximately 70â nm details were resolved in 2P-STED.