Fig 1.
Schematic showing the optical layout of the two-photon microscope developed for visualization of micro-agents along with biological tissue.
(a1) The microscope utilizes a femtosecond laser for excitation light generation while the emission light is delivered to a photomultiplier. The following micro-agents are visualized below biological tissue: (a2) a bio-inspired structure called CeFlowBot, and (a3) electrospun fibers. The colored arrows indicate structural morphologies of the micro-agents that are visible below biological tissue. (a4) Illustration of the samples used to visualize micro-agents below biological tissue. (b1)-(b2) Schematic representation of the fabricated fluorescent micro-agents.
Fig 2.
Spectrum analysis of fluorophores.
(a1) Coumarin 6 (exogenous) (442631–1G, Sigma-Aldrich, USA) and (b1) IP-Dip resin (autofluorescence) (IP-Dip, Nanoscribe GmbH, Germany). Fluorescence microscopy of electrospun fibers with the following types of morphology: (a2) beaded fibers and (a3) rod-like particles. (b2)-(b3) 3D and contour plots showing the spectrum of IP-Dip resin at varying excitation and emission wavelengths. (b4) Spectral decomposition of CeFlowBot (made from IP-Dip resin) using a fluorescence microscope (EVOS FL, Life Technologies, USA).
Fig 3.
Z-stacking, 3D reconstruction, and scanning electron microscopy of micro-agents.
(a1)-(a3) beaded fiber and (b1)-(b3) a scaled version of the bio-inspired structure (CeFlowBot), respectively. (c) Average power of femtosecond pulsed laser (pulse width of ~ 230 fs and repetition rate of 15 MHz) at the focus of the laser beam. (d1) Illustration of the laser-tissue interaction using three laser power percentages (LPPs): 40%, 50%, and 60% with the corresponding average powers of 85 mW, 92 mW, and 97 mW, respectively. (d2) Confocal microscopy of laser-affected tissue (rat liver dissection). (d3) Z-stacking of a region of interest (ROI) containing laser-affected tissue corresponding to 50% and 60% LPP. (e1) Raw images acquired from stain-free imaging of formalin-fixed rat tissues and ex ovo chorioallantoic membrane using second-harmonic generation. (e2) Magnified view of the region of interest within the raw image. The red arrows indicate renal collecting tubules and vasculatures containing erythrocytes in rat kidney cortex and ex ovo chorioallantoic membrane, respectively.
Fig 4.
Contrast analysis of the two-photon microscopy images containing micro-agents and biological tissue.
(a) Two-photon microscopy of CeFlowBot and a bifurcated vessel containing erythrocytes from ex ovo chorioallantoic membrane. (a1) Raw images acquired with 5× and 10× microscope objectives. (a2) Normalized fluorescence intensity of micro-agent and a bifurcated blood vessel along a longitudinal line. (a3) Contrast analysis to identify CeFlowBot and a bifurcated blood vessel. Two-photon microscopy and contrast analysis of CeFlowBot below rat ileal wall (fixed) using 5× (c1) and 10× (c2) microscope objectives. Two-photon microscopy and contrast analysis of electrospun fibers below rat brain tissue (fixed) (b1) and rat kidney cortex (fixed) (b2). The colored arrows indicate structural morphologies that are visible below fixed tissue.
Fig 5.
Actuation, continuous visualization, and motion analysis of magnetic electrospun fibers.
(a) Experimental validation platform for two-photon image acquisition of micro-agents below biological tissue. (b) Visualization of magnetic electrospun fibers within a microfluidic channel using 10% laser power percentage (LPP) (6 mW average power). (c) Visualization of magnetic electrospun fibers below fixed tissue (rat ileal wall) using 40% LPP (85 mW average power). The experiments are carried out with a 10× microscope objective, a scanning speed of 50 mm/s, and a sampling rate of 10 MS/s. The vector field colored in red represents the motion analysis. The continuous image acquisition experiments are shown in S1 Video.