Construction of a laser scanning microscope for multiphoton excited optical fabrication

Sridhar, Mallika; Basu, Swarna; Scranton, Victoria; Campagnola, Paul J.

doi:10.1063/1.1584079

Cited by 25 publications

(45 citation statements)

References 16 publications

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“…The optical setup and instrumentation software used for fabrication has been previously described. 40,44 We recently developed a new scanning approach known as modulated raster scanning. 44 This allows constant scanning (speed and step size) of the laser with rapid modulation of the pixel dwell time (MHz scale) to define the integrated laser exposure while maintaining constant laser peak power.…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose we use multiphoton excited (MPE) photochemistry, where the photochemical process is analogous to the two-photon excited fluorescence microscopy (TPFE) where the excitation at the focal volume results in crosslinked proteins with 3D features of submicron resolution. 34–40 We previously used the MPE technology approach to generate models consisting of LN “fibers”, i.e. linear structures of constant concentration with sub-micron height and width.…”

Section: Introductionmentioning

confidence: 99%

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Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry

et al. 2017

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Introduction Migration mis-regulation is a hallmark of cancer, and remains an important problem in cancer biology. We postulate the needs for better in vitro models to understand the details of cell-matrix interactions. Here, we utilized multiphoton excited (MPE) photochemistry to fabricate models to systematically study migration dynamics operative in breast and ovarian cancer. Gradients are a convenient means to modulate concentration and also have been implicated in metastases. Methods We specifically pattern sub-micron structured gradients from laminin and fibronectin whose up-regulation is associated with increased metastasis and poor prognosis. We developed a new continuous linear bi-directional gradient design, permitting exploration of the underlying cell-matrix interactions of migration, including speed, directness, and f-actin cytoskeleton alignment as a function of concentration. These new models provide both contact guidance and ECM binding cues, and provide a more relevant environment than possible with existing technologies such as flow chambers or 2D printed surfaces. Results We found an overall increase in these processes with increasing concentration on both laminin and fibronectin gradients for a series of ovarian and breast cancer lines. Moreover, directness was higher for more metastatic cells, indicating that epithelial or mesenchymal state of the cell type governs the dynamics. However, the specifics of the speed and directedness depend on both the cell type and protein, thus we found that we must consider these processes collectively to obtain a self-consistent picture of the migration. For this purpose, we performed a linear discriminate analysis (LDA) and successfully classified the different cell types on the two protein gradients without molecular biology analysis. Conclusions The bi-gradient structures are versatile tools to performing detailed studies of cell migration, specifically haptotxis. We further suggest the can be used in assessing efficacy of drug treatments targeted at specific matrix components.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry

et al. 2017

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“…35 Briefly, the multiphoton photochemistry is induced by a femtosecond titanium sapphire laser that is coupled to a purpose-built laser scanning microscope system mounted on an upright stand. The scanning/acquisition software is written in LabVIEW and is freely available upon request.…”

Section: Methodsmentioning

confidence: 99%

“…As previously determined by higher resolution optical microscopy measurements, this optical setup yields fiber sizes of approximately 700 nm in diameter and 2 μ m in height. 35 …”

Section: Methodsmentioning

confidence: 99%

“…This photochemical process is analogous to two-photon excited (TPE) fluorescence microscopy, where the excitation, and here, the fabrication, is confined to the focal volume, resulting in intrinsic 3D capabilities and concurrently affording sub-micron feature sizes. 2,9,22,24,26,30,31,35 Freeform 3D capabilities with submicron feature sizes are not currently possible by microfluidic and microcontact printing methods. Moreover, the crosslinked protein concentration achieved by MPE can be well controlled by tuning the laser exposure.…”

Section: Introductionmentioning

confidence: 99%

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Cell Adhesion on Micro-Structured Fibronectin Gradients Fabricated by Multiphoton Excited Photochemistry

Chen

Ajeti

et al. 2012

Cel. Mol. Bioeng.

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Concentration gradients of ECM proteins play active roles in many areas of cell biology including wound healing and metastasis. They may also form the basis of tissue engineering scaffolds, as these can direct cell adhesion and migration and promote new matrix synthesis. To better understand cell–matrix interactions on attractive gradients, we have used multiphoton excited (MPE) photochemistry to fabricate covalently linked micro-structured gradients from fibronectin (FN). The gradient design is comprised of a parallel series of individual linear gradients with overall dimensions of approximately 800 × 800 μm, where a linear dynamic range of nearly 10-fold in concentration was achieved. The adhesion dynamics of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became increasingly elongated and aligned with the direction of the gradient at increasing protein concentration. Moreover, the cell morphologies are distinct when adhered to regions of differing FN concentration but with similar topography. These results show that the fabrication approach allows investigating the roles of contact guidance and ECM cues on the cell–matrix interactions. We suggest this design overcomes some of the limitations with other fabrication methods, especially in terms of 3D patterning capabilities, and will serve as a new tool to study cell–matrix interactions.

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Properties of crosslinked protein matrices for tissue engineering applications synthesized by multiphoton excitation

Basu

Campagnola

2004

J Biomedical Materials Res

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We demonstrate the fabrication of model scaffolds and extracellular matrices using multiphoton excited photochemistry. This method is three‐dimensional in nature and has excellent biocompatibility. Crosslinked matrices were fabricated from the proteins fibrinogen, fibronectin, and concanavalin A using two‐photon rose bengal photoactivation and the relatives rates were determined. Immunofluorescence labeling of fibrinogen and fibronectin indicated retention of bioactivity following the multiphoton crosslinking process. Using the fluorescence recovery after photobleaching method, we measured the lateral mobility of fluorescent dyes of different mass and chemistry in order to model the behavior of therapeutic agents and bioactive molecules and found diffusion coefficients within these fabricated structures to be on the order of 10−9–10−10 cm2/s, or approximately three to four orders of magnitude slower than in free solution. The precise diffusion coefficients can be smoothly tuned by varying the laser exposure during the fabrication of the matrix, which results in both an increase in crosslink density as well as protein concentration in the matrix. Terminal crosslink density is achieved at integrated high exposure dose and the relative fabrication rates were determined for these proteins. For all the proteins, the range of diffusion coefficients between the threshold for fabrication and the terminal limit is correlated with the change in matrix mesh size as determined by Flory‐Rehner swelling analysis. Both normal Fickian as well as hindered anomalous diffusion is observed depending on specific molecular interactions of the tracer dyes and protein host. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 359–368, 2004

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Construction of a laser scanning microscope for multiphoton excited optical fabrication

Cited by 25 publications

References 16 publications

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry

Cell Adhesion on Micro-Structured Fibronectin Gradients Fabricated by Multiphoton Excited Photochemistry

Properties of crosslinked protein matrices for tissue engineering applications synthesized by multiphoton excitation

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