Imaging the Structure of Macroporous Hydrogels by Two-Photon Fluorescence Microscopy

Abstract

Two-photon fluorescence microscopy (TPFM) usually used to get 3-D pictures of biological systems has been applied here for the first time to macroporous hydrogels prepared by cryogelation ("cryogels"). Unlike environmental scanning electron microscopy (ESEM) which analyzes the surface of swollen samples, TPFM delivers images of successive planes in the depth of the material allowing a 3-D imaging of its structure. The macroporous hydrogels studied were poly(N-isopropylacrylamide) (pNIPA), poly(Hydroxyethyl Methacrylate-L-Lactide-Dextran) (pHEMA-LLA-D) and various co-polymeric gels of these two ones. A quantification of the macropore size distribution and the wall thickness and their modification with respect to the ratio NIPA/HEMA-LLA-D or to the temperature, in the case of pNIPA, was readily obtained.

3D ingrowth of bovine articular chondrocytes in biodegradable cryogel scaffolds for cartilage tissue engineering

“…The pore structure and morphology of HEMA–lactate–dextran‐based cryogels according to their SEM image is demonstrated in Figure 1d. In addition, we imaged the structure of the cryogels by two‐photon florescence microscopy (Chalal et al , 2009). It should be noted that the pore size distribution of the cryogels is not homogeneous.…”

Imaging Strategies for Tissue Engineering Applications

“…7A). 83,84 Also, mesenchymal stem cells embedded in various environments such as silk, 85 chitosan, 86 acellular scaffolds, 87 and rat skin 88 were monitored using two-or multiphoton fluorescence microscopy.…”

Laser scanning confocal microscopy versus scanning electron microscopy for characterization of polymer morphology: Sample preparation drastically distorts morphologies of poly(2‐hydroxyethyl methacrylate)‐based hydrogels

“…Techniques such as cryo‐scanning electron microscopy (cryo‐SEM) allow imaging of frozen hydrated samples, but these techniques are not always readily available and the procedures for sample preparation and imaging can be time consuming. Laser scanning confocal microscopy (LSCM) offers an alternative method for characterization and has been used to characterize poly(vinyl alcohol) hydrogels,16 and more recently poly( N ‐isopropyl acrylamide)17 and poly(2‐hydroxyethyl methacrylate‐ L ‐lactide‐dextran) hydrogels 18. LSCM uses a dye that is excited by light (generally from a laser source), and then emits red‐shifted light that is detected and used to construct an image.…”