Two-photon microscopy: visualization of kidney dynamics.
The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.
Ashworth
S L
SL
Department of Medicine, The Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Sandoval
R M
RM
Tanner
G A
GA
Molitoris
B A
BA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D004903:Erythrocyte Aggregation; D005456:Fluorescent Dyes / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D021621:Imaging, Three-Dimensional; D007511:Ischemia / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D007668:Kidney / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D018715:Microscopy, Video; D055095:Optics and Photonics; D012984:Software; D013997:Time Factors
D005456:Fluorescent Dyes; D008164:Luminescent Proteins