Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.
Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation.
Palmes-Saloma
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National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman, 1101, Philippines.
Saloma
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D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics; D002490:Central Nervous System / Q000196:embryology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism*; D005136:Eye Proteins; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D020869:Gene Expression Profiling; D016259:Genes, myc / Q000235:genetics; D018398:Homeodomain Proteins; D007089:Image Enhancement; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009928:Organ Specificity; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012097:Repressor Proteins; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D013997:Time Factors
D016375:Antisense Elements (Genetics); D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D015347:RNA Probes; D012333:RNA, Messenger; D012097:Repressor Proteins; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin