Dual color localization microscopy of cellular nanostructures.
The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length-scales of 30 nm.
Gunkel
Manuel
M
Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany.
Erdel
Fabian
F
Rippe
Karsten
K
Lemmer
Paul
P
Kaufmann
Rainer
R
Hörmann
Christoph
C
Amberger
Roman
R
Cremer
Christoph
C
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002477:Cells / Q000648:ultrastructure*; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure*; D042002:Chromatin Assembly and Disassembly / Q000502:physiology; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012984:Software; D000097573:Red Fluorescent Protein
D000251:Adenosine Triphosphatases; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D049452:Green Fluorescent Proteins; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C468783:H2AX protein, human; C409462:SMARCA5 protein, human