TY - JOUR
T1 - Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection
AU - Hesch, Clemens
AU - Hesse, Jan
AU - Schütz, Gerhard J
N1 - Funding Information:
This work was supported by the Austrian Science Fund (FWF) and the GEN-AU project of the Austrian Federal Ministry for Science and Research. We thank Hannes Stockinger and Vaclav Horejsi for providing mAbs, Fab fragments and plasmids, and Julian Weghuber for transfections.
PY - 2008/7/15
Y1 - 2008/7/15
N2 - We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to ∼10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.
AB - We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to ∼10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.
KW - Microscopy, Fluorescence, Multiphoton/instrumentation
KW - Equipment Design
KW - Spectrometry, Fluorescence/instrumentation
KW - Lighting/instrumentation
KW - Protein Array Analysis/instrumentation
KW - Molecular Probe Techniques/instrumentation
KW - Colocalization
KW - Biochip
KW - Fluorescence
KW - Microarray
KW - Single-molecule microscopy
UR - http://www.scopus.com/inward/record.url?scp=43449094329&partnerID=8YFLogxK
U2 - 10.1016/j.bios.2008.02.019
DO - 10.1016/j.bios.2008.02.019
M3 - Article
C2 - 18396033
SN - 0956-5663
VL - 23
SP - 1891
EP - 1895
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
IS - 12
ER -