TY - GEN
T1 - Real-time fluorescence polarization microscopy for probing local distributions of biomolecules
AU - Lee, Ji Youn
AU - Lesoine, John F.
AU - Krogmeier, Jeffrey R.
AU - Kang, Hyeonggon
AU - Clarke, Matthew
AU - Chang, Robert
AU - Sackett, Dan L.
AU - Nossal, Ralph
AU - Hwang, Jeeseong
PY - 2011
Y1 - 2011
N2 - We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)- labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.
AB - We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)- labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.
KW - BODIPY
KW - DiI
KW - absorption dipole
KW - cellular process
KW - fluorescence imaging
KW - fluorescent analog
KW - lipid domain
KW - liquid crystal variable retarder
KW - membrane dynamics
KW - polarization microscopy
UR - http://www.scopus.com/inward/record.url?scp=79955432187&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79955432187&partnerID=8YFLogxK
U2 - 10.1117/12.875612
DO - 10.1117/12.875612
M3 - Conference contribution
AN - SCOPUS:79955432187
SN - 9780819484284
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Design and Quality for Biomedical Technologies IV
T2 - Design and Quality for Biomedical Technologies IV
Y2 - 23 January 2011 through 25 January 2011
ER -