Abstract
We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidinactin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 635-643 |
| Number of pages | 9 |
| Journal | European Biophysics Journal |
| Volume | 32 |
| Issue number | 7 |
| DOIs | |
| State | Published - Oct 2003 |
| Externally published | Yes |
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Keywords
- Evanescent wave excitation
- Microscopy
- Non-linear excitation
- Total internal reflection fluorescence microscopy
- Two-photon excitation fluorescence
ASJC Scopus subject areas
- Biophysics
Cite this
Fluorescence imaging with two-photon evanescent wave excitation. / Schapper, Florian; Goncalves, J. Tiago; Oheim, Martin.
In: European Biophysics Journal, Vol. 32, No. 7, 10.2003, p. 635-643.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Fluorescence imaging with two-photon evanescent wave excitation
AU - Schapper, Florian
AU - Goncalves, J. Tiago
AU - Oheim, Martin
PY - 2003/10
Y1 - 2003/10
N2 - We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidinactin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.
AB - We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidinactin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.
KW - Evanescent wave excitation
KW - Microscopy
KW - Non-linear excitation
KW - Total internal reflection fluorescence microscopy
KW - Two-photon excitation fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0242695823&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0242695823&partnerID=8YFLogxK
U2 - 10.1007/s00249-003-0326-7
DO - 10.1007/s00249-003-0326-7
M3 - Article
C2 - 12955359
AN - SCOPUS:0242695823
VL - 32
SP - 635
EP - 643
JO - European Biophysics Journal
JF - European Biophysics Journal
SN - 0175-7571
IS - 7
ER -