Around-the-objective total internal reflection fluorescence microscopy

Thomas P Burghardt, Andrew D. Hipp, Katalin Ajtai

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within ∼100nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-theobjective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the ∼50nm deep evanescent field.

Original languageEnglish (US)
Pages (from-to)6120-6131
Number of pages12
JournalApplied Optics
Volume48
Issue number32
DOIs
StatePublished - Nov 10 2009

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Fluorescence microscopy
Evanescent fields
microscopy
fluorescence
Fluorescence
Glass
Field emission
glass
field emission
Optics
near fields
Microscopes
microscopes
optics
Laser excitation
Fluorophores
numerical aperture
conditioning
Prisms
submerging

ASJC Scopus subject areas

  • Atomic and Molecular Physics, and Optics

Cite this

Around-the-objective total internal reflection fluorescence microscopy. / Burghardt, Thomas P; Hipp, Andrew D.; Ajtai, Katalin.

In: Applied Optics, Vol. 48, No. 32, 10.11.2009, p. 6120-6131.

Research output: Contribution to journalArticle

Burghardt, Thomas P ; Hipp, Andrew D. ; Ajtai, Katalin. / Around-the-objective total internal reflection fluorescence microscopy. In: Applied Optics. 2009 ; Vol. 48, No. 32. pp. 6120-6131.
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