Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging

Abstract

We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2.

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

“…In combining high detection sensitivity, high photostability, optimal wavelength for Ti:Sapphire laser-excitation, and an estimated achievable imaging rate of B300 frames per second (under the generic conditions defined in ref. 21), pA comprehensively outperforms other FBAs. We anticipate that the application of pA in 2P microscopy will greatly enhance the capability for imaging nucleic acids with minimal structural perturbation.…”

“…18 In a very recent study, Mikhaylov et al assessed the potential utility of several established isomorphic FBAs for multiphoton microscopy. 19 As well as reporting 2P cross sections, they used a previously derived relationship between the 2P cross section and the speed of multiphoton imaging 21 to estimate the maximum frame rate that would be achievable, in a generic scanning multiphoton microscope. The best-performing FBA in this context was found to be 6-MI, with an estimated frame rate of 200 frames per second.…”

“…The best-performing FBA in this context was found to be 6-MI, with an estimated frame rate of 200 frames per second. 21 The potential for major improvements in 2P fluorescence brightness via systematic modifications was highlighted in a study of a series of structurally-related nucleosides. 20 The 1P and 2P cross sections and spectra in that report also agree well with subsequent theoretical calculations, 22 offering the prospect of the rational design of photophysical properties.…”

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

“…A possible way to overcome these limitations is multiphoton excitation, − with potential benefits including deeper penetration into tissue in the near-IR, 3D control of excitation, and reduction of photobleaching and background fluorescence. , With the aim of reaching the ultimate single-molecule level of detection, we recently reported that an adenine analogue, pA, incorporated into single-stranded oligonucleotides could undergo two-photon (2P) excitation and as few as five molecules could be detected . Moreover, pA had enhanced photostability under 2P excitation.…”