Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section

Abstract

Received October 14, 2005; revised January 7, 2006; accepted January 9, 2006; posted January 12, 2006 (Doc. ID 65391) The two-photon excitation action cross section of Hc-Red fluorescent proteins (Hc-RFPs) is measured and found to be of the same order as that of enhanced green fluorescent proteins. With a 618 nm emission wavelength in the far-red region and with an excitation wavelength around 1200 nm, Hc-RPF-based two-photon fluorescence microscopy (2PFM) can offer deep penetration capability inside live samples and is ideal for in vivo gene expression study and biomolecular imaging in live objects. In vivo 2PFM of the developing heart deep inside a transgenic zebrafish embryo tagged by Hc-RFP is also successfully demonstrated.

In vivo two-photon microscopy to 1.6-mm depth in mouse cortex

“…In the last 15 years TPM has enabled, in many fields for the first time, direct visualization of the behavior of cells in their natural environment. 2 For example, TPM has proven to be well-suited for a variety of imaging applications deep within intact or semi-intact tissues, as demonstrated in the studies of neuronal activity 3 and anatomy, 4 developing embryos, 5 and tissue morphology and pathology. 6,7 When compared to one-photon confocal microscopy, a factor of 2 to 3 improvement in penetration depth is obtained.…”

Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues

“…The imaging depth at 1280 nm excitation was deeper than at a lower wavelength (Kobat et al 2009), and lowest light attenuation occurred at 1200-1300 nm excitation (Chen et al 2002;Tsai et al 2006). Some reports showed an improved imaging depth using infrared lasers.…”

“…Some reports showed an improved imaging depth using infrared lasers. The far-red FP was excited with a Ti:sapphire laser at 1120-1230 nm excitation from an optical parametric oscillator (Tsai et al 2006) and a Cr:forsterite laser at −1300 nm excitation (Kobat et al 2009). Recent reports demonstrated high resolution of subcortical structures within an intact mouse brain using noninvasive three-photon fluorescence microscopy at 1700 nm excitation (Horton et al 2013).…”

Photonic Needles for Light Delivery in Deep Tissue-like Media

“…The most notable methods attempting to address invasiveness issues while achieving deep tissue light delivery are multiphoton stimulation 1, 23–28 , and microfabricated multisite light delivery probes 3–6 . Multiphoton light delivery is minimally invasive, but depths greater than 2 mm are not resolvable without the assistance of more invasive methods.…”