Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence

Abstract

We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

Noise characterization of broadband fiber Cherenkov radiation as a visible-wavelength source for optical coherence tomography and two-photon fluorescence microscopy

“…Thus, the performance (signal strength and σ-related SNR) of CR-High-Q in TPF is comparable to that of the spectrally sliced fs-pulse-induced SC with ~700 fs pulse-widths [9]. Using these as the references, we compare several ~600 nm sources and find that CR-LaserQuantum and CRCalmar are better alternatives to the OPO [7] (Table 2). To avoid optical alignment effects in free-space OCT [2,3], a direct comparison of various sources requires coupling them to a visible-light fiber-based OCT system.…”

“…Because most fluorophores native to biological samples can only be (efficiently) two-photon excited around or below 700 nm [6], visible ultrafast pulses are often advantageous over conventional Ti:sapphire laser pulses (700-1000 nm) in label-free TPF applications such as cancer diagnostics and in vivo imaging. For example, 590-nm pulses from an optical parametric oscillator (OPO) were used to image leukocyte trafficking in mice through tryptophan fluorescence [7]. To avoid the expensive and bulky OPO, other studies developed customized fs-pulse-induced SC sources and filtered out the visible portion to image tryptophan [8] and hemoglobin [9].…”

Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy

“…The imaging speed is 30 frames/sec and each final static image is an average of 50 frames. Details of this microscope design can be found in reference [14]. …”

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

“…The vasculature was con¯rmed by observation of leukocytes°ow in vivo and by colocalization of GFP signal in a Tie2 GFP mouse whose endothelial cells express the green°uorescent protein. 24 In Fig. 1(a) we also observed bright dermal cells residing close to the vasculature (white arrow).…”