Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

Abstract

Abstract:Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology noninvasively without breaching a blood storage bag. Phys. Lett. 208(3-4), 276-282 (1993). 38. B. Kierdaszuk, I. Gryczynski, A. Modrak-Wojcik, A. Bzowska, D. Shugar, and J. R. Lakowicz, "Fluorescence of tyrosine and tryptophan in proteins using one-and two-photon excitation," Photochem. Photobiol. 61(4), 319-324 (1995). 39. S. Yoshida, T. Iizuka, T. Nozawa, and M. Hatano, "Studies on the charge transfer band in high spin state of ferric myoglobin and hemoglobin by low temperature optical and magnetic circular dichroism spectroscopy," Biochim. Biophys. Acta 405(1), 122-135 (1975).

Report on fluorescence lifetime imaging using multiphoton laser scanning microscopy targeting sentinel lymph node diagnostics

“…32,33 However, contrary to these findings, a recent report on multiphoton excited haemoglobin in stored blood revealed a long-lifetime (∼1300 ps) component in addition to a short lifetime value (∼280 ps). 37 The fluorescence lifetime values from our study are slightly higher than the values reported previously in the literature. This discrepancy could potentially be related to the metastasis microenvironment of the LN tissue, but it is most likely related to the preceding fixation and work-up of the tissue.…”

Epi-direction detected multimodal imaging of an unstained mouse retina with a Yb-fiber laser

“…21 Hemoglobin, which is present in the choroid, fluoresces at wavelengths (λ exc =535 nm, λ em =605–615 nm) that contribute to the 2PEF red channel signal, along with A2E detected in the choroid. 6,21 Because the sclera is the fibrous protective layer of the eye, the strong emission in the range of 500–565 nm is attributed to second harmonic signal generated via interaction with collagen. 3,4 …”

“…1–6 Contrast arises from the fact that a tightly focused laser beam from ultrafast pulsed laser (< 1 ps) can generate signals through a number of nonlinear optical processes such as two-photon excited fluorescence (2PEF), three-photon excited fluorescence (3PEF), second harmonic generation (SHG), and third harmonic generation (THG). In the retina, the presence of 1.07 μm stimulated 2PEF can be attributed to visual pigments like rhodopsin present in photoreceptors outer segments and visual cycle byproducts like lipofuscin and A2E (the seventh product of the visual cycle).…”

Three‐photon‐resonance‐enhanced third‐harmonic generation for label‐free deep‐brain imaging: In search of a chemical contrast

“…As a preliminary test measurement, the absorption spectrum of erythrocytes in this geometry was found to be identical to the spectrum shown in Figure a. To discriminate the third harmonic from fluorescence emitted by erythrocytes, we first compared the signals transmitted through a filter with a ≈17‐nm passband centered at 434 nm, intended for the third harmonic from a 1,305‐nm pump, and a filter with a ≈30‐nm passband centered at 470 nm—the spectral range typical of fluorescence of Hb and other fluorophores found in erythrocytes . In transmission geometry, the signal transmitted through the narrowband filter intended for the third harmonic was found to be four orders of magnitude higher than the fluorescence signal.…”

“…To discriminate the third harmonic from fluorescence emitted by erythrocytes, we first compared the signals transmitted through a filter with a ≈17nm passband centered at 434 nm, intended for the third harmonic from a 1,305-nm pump, and a filter with a ≈30-nm passband centered at 470 nm-the spectral range typical of fluorescence of Hb and other fluorophores found in erythrocytes. [26] In transmission geometry, the signal transmitted through the narrowband filter intended for the third harmonic was found to be four orders of magnitude higher than the fluorescence signal. The longitudinal profiles of the third harmonic measured as a function of the distance from the PBS-glass interface in this experiment were found to agree very well with theoretical predictions for THG ( Figure 2d).…”