RESUMO
On the basis of an infrared femtosecond Cr:forsterite laser, we developed a semiquantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly differentiated hepatocellular carcinoma (HCC) tissues on average showed 3.7 times lower concentration of bilirubins than the corresponding nontumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than that of nontumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of nontumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.
Assuntos
Bilirrubina/análise , Bilirrubina/metabolismo , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular Tumoral , Dimerização , Humanos , Fígado/química , Fígado/patologia , Neoplasias Hepáticas/química , Microscopia de Fluorescência por Excitação Multifotônica , Técnicas de Diagnóstico Molecular , Neoplasias Bucais/diagnósticoRESUMO
Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi- THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.
RESUMO
This paper reports a facile and effortless method to realize three-dimensional (3D) molecular third-harmonic-generation (THG) microscopy through the technique of resonance enhancement with absorbing dye. Hematoxylin, a popular absorbing stain, is applied as an example to verify the multiphoton resonant enhancement based on the 1230 nm excitation light and can selectively enhance THG yield at cell nuclear sites in the studied specimens, serving as a cell nucleus contrast agent. It is concluded that combining THG microscopy with the mature staining technique can readily achieve 3D molecular imaging without using fluorescence.