Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.
Conventionally, the diagnosis of hepatocellular carcinoma (HCC) is performed by qualitative
examination of histopathological specimens, which takes times for sample preparation in fixation,
section and stain. Our objective is to demonstrate an effective and efficient approach to apply
multiphoton microscopy imaging the HCC specimens, with the advantages of being optical section,
label-free, subcellular resolution, minimal invasiveness, and the acquisition of quantitative information
at the same time. The imaging modality of multiphoton autofluorescence (MAF) was used for the
qualitative imaging and quantitative analysis of HCC of different grades under ex-vivo, label-free
conditions. We found that while MAF is effective in identifying cellular architecture in the liver
specimens, and obtained quantitative parameters in characterizing the disease. Our results demonstrates
the capability of using tissue quantitative parameters of multiphoton autofluorescence (MAF), the
nuclear number density (NND), and nuclear-cytoplasmic ratio (NCR) for tumor discrimination and that
this technology has the potential in clinical diagnosis of HCC and the in-vivo investigation of liver tumor development in animal models.
By using two-photon autofluorescence (TPAF) and second harmonics generation (SHG), we imaged hepatocellular
carcinoma (HCC) biopsies from the human patients and compared them with the conventional histological biopsies. We
found that multiphoton microscopy may be used to obtain label-free images of liver tissues and may be developed into
an effective diagnostic tool for liver diseases.
Liver is the chemical factory in body responsible for important functions such as metabolism and
detoxification. When liver can not be regenerated in time to amend damages that has occurred, failure
of hepatic functions can result. Traditionally, the study of liver pathology has depended on histological
techniques, but such methods are limited to ex-vivo observation. In order to study hepatic metabolism
in vivo, we have designed a hepatic imaging chamber made of biocompatible titanium alloy (6V4Al-Ti, ELI grade). In combination with multiphoton and second harmonic generation microscopy, our
approach allows the intravital observation of hepatic intravital activities to be achieved. Processes such as hepatic metabolism and disease progression can be studied using this methodology.
In this work, intravital multiphoton microscopy is applied to the imaging of liver metabolism with the least invasiveness.
We observed intravital dynamics of the uptake, processing and excretion of the organic anion species,
6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton
microscopy and an in vivo hepatic imaging chamber which allows us to image the dynamics of hepatic metabolism.
Multiphoton images revealed that the hepatic processing of 6-CFDA is completed within approximately 50 minutes. The
images reveal the liver metabolism of the uptake and processing of 6-CFDA from the hepatocytes, and the subsequent
excretion into bile canaliculi. Our results suggest that this approach is a promising technique for investigating intravital
hepatic physiology, diseases, and metabolism.
Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.