Extending immunofluorescence detection limits in whole paraffin‐embedded formalin fixed tissues using hyperspectral confocal fluorescence imaging

Abstract

SummaryA major problem in microscopic imaging of ex vivo tissue sections stained with fluorescent agents (e.g. antibodies, peptides) is the confounding presence of background tissue autofluorescence. Autofluorescence limits (1) the accuracy of differentiating background signals from single and multiple fluorescence labels and (2) reliable quantification of fluorescent signals. Advanced techniques such as hyperspectral imaging and spectral unmixing can be applied to essentially remove this autofluorescent signal contribution, and this work attempts to quantify the effectiveness of autofluorescence spectral unmixing in a tumour xenograft model. Whole-specimen single-channel fluorescence images were acquired using excitation wavelengths of 488 nm (producing high autofluorescence) and 568 nm (producing negligible autofluorescence). These single-channel data sets are quantified against hyperspectral images acquired at 488 nm using a prototype whole-slide hyperspectral fluorescence scanner developed in our facility. The development and further refinement of this instrument will improve the quantification of weak fluorescent signals in fluorescence microscopy studies of ex vivo tissues in both preclinical and clinical applications.

Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti‐inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia

“…Although previous reports detail an observed increase of IL‐1β IR within the dorsal horn of the spinal cord after nerve ligation with chromic gut or silk sutures (Hashizume et al 2000), detecting statistically significant changes in IL‐1β IR has been problematic. Meanwhile, the use of spectral analysis procedures in other studies has demonstrated increased accuracy and sensitivity for the detection of cell‐specific markers (Constantinou et al 2009; Mahad et al 2009; Andres et al 2010). We therefore made direct comparisons between standard fluorescent analysis with Image J software and spectral image analysis software to identify the most sensitive method to quantify immunoreactive proteins of interest.…”

Filter-based method for background removal in high-sensitivity wide-field-surface-enhanced Raman scattering imagingin vivo

“…In such cases, it will likely be necessary to apply a more rigorous linear spectral-unmixing algorithm, as we have used previously in hyperspectral fluorescence imaging. 13 We are currently investigating this for SERS imaging, using multiple discrete BP filter bands for each reporter. We are also currently implementing a modified version of the instrument, using a fast tuneable narrow BP filter with high transmission efficiency.…”

Qualitative Identification of Fish Meal and Meat Bone Meal via Fluorescence Spectral Imaging

“…Spectral imaging has been widely applied since its discovery in various fields, such as food science (Zhu et al 2013;Peng et al 2010), traditional Chinese drug detection (Hang et al 2010;Zhao et al 2010), medical molecular biology (Gendrin et al 2007), and immunofluorescent medicine (Constantinou et al 2009). FSI refers to a technique in which images are collected, displayed, processed, and analyzed through multiple spectral channels.…”