A robust and simple methodology for seven-colour fluorescence microscopy has recently been presented in a technical note. Interestingly, this has been achieved using a widely available and standard widefield microscope with a mercury lamp as excitation source. As such, this technique should be within the reach of any pathology laboratory, including in the developing nations.
This work, from scientists at Mahidol University, Bangkok and X-ZELL, Inc., uses commonly available chromophores, avoiding both tandem dyes due to their propensity to generate crosstalk between channels and secondary antibodies due to added steps and uncontrollable signal intensities between channels.
With a standard mercury lamp source, 8-position filter turret and carefully selected emission filters a 7-colour staining panel was established without resort to complex image processing and compensation which might otherwise compromise the ability to detect rare cell events such as sentinel circulating tumor or endothelial cells.
Based on their relative spectral properties for excitation, emission and overlap the following chromophores were chosen to permit the desired 7-colour analysis: (Brilliant Violet 421 (BV421), Brilliant Violet 480 (BV480), Alexa Fluor 488 (AF488), R-PE, PerCp, Alexa Fluor 594 (AF594)) and DRAQ5™.
To bench-test the in silico principle, A549 tumor cells were seeded into leukocytes from freshly lysed normal peripheral blood and an aliquots spun down onto a slide. Thereafter, the authors utilised X-ZELL's proprietary Cryoimmunostaining™ technologies to process the slide with six antibodies tagged with the chromophores above (BV421, BV480, AF488, R-PE, PerCp, AF594) and DRAQ5™ as the nuclear counterstain against antigens to differentiate all leukocytes, T-cells and granulocytes (CD45+; CD3+; CD45+, CD16+) from the tumor epithelial cells (panCK+, ALDH1+, vimentin+). These were optimised for relative brightness and enabled slide image acquisition in 10 seconds. In addition, microscopy is able to provide information on cell size, morphology and antigen distribution.
DRAQ5™ was chosen for its spectral performance and stoichiometric labeling of nuclear DNA. DRAQ5™ also proved compatible with the mounting buffer, and therefore simplifying the counterstaining and coverslip mounting to a single step. Usefully, the authors suggest that, a further chromophore in the far-infrared such as Alexa Fluor 750 or Alexa Fluor 800 could be added.
The concept that has been exemplified in this work offers new scope, with a standardized approach, for an economical and technologically-conservative approach to the multi-parametric investigation of diagnostic cell detection in complex clinical samples such as peripheral blood, pleural effusion, ascites or liquor. This should lend itself to the unanswered needs of multiplexing immunodiagnostics in pathology laboratories in both developed and developing nations.
Reference:
SC Bhakdi & P Thaicharoen
Easy Employment and Crosstalk-Free Detection of Seven Fluorophores in a Widefield Fluorescence Microscope.
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