Widefield microscopy provides high quality multi-color fluorescence imaging comparable to confocal microscopy but in a more time efficient and cost effective manner. A motorized stage and integrated software allow multi-color imaging of up to three stains on fixed bovine cells in less time than a confocal. Quantitative analysis tools within the software allow measurement of fluorescence intensity, distribution and 3D reconstruction from z-stack images. Widefield microscopy is thus a suitable alternative to confocal microscopy for quantitative fluorescence applications.

1. Getting the most from WIDEFIELD MICROSCOPY
Shagufta Naz & El-Nasir Lalani
Department of Pathology & Laboratory Medicine,
The Aga Khan University, Karachi.
Introduction
Objective
Results
• To highlight the strength of widefield microscopy.
• New generation widefield microscope with a fully motorized stage
and integrated software gives much faster result than a confocal
microscope does.
• At present widefield microscopy is not only used for multi-color
imaging and live cell imaging but also in 3D imagery where a confocal
microscope used to have the monopoly.
• Images were captured using Nikon Ti-E inverted microscope and
DS-Qi2 monochrome camera.
• For multicolor imaging: bovine pulmonary artery endothelial cells
(BPAEC) stained with Mito Tracker® Red (mitochondria), Alexa
Fluor® 488 phalloidin (F-actin) and DAPI (nuclei) were used.
• Acquisition software NIS-Elements version 4.5 was used for imaging
and post imaging quantitative analysis.
Acknowledgement: We acknowledge Qualitron corporation for providing prepared stained slides and DS-Qi2 monochrome camera.
• A widefield microscope with motorized stage and integrated software
is an ideal economical imaging tool for quantitative fluorescence.
• Multi-color
Imaging: Up
to three color
imaging was
performed in
lesser time
than a
confocal
microscope
does.
• The quality
of images
captured via
widefield
Methodology
Results
Figure 1. Three color widefield fluorescence microscopy (top) confocal microscopy (bottom).
• Z-stacking: Cells do not scatter much light resulting in clear
image at the focal plane, hence no need to deconvolve (Fig 5).
• Intensity Surface Plot: is used to
show the intensity distribution of
an image with the height of the z-
axis line (Fig 3).
Figure 3. Surface intensity plot of a 2D image.
• It is used to quantitate the
expression of cell surface marker in
terms of fluorescence in a 2D image.
• ROI statistics:
gives details
of ROI (Fig
4), is used to
quantitate the
fluorescence
in a defined
area.
Figure 4. Quantitative data of Alexa Fluor
488 stained Actin marked as red in a
defined region.
• Brown, C.M. etal. 2013. Methods Mol Biol. 931:29-59.
• An intensity line
profile
measurement
shows the intensity
distribution on a
defined line (Fig 2).
Figure 2. Measurement of fluorescence
intensity for three colors.
Figure 5. Fifteen Z-stacks arranged in tile form
(left) Seven selected Z-stacks (optical sections)
0.3μm each were used in 3-D reconstruction
(right).
microscope was comparable to confocal images (Fig 1).
• It can be performed on fixed and live cells stained with fluorescent
dyes and fluorescent labelled antibodies. However, for high resolution
imaging, dyes with high quantum efficiency are recommended such as
Alexa Fluor.
Conclusion
Reference
ID: 16.16