(683e) Comparison of Hyper-Spectral Wide-Field and Confocal Fluorescence Microscopic Techniques

COMPARISION OF HYPER-SPECTRAL WIDE-FIELD AND
CONFOCAL FLUORESCENCE MICROSCOPIC TECHNIQUES

Naga Srilakshmi
Annamdevula¹, Ashley Stringfellow², Diego Alvarez^3,2, Thomas C.Rich² , Silas J. Leavesley^1,2

¹Chemical and Biomolecular
Engineering, University of South Alabama, Mobile, AL,  ²Pharmacology, College  of Medicine, University of South Alabama,
Mobile, AL,  ³Internal
Medicine, College of Medicine, University of South Alabama, Mobile, AL

Identification
and quantification of fluorescently-labeled cells in highly autofluorescent
tissues is very difficult using single-wavelength fluorescence microscopy. We
have previously used hyper-spectral imaging and linear unmixing analysis to detect
GFP-expressing cells in autofluorescent tissues. In performing these studies,
we have found significant variations in the sensitivity and specificity of GFP
detection-dependent on the optical configuration and imaging detector. The goal
of the study was to compare the effectiveness and sensitivity of these two
spectral microscopy configurations (wide-field and confocal) for identifying
fluorescently labeled cells in highly autofluorescent environment. We studied
two different hyper-spectral fluorescence microscope systems: a wide-field
system with an acoustic-optic tunable filter and electron-multiplying charge
coupled device (EMCCD) camera and a laser scanning confocal microscope with a
diffraction grating and 32-channel photo multiplier tube (PMT). These two
systems differ in their sensitivity and signal collection, detector configuration,
depth-of-field, and spectral filtering methods. To achieve this, we performed
sensitivity analysis of multiple parameters on both systems to assess the
effects of these parameters on detection sensitivity and specificity. Confocal pinhole,
laser power, PMT gain, CCD detector gain, and rate of photobleaching were the independent
parameters studied. Dependent variables included signal-to-noise ratio (SNR),
root-mean-square error (RMS), and linear spectral unmixing analysis. Spectral
image stacks were collected from both the systems. Image analysis was performed
using MATLAB, Nikon Elements, and ENVI software packages. We have obtained preliminary
results from the wide-field spectral fluorescence microscope. Spectral analysis
is currently being performed on the spectral image stacks acquired using the
confocal spectral microscope. As spectral imaging and analysis is a new
approach to detect fluorescent cells in highly autofluorescent environment,
this study will help researchers to optimize the parameters for a given
hyper-spectral microscope system. These results should be applicable to many
types of in-vitro and ex-vivo fluorescence assays.