New Instrumentation for Use in Excitation-Emission Fluorescence-Polarization Measurements

1993 ◽  
Vol 47 (10) ◽  
pp. 1548-1554 ◽  
Author(s):  
Kevin A. Destrampe ◽  
Gary M. Hieftje
Keyword(s):  
Fluorescence Polarization ◽  
Interference Filter ◽  
Excitation Wavelength ◽  
Fluorescence Excitation ◽  
Excitation Light ◽  
Polarization Measurements ◽  
Excitation Emission Matrix ◽  
Contour Plots ◽  
New Instrumentation ◽  
Multicomponent Sample

A fluorescence excitation-emission matrix (EEM) is an excellent way to determine a specific substance in a multicomponent sample. In the present investigation, a novel, rapid-scanning spectrofluorometer has been developed for measuring an EEM; the instrument employs a 0.35-meter monochromator for choosing the excitation wavelength and a continuously variable interference filter for selecting the emission wavelength. With the system, a 51 × 93 excitation-emission matrix can be collected in 3.2 min, and detection limits of 500 ng/L are obtained for fluorescein dye in ethanol. Fluorescence polarization measurements in the plane parallel and perpendicular to the polarized excitation light adds another dimension to the instrument for characterizing a sample. The instrument is controlled by an IBM XT computer although data analysis is performed on a Macintosh computer with Spyglass software. A series of two-dimensional contour plots and multidimensional excitation-emission-polarization plots can be rapidly generated.

Excitation-Emission Matrix Scanning Spectrofluorometer

1991 ◽  
Vol 45 (10) ◽  
pp. 1721-1725 ◽  
Author(s):  
Yasuhiro Senga ◽  
Shigeo Minami
Keyword(s):  
Detection Limit ◽  
Interference Filter ◽  
Photomultiplier Tube ◽  
Excitation Source ◽  
Excitation Wavelength ◽  
Fluorescent Signal ◽  
Excitation Emission Matrix ◽  
Grating Monochromator ◽  
Lock In ◽  
Rapid Scanning

A compact rapid-scanning spectrofluorometer specifically designed to acquire an excitation-emission matrix (EEM) has been developed. A conventional grating monochromator is used for selecting the excitation wavelength. The induced fluorescence is monitored with the use of a variable circular interference filter. The EEM is acquired through rapid scanning of the fluorescent wavelength achieved via rotation of the filter in conjunction with slow scanning of the excitation wavelength using a computer. A Xe-arc lamp modulated at 540 Hz is used as an excitation source. The fluorescent signal is then detected by a photomultiplier tube whose output is integrated by a lock-in amplifier. The excitation flux is simultaneously monitored with the use of a quantum counter to compensate for the fluctuation of the lamp intensity. A 26 × 31 element EEM can be acquired within a minimum time of 4 min at a detection limit of 2 μg/L with the use of fluoresceine in ethanol as sample. The performance of our EEM scanning spectrofluorometer is further evaluated for other samples of different components.

scholarly journals Fluorescence Polarization Measurements to Probe Alignment of a Bithiophene Dye in One-Dimensional Channels of Self-Assembled Phenylethynylene Bis-Urea Macrocycle Crystals

2017 ◽  
Vol 121 (33) ◽  
pp. 18102-18109 ◽  
Author(s):  
Preecha Kittikhunnatham ◽  
Bozumeh Som ◽  
Vitaly Rassolov ◽  
Matthias Stolte ◽  
Frank Würthner ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
One Dimensional ◽  
Polarization Measurements ◽  
Self Assembled

scholarly journals Degradation of multiphoton signal and resolution when focusing through a planar interface with index mismatch: Analytical approximation and numerical investigation

2018 ◽  
Vol 11 (04) ◽  
pp. 1850020
Author(s):  
Ping Qiu ◽  
Chen He
Keyword(s):  
Spherical Aberration ◽  
Numerical Aperture ◽  
Analytical Approximation ◽  
Planar Interface ◽  
Excitation Wavelength ◽  
Physical Parameters ◽  
Approximate Analytical Expression ◽  
Excitation Light ◽  
Analytical Expression ◽  
Imaging Depth

Multiphoton microscopy (MPM) is an invaluable tool for visualizing subcellular structures in biomedical and life sciences. High-numerical-aperture (NA) immersion objective lenses are used to deliver excitation light to focus inside the biological tissue. The refractive index of tissue is commonly different from that of the immersion medium, which introduces spherical aberration, leading to signal and resolution degradation as imaging depth increases. However, the explicit dependence of this index mismatch-induced aberration on the involved physical parameters is not clear, especially its dependence on index mismatch. Here, from the vectorial equations for focusing through a planar interface between materials of mismatched refractive indices, we derive an approximate analytical expression for the spherical aberration. The analytical expression explicitly reveals the dependence of spherical aberration on index mismatch, imaging depth and excitation wavelength, from which we can expect the following qualitative behaviors: (1) Multiphoton signal and resolution degradation is less for longer excitation wavelength, (2) a longer wavelength tolerates a higher index mismatch, (3) a longer wavelength tolerates a larger imaging depth and (4) both signal and resolution degradations show the same dependence on imaging depth, regardless of NA or immersion on the condition that the integration angle is the same. Detailed numerical simulation results agree quite well with the above expectations based on the analytical approximation. These theoretical results suggest the use of long excitation wavelength to better suppress index mismatch-induced signal and resolution degradation in deep-tissue MPM.

Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy

2017 ◽  
Vol 89 (16) ◽  
pp. 8554-8564 ◽  
Author(s):  
N. L. P. Andrews ◽  
T. Ferguson ◽  
A. M. M. Rangaswamy ◽  
A. R. Bernicky ◽  
N. Henning ◽  
...  
Keyword(s):  
Hadamard Transform ◽  
Fluorescence Excitation ◽  
Excitation Emission Matrix
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