Patent Application: US-201615272086-A

Abstract:
a label free single or multi - photon optical spectroscopy for measuring the differences between the levels of fluorophores from tryptophan , collagen , reduced nicotinamide adenine dinucleotide . and flavins exist in brain samples from a of alzheimer &# 39 ; s disease and in normal brain samples with label - free fluorescence spectroscopy . relative quantities of these molecules are shown by the spectral profiles of the ad and n brain samples at excitation wavelengths 266 nm , 300 nm , and 400 nm . the emission spectral profile levels of tryptophan and flavin were much higher in ad samples , while collagen emission levels were slightly lower and nadh levels were much lower in ad samples . these results yield a new optical method for detection of biochemical differences in animals and humans for alzheimer &# 39 ; s disease .

Description:
fluorescence spectroscopy measures allowed electronic transitions of various chromophores in the complex tissue structure . there are several natural label free fluorophores that exist in tissue and cells which , when excited with ultraviolet light , emit fluorescence in the ultraviolet and visible regions of the spectrum . some of the absorption and emission spectra of these native endogenous fluorophore molecules are shown in fig2 ( a )-( b ) . the flavins and nadh show changes in the spectra between their oxidized and reduced state . the relatively large emission intensity from tissues and the need of broadly tunable excitation sources in the uv and visible has led researchers to develop lamp based fluorescence systems instead of lasers and now leds from 260 nm to 550 nm to excite the key biomolecules for i per these states can be excited by 1 pef which is more a surface process and 2 pef or 3 pef for deeper penetration . a basic fiber unit incorporates a fluorescence section and uses leds at 260 nm , 280 nm 300 nm , 350 nm , and 400 nm to excite tryptophan , collagen , elastin , nadh , and fad in brain disease . femtosecond ti lasers ( 700 nm to 1200 nm ) can be used to excite the key molecules ( 3 pef for tryptophan a 267 nm ); and 2 pef for collagen , nadh and flavins . see fig3 ( a ) - 5 . significant differences of emission peaks were found in these molecules in ad and normal ( n ) brain . the fluorescence intensity levels from tryptophan : ad & gt ; n ; from collagen : ad ˜ n ; from nadh : n & gt ; ad and from flavin : ad & gt ; n . these observation provides effective techniques to explore an optical diagnosis of alzheimer &# 39 ; s disease by examining the spectral profiles of various molecules in brain tissue , eye fluid , body fluids , and / or spinal fluid ex vivo and in vivo using optical fibers . mice were purchased from jackson laboratory and housed at the city college animal facility . a 2 - month - old triple transgenic ad mouse harboring ps1m146v , appswe and taup301l transgenes in a uniform strain background was used . another n mouse at the same age was used as control . the mouse was anesthetized with a mixture of ketamine and xylazine ( 41 . 7 / 2 . 5 mg / kg body weight ), then was decapitated and the brain was dissected and post - fixed overnight with 4 % formaldehyde in 0 . 1 m phosphate buffer ( pb ) and subsequently immersed in 30 % sucrose in 0 . 1 m pb for up to 48 hrs prior to slicing . the hippocampus of both ad and n brains was sliced coronally at a thickness of 1 mm , by using a brain matrix ( rwd life science inc , san diego , calif . ), and was placed in a cuvette ( sigma - aldrich , st . louis , mo .). it is well known that the fluorescence intensity i f depends on efficiency q from the radiative rate kr and nonradiative rate knr , the relationship can be written as : eq ( 1 ) for q equals to the ratio of numbers of photons emitted out to the numbers of photon pumped in ( nout / nin ). the intensity from excited molecules i f is where ω is the solid angle and n is the number of excited molecules . q value . the knr depends on the interaction of molecules with their host environments . weak interaction will lead to a small knr and give more emission intensity . when knr & gt ;& gt ; kr the emission is reduced . the fluorescence of alzheimer and n brain tissues was measured by a ls 50 fluorescence spectrometer ( perkinelmer , waltham , mass .). a xenon lamp was used as the discharge light source in the spectrometer . there are two monochromators , with the excitation monochromator able to detect light ranging from 200 - 800 nm and the emission monochromator able to detect light ranging from 200 - 650 nm . pulsed light from the xenon lamp hits a diffraction grating , which selects the wavelength being used . this light then enters through the excitation monochromator , at which point the light strikes the sample , which is stored in a cuvette and positioned between the two monochromators . after being struck by the light at the selected wavelength , the sample fluoresces , and the fluorescence light is collected on the other side through the emission monochromator . the wavelength accuracy is +/− 1 nm and the slit widths can be varied 2 . 5 nm - 15 nm and 2 . 5 - 20 nm for the excitation and emission slit , respectively . the ad and n brain samples were excited at wavelengths 266 nm , 300 nm , and 400 nm , to examine the fluorescence peaks of each of tryptophan , nadh , fad , and collagen . all measurements were performed by using a scanner ( at 100 nm / sec ), and the samples were held in cuvettes during the measurement . a 300 nm or 400 nm filter was placed in between the excitation monochromator and the sample for scans at 300 nm or 400 nm respectively , whereas the scan at 266 nm was done without a filter . the measurements of the ad and n brain samples were each taken twice with different slit widths at each excitation wavelength . the slit widths for the scans at 300 nm and 400 nm were 7 nm and 5 nm respectively for the first round of measurements , and 5 nm and 4 nm respectively for the second round . due to a lack of the filter at 266 nm , the excitation and emission slit widths were 4 mm and 3 mm respectively for the first round of measurements , and 3 mm excitation and 2 . 5 mm emission for the second round . the present study is aimed at detecting ad by measuring fluorescence intensities of multiple biomolecules , we used n and ad brain samples from mice . fig1 displays the fluorescence spectral profiles in ad and n brain samples at the excitation wavelengths 266 nm ( fig1 a ), 300 nm ( fig1 b ), and 400 nm ( fig1 c ). different excitation wavelengths were employed to determine the emission spectra of each biomolecule ( tryptophan , collagen , nadh , and flavin ), as shown in fig2 ( a ) and 2 ( b ) . table 1 summarizes the emission wavelengths for assigned molecules at peak emissions in ad and n brain tissues under different excitation wavelengths . one can use 1 pef , 2 pef and 3 pef to excite the molecules in table 1 , fig1 ( a ) shows that at excitation 266 nm the fluorescence peaks of ad and n brain tissues are at the same wavelengths ( ranging 365 - 385 nm and 460 - 490 nm ), corresponding to the wavelengths of emission peaks of collagen and nadh respectively ; peak intensities in ad brain are 73 % ( collagen ) and 41 % ( nadh ) respectively of those in n brain ( table 1 ). the levels of collagen in ad and n brains are relatively close , making it difficult to distinguish ad from n brain in this respect . an alternate way to differentiate the spectral profiles in ad or n brain is to compare the ratio of nadh intensity to collagen intensity , which is ˜ 1 : 1 in ad brain and 2 : 1 in n brain . comparing the spectral profiles ( peaks ) of collagen and nadh and their relative ratio may be an applicable method for diagnosing alzheimer &# 39 ; s disease . the scans at excitation wavelength 300 nm offer diagnostic possibilities for ad . the emission intensities of the ad and n brain tissues both peak in the range of 330 - 350 nm ( fig1 b ), which match the wavelength of the emission peak of tryptophan in fig2 ( b ) . in addition , the peak . intensity of tryptophan in ad brain tissue is 2 . 2 times higher than that in n brain tissue ( table 1 ). tryptophan , due to its properties of native fluorescence , has been employed in a vast array of biomedical imaging processes , including the diagnosis of breast cancer and other types of cancer . this vast disparity of tryptophan fluorescence levels in ad and n mouse brain scans proposes another method for ad diagnosis . it appears that tryptophan has more kr or less knr which may be due to the tissue environment . the scan taken at excitation wavelength of 400 nm excited flavin in ad and n brains . in both ad and n brain tissues , the wavelength of peak emissions were found in the range of 560 - 580 in ( fig1 c ), consistent with the emission wavelength of nadh and flavin in fig2 ( b ) . the peak intensity of nadh and flavin are both 3 . 3 - fold higher in ad brain compared to n brain ( table 1 ). it appears that tryptophan emission efficiency is more in ad than n which may be due to fewer interactions to the host molecules in the environment in ad brain tissue and the nonradiative knr interaction was reduced or kr was increased . the significant difference of flavin emission peaks , in addition to the fact that the excitation wavelength at 400 nm is less harmful to cells than shorter wavelength , makes scans at 400 nm another promising prospect for alzheimer &# 39 ; s diagnosis , especially in combination with the scans at excitation wavelengths 266 nm and 300 nm as discussed above . the future direction could use time resolved fluorescence which gives fluorescence rate ( k f = kr + knr ) and combines with longer wavelength multiphoton excitation which offers deeper tissue penetration . this current study is the first teaching to investigate the fluorescence spectra of collagen , nadh , tryptophan , and flavin in alzheimer and n brain tissues of a mouse model for human brain . it demonstrates significant differences of emission peaks of these molecules in ad and n brain . the fluorescence intensity levels from tryptophan : ad & gt ; n ; from collagen : ad ˜ n ; from nadh : n & gt ; ad and from flavin : ad & gt ; n . this work provides effective techniques to explore diagnosis of alzheimer &# 39 ; s disease by examining the spectral profiles of various biomolecules . 1 . alzheimer &# 39 ; s association , “ 2013 alzheimer &# 39 ; s disease facts and figures ,” alzheimer &# 39 ; 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