the only naturally 
 fluorescent amino acids are the aromatics , i.e. , 
 tryptophan , tyrosine , and phenylalanine , but in general tryptophan 
 fluorescence dominates tyrosine fluorescence , and both dominate phenylalanine 
 fluorescence . the endogenous fluorescence from 
 tryptophan has been used to study properties of proteins since the 
 original observation by weber . tryptophan usually dominates 
 protein emission , not only because it has a higher extinction coefficient 
 than tyrosine ( 5579 m cm at 
 279 nm for tryptophan versus 1405 m cm at 275 nm for tyrosine ) but also because tyrosine can be so readily 
 quenched in the protein matrix . fluorescence methodologies have been 
 used for several decades to study protein oligomerization and aggregations . a comparatively modern method to determine protein oligomerization 
 and association  dissociation equilibrium at low concentration 
 levels originates from the analysis of fluctuations in fluorescence 
 intensity . techniques based on fluctuations in fluorescence intensity , 
 namely single point fluorescence correlation spectroscopy ( fcs ) , raster 
 image correlation spectroscopy ( rics ) , photon counting histogram ( pch ) , 
 and number and brightness ( n&b ) , have been used to study protein 
 interactions both in vitro and in vivo . one of the main drawbacks to these methods , however , is that they 
 almost always require addition of an external fluorophore , either 
 an organic or inorganic molecule or a fluorescent protein . tryptophans 
 are endogenously present in almost all proteins , and hence it would 
 be useful if the fluorescence from tryptophan residues could be used 
 for the fluorescence fluctuation based techniques . the main 
 difficulties of using tryptophan as a fluorescent probe 
 in the fluctuation based techniques are severalfold . these difficulties 
 include the need for ultraviolet excitation , the optical transmission 
 properties of the components of the commercial laser scanning microscope , 
 and the relative low brightness of the tryptophan fluorescence . the 
 laser sources directly capable of excitation at 280 nm are very limited , 
 and fcs requires a highly focused excitation source , like that provided 
 by a laser . a second option involves using an optical parametric oscillator 
 ( opo ) to achieve two - photon excitation by doubling an original wavelength 
 of 560 nm . instead , 
 we chose to use three - photon excitation by utilizing the 840 nm emission 
 of a commercially available titanium the three - photon excitation has a very low probability 
 of absorption and requires higher power than the two - photon excitation . however , this power is still easily achievable with commercial ti  sapphire 
 lasers . as mentioned , the transmission 
 properties of the optics of commercial 
 laser scanning microscopes present a problem for work with ultraviolet 
 light wavelengths . the scanning mirrors and the lenses have good transmissions 
 above 400 nm , but the transmitted signal decreases dramatically in 
 the uv . the objectives usually used for the excitation and collection 
 of fluorescence in the epi configuration of a microscope do not transmit 
 below 370 nm , except for quartz objectives . combinations of these 
 optical elements result in low efficiency of photon detection in the 
 uv range of tryptophan emission . thus , for excitation using uv lasers , 
 both excitation and emission efficiencies are adversely affected . however , for multiphoton excitation , the excitation is not in the 
 uv and only the emission suffers . to circumvent this difficulty , we 
 used the home - built diver ( deep imaging via enhanced - photon recovery ) 
 microscope , originally designed for deep tissue imaging , to measure intensity fluctuations resulting from tryptophan fluorescence . the excitation part of the diver microscope has the regular upright 
 configuration of a commercial microscope . the three - photon excitation 
 of tryptophan requires an excitation wavelength near 840 nm , and this 
 wavelength transmission is not affected by the objective . the high 
 uv sensitivity of diver comes from the simplicity and lack of optics 
 in the detection part of the microscope . the sample is placed directly 
 above the filter wheel assembly and the filter wheel assembly is housed 
 on top of the wide area pmt detector . thus , the fluorescence in the 
 forward direction goes through the filter assembly and is collected 
 by the pmt ( figure 1a ) . the combination of 
 a bg39 filter ( placed to protect the pmt from direct light at 840 
 nm ) and an ug11 filter results in fluorescence detection from 300 
 to 400 nm ( magenta shaded area in figure 1b ) 
 which is suitable for tryptophan fluorescence . the simplicity of the 
 emission path of the diver results in higher sensitivity of the uv 
 emission and makes it especially suitable for uv detection , for both 
 harmonic generation and fluorescence . ( a ) the upright excitation scheme 
 and positioning of sample directly 
 on top of wide area pmt . ( b ) overlap of the transmission spectra of the filters 
 used , ug11 ( red line ) and bg39 ( black line ) , and the detection efficiency 
 of the pmt ( blue line ) . the overlapped wavelength area ( purple ) is 
 used for detection of tryptophan fluorescence . the experiments to obtain three - photon 
 excitation fcs had three 
 main objectives . the first one involved proving that three - photon 
 excitation can be achieved using a commercial ti sapphire laser 
 in the diver . to obtain evidence of the three - photon effect , log the slope 
 of the linear fit of this plot is indicative of the multiphoton effect 
 and a slope of one , two , or three indicates the one - photon , two - photon , 
 or three - photon effect , respectively . fluorescence 
 intensity traces of four different samples : n - acetyl - l - tryptophanamide ( nata ) and three tryptophan - containing protein 
 systems , -chymotrypsinogen a , rat liver phosphofructokinase , 
 and phage f1 virus , were measured as a function of increasing laser 
 power and the log  log plots of observed count rate versus excitation 
 power were constructed . figure 2 represents 
 these plots , and in each of these cases the value of the slope of 
 the linear fit has a value close to three , determining the three - photon 
 excitation . we note that the concentrations used for these experiments 
 were much higher than those used for the subsequent fcs experiments , 
 since the goal was simply to observe the effect of excitation power 
 on the count rate . excitation power dependence of ( a ) nata , ( b ) -chymotrypsinogen 
 a , ( c ) phosphofructokinase , and ( d ) phage f1 virus . the slopes of 
 the linear fits were around 3 , demonstrating three - photon excitation 
 of these molecules . once it was determined 
 that the fluorescence from the three - photon 
 excitation effect can indeed be achieved in the diver microscope the 
 next step was to determine the point spread function ( psf ) for the 
 three - photon excitation . unlabeled polystyrene beads ( d = 100 nm ) were embedded in a gelatin matrix and excited with 1038 
 nm ( /3 = 1038/3 = 346 nm ) . these beads absorb at 840 nm ( wavelength 
 of tryptophan excitation ) , but the gelatin matrix melts due to a local 
 heating effect and local motions start complicating the measurements . 
 to obtain the psf , z - stack images of these beads were measured with a pixel size of 0.012 m and z steps of 0.20 m . prior to imaging enrico gratton at the laboratory for 
 fluorescence dynamics at the university of california , irvine . simfcs 
 was also used for the data acquisition by the diver and for further 
 analysis described in this study . the size of the bead was smaller 
 than the psf , and thus deconvolution of the intensity image with the 
 size of the bead resulted in the calculation of the size of the psf , 
 0.2 m in the xy and 0.5 m at the z axis . 3d reconstruction of the z stack image of polystyrene 
 beads embedded in gelatin matrix when excited with 1038 nm laser line . the sizes of the images of beads were measured using the measuring 
 cuboid ( shown in red in the zoomed in image ) . the next step was to obtain three - photon excitation fcs and 
 study 
 the oligomerization equilibrium of the rat liver phosphofructokinase 
 ( pfk ) . rat liver phosphofructokinase is believed to exist as a tetramer 
 in the presence of mgatp and in the absence of fructose-6-phosphate 
 ( f6p ) . in the presence of f6p , the tetramers associate and create 
 larger oligomers , believed to be an octamer of tetramers . there are ten tryptophans present in each monomeric unit of the 
 phosphofructokinase , and thus the tetramer and the octamer of tetramers 
 contain 40 and 320 tryptophan residues , respectively . the large number of tryptophans increases the particle 
 brightness , defined as counts per protein ( molecule ) per second , and 
 makes the fluorescence easier to detect at the low concentration of 
 protein required to have sufficient fluctuations in the excitation / emission 
 volume . the three - photon excitation fcs decays 
 were measured for the 20 and 7 m subunit ( i.e. , monomer ) concentration 
 of pfk in 20 mm mops buffer in the presence of 5 mm f6p and after 
 substitution of f6p with 5 mm atp . 5 mm mgcl2 was present 
 in all samples . the correlation is much more prominent 
 than the one in the presence of atp , and the ratio of the two g(0 ) values are 6.2 . this observation is due to 
 the fact that in the presence of f6p the major contributing species 
 to the correlation function is the octamer of tetramers . in a solution 
 containing just the octamer of tetramers , the concentration of fluorescent 
 species is one - eighth compared to the same solution containing only 
 tetramers . the amplitude of the correlation function g(0 ) is inversely proportional to the concentration , and thus the 
 octamer of tetramers in the presence of f6p has a higher correlation 
 amplitude . when f6p is substituted with atp , the large oligomers dissociate 
 and the major contributing species becomes the tetramers . thus , the 
 concentration increases and the amplitude of the autocorrelation decreases . 
 the ratio of g(0 ) values indicates this change in 
 concentration . ( a ) single point fcs decays of the phosphofructokinase 
 ( pfk ) in 
 the presence of fructose 6 phosphate ( f6p ) ( black ) and in the presence 
 of atp ( blue ) . ( b ) overlap of normalized autocorrelation functions 
 of pfk ( red ) , 100 nm polystyrene beads ( black ) . to compare the time scales of autocorrelation decays of biological 
 systems with different sizes , three - photon excitation fcs decays the normalized autocorrelation decays of these two samples are plotted 
 in figure 4b , and the red and black colors 
 represent the decays for pfk and beads , respectively . these correlation 
 decays were fitted with the following diffusion equation to obtain 
 characteristic diffusion time of these two samples:1where d is the characteristic 
 diffusion time of a biomolecule and s is the shape 
 factor and has a value of 2.5 . the octamer of tetramers are the main 
 contributors toward the fcs decays of pfk in the presence of f6p and 
 have a characteristic diffusion time of 1.45  0.1 ms . this value 
 is similar to that obtained for the correlation decays of the 100 
 nm polystyrene beads ( 1.65  0.1 ms ) . these results demonstrate 
 that three - photon excitation fcs can indeed be achieved and used to 
 study oligomerization equilibrium using the commercially available 
 ti  sapphire laser and the high sensitivity and forward geometry 
 of the diver microscope . in this study we have shown and expanded 
 the idea of using three - photon 
 excitation fcs to look at the oligomerization equilibria of proteins 
 using their intrinsic tryptophan fluorescence . we have demonstrated 
 that the combination of the uv sensitivity of the diver microscope 
 and three - photon excitation achieved by the ti multiphoton excitation was 
 used before to excite tryptophan , for fcs measurements and for imaging . the detection limits for the two - photon excitation fcs varied from 
 a few million ( highly unlikely in biological systems ) to a few hundred 
 tryptophans in each unit of the fluorescent species . in the most recent 
 paper involving measurement of fcs curves in proteins using tryptophan 
 fluorescence by sahoo et al . , the authors 
 looked at aggregated proteins containing greater than 300  000 
 tryptophan residues . in our system , to obtain a clean correlation 
 decay in 5 min acquisition time , the number of tryptophan 
 residues in a protein complex present at submicromolar oligomer concentration 
 is required to be 200 . this observation shows the extraordinary 
 sensitivity of diver combined with three - photon excitation can lead 
 to a single - point fcs measurement of proteins in buffer . this study used extrinsic label of dna base 
 analogue 2-aminopurine , and the corresponding correlation decay was 
 very noisy and was not used by the authors to calculate diffusion . currently we are trying to improve the sensitivity of the system and 
 to use this system to look at virus particles in more detail . the 
 final goal is to follow the infection process of virus particles without 
 labeling and to monitor the real time state of infection . the very 
 large number of tryptophans in viruses can be useful for the strong 
 signal - to - noise ratio and identification of virus capsid inside the 
 cells . the three - photon excitation fcs can also be extended to study 
 other protein oligomerization processes , provided there are enough 
 tryptophan to give rise to significant signal - to - noise ratio .