Compact, non-invasive frequency domain lifetime differentiation of collagens and elastin

Abstract

a b s t r a c tChanges in the composition of type I and type III collagen in tissue can shed light on various diseases. However, many of the current collagen detection techniques require invasive and destructive tissue sampling. In this study, a low cost, low complexity light emitting diode (LED) based system was developed to realize both non-invasive detection and specific discrimination of collagen and elastin variations in tissue based on fluorescence lifetimes. Modulated LED excitation was applied to frequency domain (FD) fluorescence lifetime spectroscopy to calculate tissue autofluorescence lifetimes. Using this method, fluorescence lifetimes from collagen type I versus type III were clearly separated at 3.95 ns and 5.01 ns, respectively, distinct from the elastin lifetime at 6.78 ns. The probe was tested on bovine ocular tissues, with cornea showing much shorter average lifetime of 4.27 ns than sclera at 7.48 ns. Furthermore, measurements of an 8 mm murine skin wound at 14 days post-wounding also showed distinct, longer average lifetimes at 9.74 ns versus normal skin at 6.72 ns. This FD tissue detection technique can potentially offer a way to examine tissue structures and discern the underlying pathology nondestructively.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques

“…A few of the more recent studies of this type specifically concerned with eye tissue are [2, 8–12]. Imaging tissue microstructure that is under load and deforming is more difficult and fewer works are available.…”

Three-dimensional printed miniaturized spectral system for collagen fluorescence lifetime measurements

“…Spectral-lifetime results in Table 3, which ranged between 2 and 4 ns for type I and 3 and 5 ns for type III, compared well with lifetime measurements from other collagen autofluorescence reports. [16][17][18]46,47 Lifetimes increased from 400 nm, to 420 nm, and to above 435 nm for both collagens. Collagen III had a longer lifetime Fig.…”

“…After the phase angles and modulation depth ratios were calculated from 10 through 60 MHz, the data were fitted to a multiexponential model, 18,45 where the fluorescence impulse response function was described in Eq. (1).…”

“…This impulse response function can be measured in time domain using a short excitation pulse, or using a time-varying sinusoidal intensity in frequency domain. 18,35,36 The frequency domain method preserves the modulation frequency of the sinusoid, while changing the modulation amplitude and phase as a function of the specific fluorophore's impulse response decay. This is analogous to a linear time-invariant system encountered in electrical circuits.…”

“…[5][6][7][8] Unique methods such as laser-induced fluorescence and multiphoton excitation are becoming popular as label-free detection techniques, which have already been applied to ocular, skin, and liver tissue microscopies, [9][10][11][12][13][14][15][16][17] in addition to our previous work on differentiating collagen types via fluorescence lifetimes. 18 However, these aforementioned techniques are not designed for clinical use, and their bulk and complexity reduce their usability in applications such as wound monitoring and noninvasive detections. Motivated by the benefits and limitation of noninvasive techniques, we designed a compact, low-cost optical system based on frequency domain fluorescence lifetimes to measure tissue collagen ( Fig.…”