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. 2024 Jun;29(Suppl 2):S22707.
doi: 10.1117/1.JBO.29.S2.S22707. Epub 2024 Jul 3.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity

Karteek Kunala  1 , Janet A H Tang  2   3 , Keith Parkins  2 , Jennifer J Hunter  2   3   4
Affiliations

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity

Karteek Kunala et al. J Biomed Opt. 2024 Jun.

Abstract

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE).

Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO.

Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter.

Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters.

Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

Keywords: adaptive optics; fluorescence lifetime imaging; in vivo imaging; lipofuscin and melanin; multispectral imaging; retinal pigment epithelium.

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Figures

Fig. 1
Fig. 1
An example of segmentation (b) of the fluorescence intensity image (a) in a 23 YO male at 12°T collected using the 532/LSC where all of the histograms within a cell are combined, and an exponential fit performed to give a single mean fluorescence lifetime in a single cell (d) compared to the mean fluorescence lifetime processed with a kernel of 7 pixels across the image and overlaid with cell borders for visual guide (c); the square shows the kernel size of 7 pixels (c). The phasor has a similar mean but the standard deviation is reduced with segmentation (e). (f) The normalized histogram of the cell-encircled and pixel-based lifetimes. (g) and (h) τm versus photon count contour plots for the unbinned pixel-wise and cell-encircled analyses, respectively.
Fig. 2
Fig. 2
A comparison of the forward and backward scans of a single acquisition in a 28 YO female at 12°T. (a) and (b) The fluorescence intensity of the forward and backward scan, respectively. (c) and (d) The segmented fluorescence intensity and (e) and (f) the τm values. (g) The phasor for the forward and backward scan. (h) The histogram of τm and (i) the difference of the forward minus the backward scan.
Fig. 3
Fig. 3
Fluorescence lifetime parameters change with age in the 765/NIR channel. (a) τm, (b) τ1 and (c) τ2 tending toward a longer lifetime with age. a1 (d) tends toward a shorter lifetime with age.
Fig. 4
Fig. 4
Lifetime parameters change with eccentricity in the 765/NIR channel. A clear increase in (a) τm, (b) τ1, and (c) τ2 and a decrease in panel (d) a1 was observed. All were statistically significant with eccentricity with p<0.001 for all parameters.
Fig. 5
Fig. 5
Phasor location moves up and to the left toward longer lifetimes with age in the 765/NIR channel.
Fig. 6
Fig. 6
Fluorescence lifetime parameters with eccentricity in the 532/LSC channel. (a) τm, (b) τ1, and (c) τ2. * denotes a statistical difference with a corrected Student’s t-test.
Fig. 7
Fig. 7
Fluorescence lifetime parameters with age in the 532/LSC channel. (a) τm, (b) τ1, and (c) τ2 shows the clear increase in lifetime with age. (d) The a1 reduce with age, which has a similar effect of increasing the mean lifetime with age.
Fig. 8
Fig. 8
Fluorescence lifetime parameters in 473/LSC channel changes with eccentricity. A clear increase in (a) τm, (b) τ1 and decrease in panel (d) a1 can be seen while (c) τ2 tends toward a longer lifetime, but the effect is weak.
Fig. 9
Fig. 9
Fluorescence lifetime parameters in 473/SSC channel with eccentricity. (a) τm tends toward longer lifetime and (b) τ1 shows a clear increase in lifetime with eccentricity. (c) τ2 does not a particular trend. And a decrease in panel (d) a1 can be seen with eccentricity.
Fig. 10
Fig. 10
In 473/SSC channel, no eccentricity effect was seen in the phasor plot. The phasor fingerprint from our AOFLIO system (taken from Tang et al.47) is also shown for comparison with the in vivo cell analysis data.
Fig. 11
Fig. 11
A comparison across the four spectral channels in a 28 YO female at 12°I (S3). (a)–(d) The fluorescence intensity for the 765/NIR, 532/LSC, 473/LSC, and 473/SSC, respectively. (e)–(h) The corresponding fluorescence lifetime. (i) The histogram of the τm values for each channel and (j) is the phasor data for each spectral channel.
Fig. 12
Fig. 12
A comparison of phasor coordinates across the four spectral channels reveals the difference with eccentricity and wavelength. The phasor data shifted up and to the left with eccentricity in 765/NIR, 532/LSC and 473/LSC while 473/SSC did not show any clear trend. The 532/LSC and the 473/LSC lie along the same line just shifted showing the correlation between the two channels.
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