Biomedical Engineering Reference
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The toxicity of glycerol in rat skin in vivo was assessed by Mao et al. [29]. While 75% glycerol caused local
skin edema (a condition of abnormal accumulation of liquid under the skin) in 24 h, followed by tissue
suppuration and necrosis, 30% glycerol cleared the skin without an injury. Thus, it is important to balance
between concentrations of OCAs strong enough for sufficient clearing, yet gentle on tissues in vivo .
8.7 optical clearing in Kidney tissue
As a final example, we look at optical clearing of the kidney investigated by MPM fluorescence micros-
copy. The kidney tissue is composed of vast vasculatures, renal corpuscules, and tubules, all covered by
fibrous capsules. These numerous interfaces of different refractive indices result in strongly scattering
environment, where image contrast deteriorates with depth due to signal attenuation and spherical
aberrations. The latter results from yet another refractive index mismatch that we address for the first
time in this chapter—between the immersion fluid and the sample [40]. Indeed, although the sample
is homogenized in terms of the effective refractive index by the clearing agent, we now have a strong
mismatch if a water immersion objective is used. This can be improved with oil immersion objective
as demonstrated by Young et  al. [41], who investigated clearing capabilities of several glycerol-based
agents with refractive indices ranging from 1.3386 to 1.5297. These were prepared by the addition of 2%
of 1,4-diazabicyclo [2,2,2] octane (DABCO) and either PBS or benzyl alcohol in various proportions.
Additionally, kidneys were intravenously labeled with 0.2 μm fluorescent microspheres distributed
through the volume of the tissue to assess the axial resolution with depth. As expected, the penetration
depth increases with refractive index of the OCA used due to better index matching within the sample
and optimal reduction of scattering. This effect is clearly demonstrated in Figure 8.10 by XY images
taken at 25, 75, and 125 μm deep, and the reconstructed XZ projection, as imaged with 60× NA1.4 oil
(a)
(b)
XY at 25 µm
depth
XY at 75 µm
depth
(c)
(d)
XY at 125 µm
depth
Z
XZ
X
FIgurE 8.10 Two-photon microscopy of the kidney tissue labeled with Hoechst, lens culinaris, agglutinin-
fluorescein and phalloidin-rhodamine, and mounted in the media with refractive index 1.51. The image volume
is collected with Olympus 60 × NA 1.4 oil immersion objective. The pixel dimensions are 0.345 × 0.35 μm. Scale
bar = 30 μm. (Young, P. A. et al. The effects of spherical aberration on multiphoton fluorescence excitation micros-
copy. J. Microsc. 2011. 242: 157-165. Copyright Wiley-VCH verlag GmbH & co. KGaA Reproduced with permission.)
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