Abstract
We built a simple and versatile setup to measure tissue ballistic and total transmission with customizable wavelength range, spatial resolution, and sample sizes. We performed ballistic transmission and total transmission measurements of overlying structures from biological samples ex vivo. We obtained spatially resolved transmission maps to reveal transmission heterogeneity from five microscale tissue samples: Danionella skin, mouse skull bone, mosquito cuticle, wasp cuticle, and rat dura over a wide spectral range from 450 nm to 1624 nm at a spatial resolution of ∼25 µm for ballistic transmission measurements and ∼50 µm for total transmission measurements. We expect our method can be straightforwardly applied to measuring the transmission of other samples. The measurement results will be valuable for multiphoton microscopy. The total transmission of a sample is important for the collection of multiphoton excited fluorescence and the assessment of laser-induced sample heating. The ballistic transmission determines the excitation power at the focus and hence the fluorescence signal generation. Therefore, knowledge of ballistic transmission, total transmission, and transmission heterogeneity of overlying structures of animals and organs are essential to determine the optimal excitation wavelength and fluorophores for non-invasive multiphoton microscopy.
Highlights
Optical imaging with near-infrared (NIR) light [1,2,3,4,5] reduces light scattering in biological tissue
The results shown here will provide valuable guidance for high-resolution optical imaging such as Multiphoton microscopy (MPM), where the ballistic transmission and total transmission are essential for determining the optimal excitation wavelength and fluorophores
The Danionella was euthanized in benzocaine solution and was completely submerged in phosphate-buffered saline (PBS)
Summary
Optical imaging with near-infrared (NIR) light [1,2,3,4,5] reduces light scattering in biological tissue. Non-invasive MPM of intact animals or organs [2,7] requires knowledge of optical transmissions of the overlying structures over a wide spectral range: ballistic transmission at the NIR excitation wavelength (∼800 nm to ∼1700 nm) determines the amount of excitation light at the focus and the multiphoton excited signal [4]; total transmission at the excitation wavelength governs sample heating [8,9]; total transmission in the visible range (∼400 nm to ∼700 nm) or at the fluorescence emission wavelengths affects fluorescence signal collection efficiency [10,11,12]. While optical properties for some tissues at NIR have been reported [13,14,15,16], optical transmission of a large number of biological samples, at the long wavelengths (>1100 nm), are often not readily available [15,17]
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