Measurement of intrinsic optical backscattering characteristics of cells using fiber-guided near infrared light

Abstract

Background: Intrinsic optical signals (IOS), which reflect changes in transmittance and scattering light, have been applied to characterize the physiological conditions of target biological tissues. Backscattering approaches allow mounting of the source and detector on the same side of a sample which creates a more compact physical layout of device. This study presents a compact backscattering design using fiber-optic guided near-infrared (NIR) light to measure the amplitude and phase changes of IOS under different osmotic challenges.

Methods: High-frequency intensity-modulated light was guided via optic fiber, which was controlled by micromanipulator to closely aim at a minimum cluster of cortical neurons. Several factors including the probe design, wavelength selection, optimal measuring distance between the fiber-optical probe and cells were considered. Our experimental setup was tested in cultured cells to observe the relationship between the changes in backscattered NIR light and cellular IOS, which is believed mainly caused by cell volume changes in hypo/hyperosmotic solutions (+/- 20, +/- 40 and +/- 60 mOsm).

Results: The critical parameters of the current setup including the optimal measuring distance from fiber-optical probe to target tissue and the linear relationship between backscattering intensity and cell volume were determined. The backscattering intensity was found to be inversely proportional to osmotic changes. However, the phase shift exhibited a nonlinear feature and reached a plateau at hyperosmotic solution.

Conclusions: Our study indicated that the backscattering NIR light guided by fiber-optical probe makes it a potential alternative for continuous observation of intrinsic optical properties of cell culture under varied physical or chemical challenges.

Figure 1

The overall block diagram of the high-frequency modulation optical system operated with near infrared light source and PMT detector.

Figure 2

Schematic diagram of a fiber-optical probe made by binding two parallel optical fibers together for detecting the optical changes of overlapping area of cortical neurons. (a) (upper part) The half-angle of light cone of a fiber is characterized by the numerical aperture (N.A.). With the half-angle and source/detector distance determined, the minimum distance from probe tip to dish that makes two light cones overlapping can be observed as the detection area. (b) The system setup is designed for manipulating the fiber-optical probe at varied distances from the probe tip to the culturing cells as well as for cellular experiment under varied osmotic pressures. The scattering photons are captured by PMT which can be correlated with the cell coverage of the cellular image capturing by CCD (shown at left lower corner) for calibration curve. (c) Expanded view and labeling of overlapping area for calculating the optical detection region.

Figure 3

The relationship between the averaged intensity and phase data measured at various distances from the tip of fiber-optical probe to the surface of culture dish. The sigmoid-like trend in the intensity (-o-) of optical output is found with the increase of measuring distance, which peaks around 1250 μm. Opposite trend can be observed in the phase information, with a minimum around 1250 μm.

Figure 4

The linear relationship between the mean light intensity versus the cell coverage of cellular image within the overlapping detection area.

Figure 5

The changes of scattering light in response to a 7-min exposure to ± 20, ± 40 and ± 60 mOsm of osmotic challenges. The time-course plots of (a) the relative amplitude (ΔRa) and (b) relative phase shift (ΔRp) under different osmotic conditions. The negative ΔRp indicates the leading in phase compared to that of cells before osmotic challenge.

Figure 6

The largest change in light intensity and phase shift to the different levels of osmotic change. (a) shows an inverse relationship between osmolality and relative intensity (ΔRa) (b) The relative phase shift (ΔRp) decreases with the decrease of osmotic pressure but reaches a plateau at high osmotic pressure.