Photonic engineering for biological study
Open Access
- Author:
- Wu, Fei
- Graduate Program:
- Electrical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 22, 2006
- Committee Members:
- Shizhuo Yin, Committee Chair/Co-Chair
Karl Martin Reichard, Committee Member
Timothy Joseph Kane, Committee Member
Zhiwen Liu, Committee Member - Keywords:
- calcium ion
confocal microscope
optical trapping
cardiac
retina - Abstract:
- My dissertation focuses on designing and developing prototypes of optical tools in the laboratory that can facilitate practical medical therapies. More specifically, this dissertation examines two novel biophotonic techniques: 1) a frequency multiplexed confocal microscope with the potential to provide rational therapy of congestive heart failure (CHF), and 2) the ¡°optical comb¡± with the potential to improve results of retina reattachment surgery and accelerate post surgical recovery. Next, I will discuss the background, design and initial experimental results of each study individually. Part I: The Frequency Multiplexed Confocal Microscope CHF is one of the largest threats to human health currently. Nearly 5 million Americans are living with heart failure, and 550,000 new cases are diagnosed each year. Observations on humans as well as experimental animal models indicate that heart cell (myocyte) contractile abnormalities partly account for pump dysfunction observed in the heart disease state which leads to CHF. Therefore, understanding the mechanisms by which contraction in a single myocyte is regulated is important in our quest for effective therapy for CHF. Recently high-resolution imaging suggests that the ion transporters involved in cardiac excitation-contraction coupling are grouped together to defined regions (t-tubules and triads) of the cardiac cell membrane. Despite the important question of whether these small domains do indeed exist in the cardiac myocytes, there is no straight-forward approach published to-date to quantify and to track simultaneous changes of calcium ion and sodium ion concentration in a living cardiac myocyte during an action potential. Fluorescence confocal microscopy is a powerful tool for life science because of its capability to optically section a thick specimen and obtain the 3-D image of that sample. However, a conventional confocal microscope requires pixel-by-pixel scanning, and as a result, has poor temporal resolution (i.e. slow imaging speed), which makes it difficult to monitor the fast dynamics in cells. To overcome the limitations of existing confocal microscope technology, this dissertation proposes a non-scanning, real-time, high resolution technique (a multi-point frequency multiplexed confocal microscope) to measure 3-D intracellular calcium ion concentration in a living cardiac myocyte. This method can be also applied to measure the intracellular sodium ion concentration, or other ions in which high quantum-yield fluorescent probes are available. The novelty of the proposed research lies in the introduction of carrier frequency multiplexing techniques which can differentiate fluorescence emitted at different spatial locations in cardiac myocyte by their modulated frequency. It therefore opens the possibility to visualize the transient dynamics of intracellular dynamics at multiple locations in cells simultaneously, which will shine a new light on our understanding of CHF. The procedure for frequency multiplexing proposed is described below. Multiple incident laser beams are focused onto different locations in an isolated rat cardiac myocyte with each beam modulated at a different carrier frequency. The fluorescence emission at each location therefore bears the same modulated frequency as the stimulation laser beam. Each fluorescence signal is sent to the photo multiplier tube (PMT) after being spatially filtered by a single mode fiber (functioning as a pinhole). Since each signal has a different carrier frequency, only one signal detector is required to collect multiple signal streams which eliminates the errors introduced by difference of multiple detectors. After taking the Fourier Transform of the collected data, multiple peaks can be found in the frequency domain. Each peak refers to a corresponding location in the sample. The temporal information of the fluorescence signal variation at each location can be obtained by demodulating the low frequency information from the carrier frequency, followed by an inverse Fourier transform. Part II: The ¡°Optical Comb¡± Retinal detachment refers to separation of the inner layers of the retina from the underlying retinal pigment epithelium. It can cause degeneration of the retina and may lead to permanent vision loss if not promptly treated and hence is considered an ocular emergency. Currently, the only treatment available for retinal detachment is surgical reattachment. Recent research findings provide a new explanation for the mechanism of visual loss due to detachment. Diffusion caused by the detached retina is but one of the factors of visual impairment, another factor could be the misalignment of the photoreceptors. During post surgery recovery, the photoreceptors and pigment epithelium regenerate and regain original contour; thus the vision may continue to improve over many months. To accelerate the recovery, ways to enhance photoreceptor realignment are required. In the second part of my dissertation, a novel technique called ¡°optical comb¡± is proposed to tackle the problem. The idea of an ¡°optical comb¡± is developed from the general working principle of the well known ¡°optical tweezers¡± in the optical literature, which can pull micro-objects through the trapping force produced by a focused laser beam. If we can manage to incident the focused laser beam onto the misaligned photoreceptors and further scan it back and forth, trapping forces that produced may be able to ¡°comb¡± the photoreceptors to be aligned, and thereby help with post surgery recovery. A series of experiments have been carried out to demonstrate the plausibility of this idea. First, several micro glass rods with size similar to human¡¯s photoreceptors (6 microns in diameter and 30 microns in length) were used. We observed that when the laser beam is focused close to one end of the micro rod originally laid on a glass coverslip, the rod is pulled to stand upright successfully, and we can manipulate the direction it faces by controlling its relative position to the laser beam. We are now experimenting with this combing technique with detached bovine retina samples to further verify its feasibility over live animal cells.