We describe an 'open' design methodology for wide-field fluorescence, confocal and fluorescence lifetime imaging microscopy (FLIM), and how the resulting microscopes are being applied to radiation biology and protein activity studies in cells and human tissue biopsies. The design approach allows easy expansion as it moves away from the use of a monolithic microscope body to small, commercial off-the-shelf and custom made modular components. Details have been made available under an open license for non-commercial use at http://users.ox.ac.uk/~atdgroup. Two radiobiology 'end-stations' have been constructed which enable fast radiation targeting and imaging of biological material opening up completely novel studies, where the consequences of ionising radiation (signaling and protein recruitment) can be studied in situ, at short times following irradiation. One is located at Surrey University, UK, where radiation is a highly focused in beam (e.g. protons, helium or higher mass ions). The second is installed at the Gray Institute linear accelerator facility, Oxford University, which uses sub-microsecond pulses of 6 MeV electrons. FLIM capabilities have enhanced the study of protein-protein interactions in cells and tissues via Förster Resonance Energy Transfer (FRET). Extracting FRET signals from breast cancer tissue is challenging because of endogenous and fixation fluorescence; we are investigating novel techniques to measure this robustly. Information on specific protein interactions from large numbers of patient tumors will reveal prognostic and diagnostic information.
We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery
(FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a
widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2)
as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple
wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2
mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical
working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized
color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image
alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous
detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both
fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the
advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the
laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node
detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue
and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of
surgical procedures.
Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in
efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map.
Near Infrared Spectroscopy (NIRS) is a powerful method for non-invasive mapping of cerebral functional activation. We have developed an NIRS instrument that is portable, inexpensive and lightweight consisting of an array of light emitting diodes (LEDs) and photodiodes (PDs) mounted on a flexible printed circuit board (PCB). The flexibility and portability of the instrument makes it easy to apply to subjects ranging from premature babies in intensive care to adults. The flexible PCB array consists of 48 LEDs operating at two different wavelengths (780nm & 880nm) together with 14 photodiodes. Transimpedence amplifiers for each of the PDs are located on the flexible pad to minimise noise pick up. The LEDs are pulsed (10ms) at a peak optical power of 20mW, while a sample and hold circuit monitors the voltages at all of the PDs. The array is also encapsulated in black silicon rubber, except for the regions directly above the LED's and PD's, which have a clear silicone rubber cover. The sensor array is attached to the electronics by two one metre long flexible ribbon cables. The monitoring circuit provides medical grade electrical isolation between the patient and computer. Studies have been conducted on phantoms to test the penetration depth of the sensor array for two different separations of LEDs and PDs (11mm and 27mm). The maximum depths that can be probed are 5mm and 11mm respectively. This makes it suitable for studying cortical activation in babies.
Many commercial clinical Near Infrared Spectroscopy (NIRS) instruments rely upon estimates of the optical pathlength of tissue based upon the optode spacing multiplied by an average differential pathlength factor(DPF) for the tissue under investigation. Significant variation in the published DPF is the major source of error in the NIRS data quantification. To reduce this error, an inexpensive, stand-alone, single wavelength, single frequency optical pathlength meter based on the homodyne phase method was constructed for clinical use with an existing NIRO 300 instrument (Hamamatsu Photonics KK). The construction and testing of the optical pathlength meter is discussed.
KEYWORDS: Sensors, Tissue optics, Signal detection, Modulation, Spectroscopy, Semiconductor lasers, Digital signal processing, Optical amplifiers, Avalanche photodetectors, Near infrared
We describe the development of a modular, multiwavelength, wideband intensity-modulated near infrared spectrometer (100MHz -1GHz) capable of simultaneous measurement of three parameters - the dc intensity, the ac intensity and the phase shift of light transmitted through tissue at a high sampling rate. The intended purpose of this instrument is the detection of fast dynamic changes in the reduced scattering coefficient of tissue due to cellular depolarization. The instrument requires sensitivity to small changes in optical pathlength and absorption in small volumes of tissue.
The laser torquemeter is an instrument designed to measure time-resolved torque on a rotating shaft. The torquemeter uses two probe laser beams, axially separated on a shaft of known mechanical properties. When the shaft rotates, the backscattered speckle patterns, observed by a photodetector, change continuously but repeat exactly with each revolution of the shaft. The shaft is rotated at a low torque level and the photodetector signal is recorded. Measurements of shaft twist are obtained by comparing the photodetector signals from a torsionally-loaded rotating shaft with the previously recorded photodetector signal. Reliable operation of the laser torquemeter depends on the repeatability of the speckle patterns scattered from the surface of the shaft. If the shaft tilts - pitch and yaw motion - as it rotates, the backscattered speckle pattern moves at a velocity proportional to twice the shaft tilt rate and the similarity between the photodetector output signal and the recorded signal is reduced. To determine typical values of tilt, measurements are taken from the drive shaft of a 4 cylinder diesel engine. In order to design a reliable torquemeter, knowledge is required of how the photodetector output and the stored reference signal de-correlate as a function of shaft tilt. This paper examines several optical configurations and an optimum configuration for achieving resistance to decorrelation due to shaft tilt is recommended. This competes an earlier study which optimized the resistance to decorrelation due to in-plane motion of the shaft. Both issues must be addressed in the design of a robust torquemeter.
Laser speckle produced from a diffuse object may be used in determining the angular position of a rotating object. When the object rotates the backscattered speckle patterns are sampled by a suitably positioned photodetector. The photodetector output signal is periodic and one period is stored in memory as a reference. Shaft position is then determined by cross-correlating this stored reference signal with the current photodetector output signal. When the shift is axially displaced the back scattered speckle patterns change and the cross-correlation between the reference signal and the current signal is reduced. The Laser Torquemeter is an instrument designed to measure the time- resolved torque transmitted in a rotating shaft and uses two probe laser beams, axially separated on a shaft of known mechanical properties. In order to design a robust Laser Torquemeter it is necessary to determine the cross- correlation of the current photodetector output signal and the stored reference signal as a function of axial shift position because the reduced cross-correlation due to axial motion results in a loss of tracking which is determined to the performance of the Torquemeter. Experiments conducted to measure the photodetector output cross-correlation show that the ratio of decorrelation displacement to beam diameter can achieve a maximum value of 0.7. For a typical axial shaft vibration of 0.6 mm peak to peak, a minimum beam diameter of 1 mm is recommended and further details of the optical configuration necessary to achieve this optimum resistance to decorrelation are suggested.
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