Equipment at Biophotonics Lab
An optoelectronic instrument called a fluorometer was designed in our lab. This device is equipped with two synchronized filter wheels, one to accommodate four excitation wavelengths, the other one to house four emission filters, a bifurcated fiber optic bundle, and a photomultiplier tube for signal detection. The filter wheels communicate with the computer through a control box (Lambda- 3, Sutter instrument, CA). To control PMT gain, wheel synchronization, and data acquisition, we developed a LabView program. The bifurcated fiber bundle consists of high-grade fused silica fibers for UV transmission (Newport instrument, NJ) with distal end of 3mm inner diameter. For fluorescence spectroscopy, the peak energies of the NADH and FAD excitation spectra occur at 365nm and 436nm, respectively. The appropriate bandpass filters for NADH/FAD excitation are 365HT25/440DF20. The emission filters for NADH and FAD fluorescence are 455DF70 and 525DF50, respectively. A schematic of this instrument and a photo of it are presented in the following figure.
The cryo imaging instrument is a 3-D fluorescence imaging system for frozen tissues. Tissue status is preserved in low temperatures such as liquid Nitrogen (LN2) and the time is also captured at the freezing moment as a snapshot. This instrument is an automated image acquisition and analysis system consisting of hardware and software designed to compute fluorescence images of tissue sections. Fluorescence images of the frozen tissue block are obtained using computer-controlled microtome for slicing the tissue and a CCD camera (EXi Aqua, Qimaging, 14 bit, 1392 x 1040 resolution) to capture the images. The excitation light source is a 200W mercury arc lamp filtered at the excitation wavelength of NADH and FAD. The excitation band pass filters used for NADH is 350 nm (80 nm bandwidth, UV Pass Blacklite, HD Dichroic, Los Angeles, CA) and for FAD is 437 nm (20 nm bandwidth, 440QV21, Omega Optical, Brattleboro, VT) and the emission filters for NADH are 460 nm (50 nm bandwidth, D460/50M, Chroma, Bellows Falls, VT) and for FAD are 537 nm (50 nm bandwidth, QMAX EM 510-560, Omega Optical, Brattleboro, VT).
The schematic of this instrument and a photo of it in the lab are presented below. A motor-driven microtome sequentially sections frozen tissue whereas a mercury filtered light excites fluorophores in the exposed surface of the tissue block. At each slice, the CCD camera records fluorescence images to be later analyzed for fluorescence image display. The microtome is housed in an ultralow freezer unit that maintains the sample at -80 degrees C during sample slicing and image acquisition. The resolution of microtome in slicing the tissue in z direction is 10μm. This cryoimaging enables high resolution 3-D fluorescence imaging of redox ratio in frozen tissue blocks by sectioning slice by slice. The scanning process is fully computerized by a homemade labview module, and programs will be developed for 3-D rendering of the redox ratio data.
3. Automated Time-Lapse Flourescence Microscopy:
We are also equipped with an automated Nikon microscope for cell studies. This system is an inverted TiE fluorescence microscope for automated time lapse, multi-channel fluorescence, phase and DIC imaging. The system is equipped with motorized stage, intesilight light source, environmental control chamber (37°C, a gas mixture of 95% O2 and 5% CO2), and an EXi Aqua CCD camera (Qimaging, 14 bit, 1392 x 1040, 6.45 μm pixel size).
Image cytometry software will be developed using Matlab, for microscopy images. Deconvolution of the images can be carried out on a dedicated 8-CPU Dell server. The automated microscope is controlled by its own PC; however, a supercomputer is available in the College of Engineering & Applied Science for viewing, manipulating and analyzing large datasets. An Aqua Exi CCD camera (Qimaging, 1392 x 1040 pixels, 6.45 μm pixel size) records fluorescence images from the microscope.
4. Organ Perfusion System:
The Biophotonics Lab perfusion system is an ADInstruments apparatus consisting of a complete chamber, reservoirs, tubing, pump, temperature control and Powerlab acquisition. The PowerLab 4/35 data acquisition system is a four- channel, 16 bit resolution recorder with programmable gain, four optional single-ended (BNC)/differential (Pod port) analog inputs, two independent stimulator outputs and an external trigger input and signal triggering. The unit also features a wide range of low-pass filters, AC or DC coupling and digital inputs and outputs for external instrument control. The PowerLab uses a high-speed USB 2.0 interface for connection to Windows and Mac OS computers. The lung can be attached to this ventilation and perfusion system. The control perfusate consists of (in mM) 4.7 KCl, 2.51 CaCl2, 1.19 MgSO4, 2.5 KH2PO4, 118 NaCl, 25 NaHCO3, 5.5 glucose, and 5% BSA. Control perfusate is pumped through the lung until the lung is evenly blanched and venous effluent is clear of blood by visual inspection. The lung is ventilated with end-inspiratory and end-expiratory pressures of ∼6 and 3 mmHg, respectively. The pulmonary arterial pressure is referenced to atmospheric pressure at the level of the left atrium and monitored continuously during the course of the experiments. The venous effluent pressure is atmospheric pressure.
5. Fluorescence Imager
Another instrument we designed is the in vivo fluorescence imager. This instrument contains a fluorescence LED light source (Excelitas Technologies), an emission filter wheel to house different emission filters that is controlled by a control box (ThorLabs) that allows it to communicate with a computer, a lens (Edmund Optics), and a CCD camera (Retiga R6). The Xcite LED1 has four switchable wavelengths, and is used to excite specific fluorophores of NADH and FAD from the surface of wounds. Then, on the emission path, we have a filter wheel to pass the emitted photons from NADH and FAD. The emission filters for NADH and FAD are set at 460 nm (50-nm bandwidth, D460/50M, Chroma, Bellows Falls, VT) and 537 nm (50-nm bandwidth, QMAX EM 510-560, Omega Optical), respectively. A one-channel stepper motor is used as the controller for the emission filter wheel (BSC201, ThorLabs). At the end of the emission path, the CCD camera is used to save the real-time images of surface fluorescence. This instrument is able to move and rotate sufficiently so that a patient with diabetic ulcers on their lower extremities would not have to move their position, resulting in any discomfort. A schematic of this instrument and a photo of it are presented in the following figure.
- Electronics: High-frequency oscilloscopes, arbitrary waveform generators, low-noise amplifiers, digitizers, DC power supplies
- Photonics: Several laser systems and drivers, fast switchable incoherent sources, spatial light modulators, ultra-sensitive solid-state detectors and photomultipliers, several anti-vibration optical tables, optical power meters, optical/optoelectronic components, optical fibers
- Microprocessor and data acquisition systems: Microcontroller boards, NI high speed DAQ systems
- Machining: CNC mill, CNC lathe, powered saw/drill, a complete mechanical bench
- Biology/neuroscience/molecular genetics: Olympus BX51 fluorescence microscope with motorized stage; patch clamp facility, including Multiclamp 700B amplifier; Digidata 1440 digitizer; perfusion systems; micropipette puller; AE31 inverted fluorescence microscope; stereo microscope; motorized micromanipulators; high-power light sources, including X-cite and Lambda DG-4; Class II bio-safety cabinet; CO2 incubator; Autoclave; ultra-low freezer; electroporation; sensitive CCD cameras
- Software: MATLAB, Mathematica,Labview, Comsol, Optiwave,Pclamp,Zemax,ProE
For our cell studies we have acquired equipment such as ultra low freezers, micro centrifuges, bio safety cabinets, an incubator and supplies. In addition to the major equipment in the lab, available common-use equipment in the shared lab spaces of multiple PI’s in College of Engineering & Applied Science includes,
- Confocal microscope in the biology department,
- Fiber optics, lasers
- Laser diodes
- High-speed- and ultra-centrifuges
- Bacterial incubators
- Sonicators, and scintillation counters
- Data A2D converter boards (National Instruments, Inc.)
- Labview Licences (National Instruments, Inc.)
- MATLAB workstations