Schematic diagram of the imaging system. Minor subsystems such as oxygen sensor, pressure monitor, and temperature were omitted from the diagram.
(a) A drawing of the main camera system. For clarity, all USB hubs and relay circuits were removed from this drawing. (b) Overall optical design. L1 = 720 mm, L2 = 130 mm, L3 = 527 mm, L4 = 383 mm, L5 = 18 mm, L6 = 563 mm, L7 = 78 mm, L8 = 260 mm. Distances listed are approximate and adjustable. With these parameters, the camera system has a demagnification factor of 0.62. Detail for prism/camera assembly is shown in Fig. 3 .
Camera optics. CCDs were arranged in banks of 4, with each CCD in the bank following a different optical path. (a) Front view as observed from the travel path of the image. (b) Top view showing the different light paths for each image. In this drawing, the outer casings of the cameras were removed to reveal the board level CCDs.
Microscope coupling to camera system. The camera is mounted on a custom optical table. The microscope is mounted on an extension table off the optical table with an x-y translation stage to allow alignment of the image from the microscope to the camera optics. Additional alignment in the vertical dimension is accommodated with a second focusing block on the microscope.
Laser alignment and imaging light path. A 5-axis fiber alignment system (3 for x-y-z position adjustment and 2 for tilt angle adjustment–details not shown) is used to optimize the delivery efficiency through the microscope and adjust the spot size of the laser on the sample.
Abbreviated timing diagram for image capture. The dual photodiode detector circuits provide redundant measurement of the speed and location of the rotating mirror.
Estimated number of photons available for image formation in high speed fluorescence mode, for several surface density of the dye (100, 1000, 2500, and 10 000 molecules/μm2). For this calculation, the fluorophore is R-PE, with extinction cross section of 3.26 × 10−15 cm2 and quantum efficiency of 0.82. Other parameters used: pixel size 6.45 μm, illumination size 50 μm, X = 62, Q 0 = 0.20, t E = 20 ns.
Testing procedure for relative sensitivity of candidate CCDs. (a) A TV chart was imaged with a single light pulse at various durations with all candidate cameras. (b) and (c) Linear fits of normalized image brightness in selected region of interest vs. light level for various gain/contrast settings. (b) Data from the Lumenera camera ultimately chosen for our system. (c) Data from the TI EMCCD chip contained in the Hamamatsu C9100-02 camera.
Spatial resolution of the imaging system. (a) High resolution target under the microscope. (b) Horizontal and vertical profiles of this area demonstrate the resolving power of the system.
Selected frames from fluorescence movies of reference beads at 25 Mfps. (a) 1.01 μm beads; (b) 1.90 μm beads; (c) 4.16 μm.
A bright field movie of lipid MBs under US excitation (f = 2.25 MHz, P a = 1.0 MPa), demonstrating US-induced MB vibration and breaking. Imaging is at 25 Mfps and playback is at 16 fps. Cropped to 256 × 256 pixels. Frame size is 27 μm × 27 μm (enhanced online). [URL: http://dx.doi.org/10.1063/1.4809168.1]doi: 10.1063/1.4809168.1.
A bright field movie of polymer MBs under US excitation (f = 1 MHz, P a = 2.0 MPa) demonstrating violent MB oscillations. Imaging is at 25 Mfps and playback is at 16 fps. Cropped to 512 × 512 pixels. Frame size is 89 μm × 89 μm (enhanced online). [URL: http://dx.doi.org/10.1063/1.4809168.2]doi: 10.1063/1.4809168.2.
A fluorescence movie of fluorescently labeled polymer MBs under US excitation (f = 1 MHz, P a = 2.0 MPa) showing lower amplitude oscillations of the fluorescent MB shell, despite violent oscillations of the gas during bright field imaging (Fig. 12 ). Imaging is at 25 Mfps and playback is at 16 fps. Cropped to 512 × 512 pixels. Frame size is 89 μm × 89 μm (enhanced online). [URL: http://dx.doi.org/10.1063/1.4809168.3]doi: 10.1063/1.4809168.3.
Comparison of sensor sensitivity.
Comparison of main characteristics of UPMC Cam to other high speed camera types.
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