Two types of optical systems were used in this study. One optical system observed the fluorescence of QDs and consisted primarily of an epi-fluorescence microscope (IX-71, Olympus) with modifications, a Nipkow disk-type confocal unit (CSU10, Yokogawa), and an electron multiplier-type charge-coupled device camera (EM-CCD, Ixon DV887, Andor Technology), which is a highly sensitive camera24 (link)34 (link)35 (link). A PlanApo (X60, 1.40 NA, Olympus) objective lens was used for imaging. QDs were illuminated using a blue laser (488-nm wavelength, CrystaLaser). The laser-excited fluorescence was filtered with a 695–740-nm bandpass filter to image the QDs and autofluorescence of the tissues or a 640–690-nm bandpass filter to image the autofluorescence of tissues in an identical focal plate and field. Images were obtained at a rate of 5–10 frames per second. The other optical system was a Zeiss Pascal Confocal Microscope System (Zeiss). This system was used to observe cell movement for the cell migration assay or to measure the cell number for the cell-invasion assay.
Ixon dv887
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon DV887 is a scientific camera designed for low-light imaging applications. It features a back-illuminated electron-multiplying CCD (EMCCD) sensor that provides high quantum efficiency and ultra-low noise performance. The camera is capable of capturing images and videos at high frame rates, making it suitable for a range of scientific research and industrial applications.
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6 protocols using ixon dv887
1
Dual-Optical System for Fluorescence Imaging
2
Dual-color FRAP Microscopy Setup
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The microscopy setup consisted of an epifluorescence microscope (Ti-E, Nikon), an objective lens (40× CFI Plan Apo Lambda, 0.95 NA, Nikon), a relay optics box for dual-color imaging (GA03; G-Angstrom, Japan), and an electron multiplier-type CCD camera (EM-CCD, iXon DV887 or DU897; Andor Technology PLC, UK). A slit was placed at the imaging surface of the microscope in the relay optics. A dichroic mirror (FF458-Di02, Semrock) located just outside the slit split the optical pathway after the imaging surface into two pathways for cyan (CFP) and yellow (YFP1G) fluorescence. The two pathways converged on the acceptance surface of the EM-CCD camera side by side. Band-pass filters were set for each pathway (467–499 nm for CFP and 510–560 nm for YFP). Multiphoton fluorescence recovery after photobleaching (MP-FRAP) experiments were conducted as described elsewhere [17 (link)]. Image analyses were carried out in the ImageJ software (NIH, USA).
3
Fluorescence Imaging of Filament Persistence
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Fluorescence imaging was carried out using an epi-fluorescence Leica DM IRB microscope equipped with a 100× oil-immersion objective (Leica 11506168) and an iXon DV887 back illuminated EMCCD camera (Andor Technology). Fluorescence excitation was induced with a mercury vapor lamp and a N2.1 filter cube (Leica 11513882, excitation filter from 515 to 560 nm) transmitting only the wavelength exciting Cy3 to the sample. Images were recorded as grayscale pictures with the camera-associated Andor SOLIS software. For determination of the persistence length and contour length filaments were absorbed to a glass surface. For the persistence length evaluation it was ensured that the absorbed filaments were not influenced by surface-filament interactions via kurtosis analysis as previously described.13 (link)
4
Electrophoretic Manipulation in Microfluidics
5
Fluorescence Imaging of Quantum Dots
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The optical system for observing the fluorescence of QDs consisted primarily of an epi‐fluorescent microscope (IX‐71; Olympus, Tokyo, Japan) with modifications, a Nipkow disk‐type confocal unit (CSU10; Yokogawa, Tokyo, Japan), and an electron multiplier type charge‐coupled device camera (EM‐CCD; Ixon DV887; Andor Technology, Belfast, UK) 27 , 28 . A PlanApo (X60, 1.40 NA; Olympus) objective lens was used for imaging. QDs were illuminated by a blue laser (488 nm wavelength, 50 mW, Spectra‐Physics). The laser‐excited fluorescence was filtered with a 695–740 nm band‐pass filter for imaging QDs and auto‐fluorescence of tissues or a 640–690 nm band‐pass filter for imaging auto‐fluorescence of tissues at identical focal plate and field. Images were obtained at exposure times of 200 msec or 20 sec.
6
Visualizing Actin Filament Dynamics
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Movement of fluorescently labeled actin or native thin filaments was recorded using a custom-made TIRF microscope with single-fluorophore sensitivity (Amrute-Nayak et al., 2008 (link); Rump et al., 2011 (link)) and modifications as described below. In our inverted objective-type TIRF microscope, rhodamine phalloidin–labeled actin or thin filaments were excited by light of 532 nm wavelength produced by a Nd:YAG laser (Compass 315M-150 SL; Coherent). Both exciting laser light and emitted fluorescence light passed through a high numerical aperture 60× oil immersion objective lens (Plan Apo 60×, NA 1.45, oil; Olympus). Via a QV2 four-channel simultaneous-imaging system (Photometrics) with a respective dichroic mirror (T585 LPXR; Chroma), rhodamine fluorescence signals were projected onto a back-illuminated EMCCD camera (iXon DV887; Andor Technology, cooled to −80°C) and recorded with the software Andor SOLIS for imaging (version 4.15.30000.0). The videos were recorded at a constant sample temperature of 23°C with a frame rate of 5 Hz and converted by Andor SOLIS to 16-bit grayscale. TIFF-stacks for analysis with the computer program ImageJ (W.S. Rasband, National Institutes of Health) as described below.
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