3D imaging is demanding technology required in fluorescence microscopy. Even though holography is a powerful technique, it could not be used easily in fluorescence microscopy because of low coherence of fluorescence light. Lately, several incoherent holographic methods such as scanning holography, Fresnel in coherent correlation holography (FINCH), and self-interference incoherent digital holography (SIDH) have been proposed. However, these methods have many problems to be overcome for practical applications. For example, DC term removal, twin image ambiguity, and phase unwrapping problems need to be resolved. Off-axis holography is a straightforward solution which can solve most of these problems. We built an off-axis SIDH system for fluorescence imaging, and investigated various conditions and requirements for practical holographic fluorescence microscopy. Our system is based on a modified Michelson interferometer with a flat mirror at one arm and a curved mirror at the other arm of the interferometer. We made a phantom 3D fluorescence object made of 2 single-mode fibers coupled to a single red LED source to mimic 2 fluorescence point sources distributed by a few tens of micrometers apart. A cooled EM-CCD was used to take holograms of these fiber ends which emit only around 180 nW power.
Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 µg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.
The use of AgNP is becoming more and more widespread in biomedical field. But compared with the promising
bactericidal function, other physiological effects of AgNP on cells are relatively scant. In this research, we propose
quantitative phase microscopy (QPM) as a new method to study the degranulation, and AgNP-induced RBL-2H3 cell
degranulation is studied as well. Firstly, HeLa cells as the cell control and PBS as the solvent control, we measured the
cell volume and cross section profile (x-z plane) with QPM. The results showed that the volume and cross section profile
changed only the RBL-2H3 cells exposed to calcium ionophore A23187, which demonstrates the validity of QPM in
degranulation research. Secondly, 50μg/mL of AgNP was used instead of A23187, and the measurement of cell volume
and cross section profile was carried out again. RBL-2H3 cell volume increased immediately after AgNP was added, and
cross section profile showed that the cell surface became granulated, but HeLa cell was lack of that effect. Phase images
obviously indicated the RBL-2H3 cell deformation. Thirdly, stained with Fluo-3/AM, intracellular calcium Ca<sup>2+</sup>]<sub>i</sub> of
single RBL-2H3 cell treated with AgNP was observed with fluorescent microscopy; incubated with AgNP for 20min, the
supernatant of RBL-2H3 cells was collected and reacted with o-phthalaldehyde (OPA), then the fluorescent intensity of
histamine-OPA complex was assayed with spectrofluorometer. The results of Ca<sup>2+</sup>]<sub>i</sub> and histamine increase showed that
degranulation of AgNP-induced RBL-2H3 cell occurred. So, the cell volume was used as a parameter of degranulation in
our study and AgNP-induced RBL-2H3 cells degranulation was confirmed by the cell volume increment, cross section
profile change, and [Ca<sup>2+</sup>]<sub>i</sub> and histamine in supernatant increase.
Three dimensional particle tracking is useful technology to characterize live cell or surrounding environment by tracing
small particles such as fluorescence beads or polystyrene beads which adhered to objective samples. In microscopy
imaging system, the longitudinal(z axis) tracking of the particle is essential for implementation of three-dimensional
particle tracking, however it's been still a challenging topic to find the exact position of the particle in z axis with high
In this study, we present that a novel technique to find the longitudinal position of the particle, as well as the transverse
position(x,y axis) by applying the numerical reconstruction and focusing with digital holographic microscope.
Transmission type off-axis digital holographic microscope is implemented for this experiment, based on Mach-Zehnder
interferometer and 632.8nm HeNe laser is used as a coherent light source of the microscope and high-speed CMOS
camera is utilized for acquiring the hologram. Digital holographic microscope makes it possible to record and reconstruct
the phase and amplitude image of the sample. In order to find the position of the particle in z axis, we apply the
numerical focusing algorithm, which enables the translation of the imaging focus without actual longitudinal movement
of the sample. To demonstrate the presented method, Brownian movement of 3μm polystyrene sphere suspended in
water is investigated in this experiment.
An all-purpose multifunctional optical microscope based on digital holographic microscopy (DHM) is proposed, which enables DHM to be practically suitable for bioimaging applications. With our simple suggested digital signal-processing scheme, we have demonstrated that various optical images, including pure amplitude, pure phase, dark-field, phase-contrast, and differential interference contrast images, can be obtained from a single DHM measurement. Because these images obtained with our method are essentially the same as those obtainable with conventional optical imaging instruments, biologists can easily analyze them with their previous knowledge and experience.
We present a simple 2D image acquisition technique electronically implemented for laser scanning confocal microscope
using galvanometer scanners. In order to synchronize image acquisition process with the movement of the galvanometer
scanner, position signal of the mirror of the galvanometer scanner is used and manipulated for generating of sync-signals.
This is achieved using an analog-digital converter to read a position signal from the scanner which tells about its position
and to generate a trigger signal (or pixel clock) which tells the moment of digitizing the received analog signal from the
photo-detector. This facilitates processing the image in synchronization with the actual motion of the scanning laser
beam scanner. Image construction is performed by a video acquisition board (frame grabber). The newly developed
scanning and image acquisition systems are implemented in a confocal microscope with fiber-optic components for
compact configuration and flexible light path.
We present a fluorescence lifetime imaging microscope (FLIM) based on a real-time waveform acquisition method. The
fluorophores were excited by a 635-nm gain-switched laser diode, which produced short pulses with duration ~50 ps in a
20-MHz repetition rate. The fluorescence signals were detected by a silicon avalanche photo-diode (APD) in addition to
a wide-band electric amplifier. The converted electric pulses were sampled by a high-speed digitizer of which sampling
rate was 2 GS/s. In order to reduce the sampling interval for analyzing sub-nanosecond lifetimes, an interleaved data
acquisition technique was used. The effective sampling rate was increased to 10 GS/s. In addition, the impulse response
was measured simultaneously with the lifetime signals by an interleaving manner and was used in calibration of the
system. By using these methods, accurate lifetime information was acquired in a short time less than 8 μs.