Bessel beams have been used in many applications due to their unique optical properties of maintaining their intensity profiles unchanged during propagation. In imaging applications, Bessel beams have been successfully used to provide extended focuses for volumetric imaging and uniformed illumination plane in light-sheet microscopy. Coupled with two-photon excitation, Bessel beams have been successfully used in realizing fluorescence projected volumetric imaging. We demonstrated previously a stereoscopic solution–two-photon fluorescence stereomicroscopy (TPFSM)–for recovering the depth information in volumetric imaging with Bessel beams. In TPFSM, tilted Bessel beams were used to generate stereoscopic images on a laser scanning two-photon fluorescence microscope; upon post image processing we could successfully provide 3D perception of acquired volume images by wearing anaglyph 3D glasses. However, tilted Bessel beams were generated by shifting either an axicon or an objective laterally; the slow imaging speed and severe aberrations made it hard to use in real-time volume imaging. In this article, we report recent improvements of TPFSM with newly designed scanner and imaging software, which allows 3D stereoscopic imaging without moving any of the optical components on the setup. This improvement has dramatically improved focusing qualities and imaging speed so that the TPFSM can be performed potentially in real-time to provide 3D visualization in scattering media without post image processing.
Structured-illumination microscopy (SIM) is an efficacious tool to decrease the contribution of the out-of-focus light to images of specimens. However, in SIM, the frequency of the spatial modulation applied to specimens should be adjustable according to the optical properties of the specimens to reach the optimal contrasts. Hence, a common theme in SIM is how the flexibility and quality of modulations at different frequencies can be improved. Digital scanned laser light-sheet microscopy with structured illumination (DSLM-SI) has been the most flexible means for generating modulation and optical sectioning. The complexity of synchronization between the temporal modulation and the beam scanning makes it hard to use and less stable; it also takes more time to acquire images for one plane than selective plane illumination microscopy (SPIM). In this report, we present a recent effort to use a spatial light modulator (SLM) to provide spatial modulation in SPIM. With the SLM, both of the frequency and phase of lateral modulation can be changed rapidly; moreover, this SLM-based SPIM can achieve fast imaging without mechanical moving parts.
Bessel beams have been proved to have the ability to extend the depth of focus in fluorescence microscopy. But the depth discrimination was not investigated thoroughly. Following our previous work2, we investigated focal fields of Bessel-Gauss beams at different scanning angles. We found that the central focusing lines were tilted differently at different scanning angles. This effect manifests the ability of the true perspective view in the fluorescence stereomicroscopy.
Confocal laser scanning microscopy (CLSM) has become one of the most important biomedical research tools today due
to its noninvasive and 3-D abilities. It enables imaging in living tissue with better resolution and contrast, and plays a
growing role among microscopic techniques utilized for investigating numerous biological problems. In some cases, the
sample was phase-sensitive, thus we introduce a novel method named laser oblique scanning optical microscopy
(LOSOM) which could obtain a relief image in transparent sample directly.
Through the LOSOM system, mouse kidney and HeLa cells sample were imaged and 10x, 20x and 40x magnify
objective imaging results were realized respectively. Also, we compared the variation of pinhole size versus imaging
result. One major parameters of LOSOM is the distance between fluorescence medium and the sample. Previously, this
distance was set to 1.2 mm, which is the thickness of the slide. The experiment result showed that decreasing d can
increase the signal level for LOSOM phase-relief imaging. We have also demonstrated the application of LOSOM in
absorption imaging modality, when the specimen is non-transparent.
We reported recently that laser oblique scanning optical microscopy (LOSOM) is able to obtain a relief image in
transparent sample directly. To optimize the performance of LOSOM, the parameters such as numerical aperture, the
distance between the specimen and the fluorescent medium and the pinhole size are investigated in this work. A beam
blocker is introduced in light path which enhances dramatically the visualization of local phase difference.