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We apply a coded aperture snapshot spectral imager (CASSI) to fluorescence microscopy. CASSI records a two-dimensional (2D) spectrally filtered projection of a three-dimensional (3D) spectral data cube. We minimize a convex quadratic function with total variation (TV) constraints for data cube estimation from the 2D snapshot. We adapt the TV minimization algorithm for direct fluorescent bead identification from CASSI measurements by combining a priori knowledge of the spectra associated with each bead type. Our proposed method creates a 2D bead identity image. Simulated fluorescence CASSI measurements are used to evaluate the behavior of the algorithm. We also record real CASSI measurements of a ten bead type fluorescence scene and create a 2D bead identity map. A baseline image from filtered-array imaging system verifies CASSI's 2D bead identity map. OCIS codes: 100.3010, 100.3190, 110.0180, 110.1758, 110.4234, 180.2520.

We present a diffraction analysis for image formation in fluorescence confocal microscopy with divided apertures. The three-dimensional optical transform function is given, and axial resolution and transverse resolution are investigated. The results show that the employment of a divided-aperture technique in fluorescence confocal microscopy can enhance the rejection of background scattering. In addition, the axial resolution and transverse resolution can be improved by adjusting the width of the divider strip. For a given detector size, an optimum value of the divider strip width is given to obtain the best axial resolution or transverse resolution. The integrated intensity and signal-to-background ratio are also discussed. [c] 2010 Optical Society of America OCIS codes: 170.3660, 170.4580, 170.6510, 170.1530, 290.0290.

In-phase focal modulation microscopy (IPFMM) with single photon excited fluorescence is presented. Optical transfer functions and images of thin and thick fluorescent edges in IPFMM are investigated. The results show that, compared with confocal microscopy, using IPFMM can result in a sharper image of the edge, and the edge gradient can be increased up to 75.4% and 58.9% for a thick edge and a thin edge, respectively. Signal level is also discussed, and the results show that, to obtain high transverse resolution with IPFMM, the normalized detector pinhole radius should not exceed 2.8. OCIS codes: 170.3660, 170.4580, 170.6510, 170.1530, 290.0290.

Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within ~100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the ~50 nm deep evanescent field. OCIS codes: 260.6970, 180.2520, 180.4243, 170.2520, 170.0180, 110.0180.

Focal modulation microscopy (FMM) is an emerging microscopy technique for fluorescence imaging of thick biological tissue in vivo. A spatial phase modulator is a critical component whose characteristics have a significant impact on the performance of a FMM system. We have designed a simple spatial phase modulator based on a tilting glass plate that provides superb modulation stability. Image quality has been improved remarkably after integrating such a modulator into a FMM system. OCIS codes: 180.2520, 110.0113, 120.5060, 230.4110.

We report on a scanning total internal reflection fluorescence microscope with improved imaging properties. In the illumination system of the developed microscope, radial polarization is employed to obtain the point spread function with a single and circular peak. By using radial polarization, the confocal detection method, which can reduce the background noise of the image, is effectively applicable in the optical system. The observed image of a fluorescent particle with the size of 200 nm reveals the improvement of the imaging properties. OCIS codes: 110.0180, 180.2520.

A new optical-fluorescence microscopy technique, called HR-OPFOS, is discussed and situated among similar OPFOS-implementations. OPFOS stands for orthogonal-plane fluorescence optical sectioning and thus is categorized as a laser light sheet based fluorescence microscopy method. HR-OPFOS is used to make tomographic recordings of macroscopic biomedical specimens in high resolution. It delivers cross sections through the object under study with semi-histological detail, which can be used to create three-dimensional computer models for finite-element modeling or anatomical studies. The general innovation of this class of microscopy setup consists of the separation of the illumination and observation axes, but now in our setup combined with focal line scanning to improve sectioning resolution. HR-OPFOS is demonstrated on gerbil hearing organs and on mouse and bird brains. The necessary specimen preparation is discussed. OCIS codes: 110.2945, 110.6880, 110.6955, 120.4570, 170.2520, 170.3880.

We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing widefield fluorescence lifetime imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications. OCIS codes: 170.0180, 170.1530, 170.2520, 170.6920, 260.6970.

We demonstrate the observation of gold-nanoparticle internalization in membranes of living cells by using noninterferometric widefield optical profilometry (NIWOP). The NIWOP technique can trace the height of an 80 nm gold particle on the membrane by calibrating the change of light intensity scattered from the particle along the optical axis. On the membrane, the depth resolution based on the scattering signal is similar to that based on the reflection signal, nearly 20 nm. Comparing the heights of the nanoparticle and the nearby cell membranes, we can identify the occurrence of particle internalization. Combining fluorescence microscopy with NIWOP, we also find actin aggregation around the site of the internalization process, which is an indication of endocytosis. OCIS codes: 170.1530, 170.3880, 180.6900, 290.5850.

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity. OCIS codes: 180.2520, 170.3650, 180.4315, 180.6900.

A new approach to measuring the sizes of small fluorescent objects by use of spatially modulated illumination (SMI) far-field light microscopy is presented. This method is based on SMI measurements combined with a new SMI virtual microscopy (VIM) data analysis calibration algorithm. Here, experimental SMI measurements of fluorescent objects with known diameter (size) were made. From the SMI data obtained, the size was determined in an independent way by use of the SMI VIM algorithm. The results showed that with SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy. OCIS codes: 100.0100, 110.0180, 180.0180, 180.2520, 180.3170, 180.6900.

We present an experimental investigation to evaluate fluorescence microscopy as a tool to detect surface contamination as well as reveal surface damage precursors on optical components for large-aperture laser systems. We performed fluorescence imaging experiments using 351-nm laser excitation, whereas in situ damage testing was performed at laser fluences well below the dielectric breakdown threshold of the pure material. The experimental results demonstrated the potential of this technique to address both aforementioned technical issues. OCIS codes: 180.2520, 140.3330, 140.3380.

The dependence of spectral broadening of an ultrashort-pulsed laser beam on the fiber length and the illumination power is experimentally characterized in order to deliver the laser for two-photon fluorescence microscopy. It is found that not only the spectral width but also the spectral blue shift increases with the fiber length and illumination power, owing to the nonlinear response in the fiber. For an illumination power of 400 mW in a 3-m-long single-mode fiber, the spectral blue shift is as large as 15 nm. Such a spectral blue shift enhances the contribution from the short-wavelength components within the pulsed beam and leads to an improvement in resolution under two-photon excitation, whereas the efficiency of two-photon excitation is slightly reduced because of the temporal broadening of the pulsed beam. The experimental measurement of the axial response to a two-photon fluorescence polymer block confirms this feature. OCIS codes: 170.2520, 060.2430, 060.4370, 110.2350, 190.4180.

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15 to 5 aL. Amixed setup with an annular illumination aperture and a circular detection aperture is also considered. Key words: Confocal fluorescence microscopy, theta microscopy, annular apertures, oblique illumination.