Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb(3+)-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.

Achieving a greater imaging depth with two-photon fluorescence microscopy (TPFM) is mainly limited by out-of-focus fluorescence generated from both ballistic and scattered light excitation. We report on an improved signal-to-noise ratio (SNR) in a highly scattering medium as demonstrated by analytical simulation and experiments for TPFM. Our technique is based on out-of-focus rejection using a confocal pinhole. We improved the SNR by introducing the pinhole in the collection beam path. Using the radiative transfer theory and the ray-optics approach, we analyzed the effects of different sizes of pinholes on the generation of the fluorescent signal in the TPFM system. The analytical simulation was evaluated by comparing its results with the experimental results in a scattering medium. In a combined confocal pinhole and two-photon microscopy system, the imaging depth limit of approximately 5 scattering mean free paths (MFP) was found to have improved to 6.2 MFP.

We designed and fabricated a telecentric f-theta imaging lens (TFL) to improve the imaging performance of spectral domain optical coherence tomography (SD-OCT). By tailoring the field curvature aberration of the TFL, the flattened focal surface was well matched to the detector plane. Simulation results showed that the spot in the focal plane fitted well within a single pixel and the modulation transfer function at high spatial frequencies showed higher values compared with those of an achromatic doublet imaging lens, which are commonly used in SD-OCT spectrometers. The spectrometer using the TFL had an axial resolution of 7.8 μm, which was similar to the theoretical value of 6.2 μm. The spectrometer was constructed so that the achromatic doublet lens was replaced by the TFL. As a result, the SD-OCT imaging depth was improved by 13% (1.85 mm) on a 10 dB basis in the roll-off curve and showed better sensitivity at the same depth. The SD-OCT images of a multi-layered tape and a human palm proved that the TFL was able to achieve deeper imaging depth and better contrast. This feature was seen very clearly in the depth profile of the image. SD-OCT imaging performance can be improved simply by changing the spectrometer’s imaging lens. By optimizing the imaging lens, deeper SD-OCT imaging can be achieved with improved sensitivity.

We present the development and implementation of a spatially and spectrally resolved multipoint fluorescence correlation spectroscopy (FCS) system utilizing multiple end-capped optical fibers and an inexpensive laser source. Specially prepared end-capped optical fibers placed in an image plane were used to both collect fluorescence signals from the sample and to deliver signals to the detectors. The placement of independently selected optical fibers on the image plane was done by monitoring the end-capped fiber tips at the focus using a CCD, and fluorescence from specific positions of a sample were collected by an end-capped fiber, which could accurately represent light intensities or spectral data without incurring any disturbance. A fast multipoint spectroscopy system with a time resolution of ∼1.5 ms was then implemented using a prism and an electron multiplying charge coupled device with a pixel binning for the region of interest. The accuracy of our proposed system was subsequently confirmed by experimental results, based on an FCS analysis of microspheres in distilled water. We expect that the proposed multipoint site-specific fluorescence measurement system can be used as an inexpensive fluorescence measurement tool to study many intracellular and molecular dynamics in cell biology.

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