Multiphoton absorption of femtosecond laser pulses focused through an objective with high numerical aperture (NA) can be used to image and manipulate cellular and intracellular objects. This review highlights recent advances in intracellular manipulation, including nanosurgery and labeling in living cells with femtosecond lasers.

We are pleased to publish the third issue (Vol. 2, No. 1) of Journal of Innovative Optical Health Sciences (JIOHS) which focuses on the developments and biomedical applications of nonlinear optical (NLO) microscopy. NLO microscopy is becoming a powerful tool for bioimaging due to several unique advantages over traditional methods. Nonlinear dependence on excitation intensity gives NLO microscopy inherent three-dimensional (3D) imaging capability without the need for a confocal pinhole. This is particularly advantageous in the case of tissue imaging where significant scattering can reduce the signal collection efficiency by confocal detection. Laser scanning facilitates real-time NLO imaging of live tissues and animals. NLO microscopy utilizes near IR excitation which provides both superior optical penetration into tissues as well as reduced photodamage due to reduced interaction with endogenous molecules. This issue includes seven original papers and five review articles.

This review highlights the recent applications of non-linear optical (NLO) microscopy to study obesity-related health risks. A strong emphasis is given to the applications of coherent anti-Stokes Raman scattering (CARS) microscopy where multiple non-linear optical imaging modalities including CARS, sum-frequency generation (SFG), and two-photon fluorescence are employed simultaneously on a single microscope platform. Specific examples on applications of NLO microscopy to study lipid-droplet biology, obesity-cancer relationship, atherosclerosis, and lipidrich biological structures are discussed.

Ultrashort pulse, multispectral non-linear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen. A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.

Coherent anti-Stokes Raman scattering (CARS) microscopy is used to visualize the release of a model drug (theophylline) from a lipid (tripalmitin) based tablet during dissolution. The effects of transformation and dissolution of the drug are imaged in real time. This study reveals that the manufacturing process causes significant differences in the release process: tablets prepared from powder show formation of theophylline monohydrate on the surface which prevents a controlled drug release, whereas solid lipid extrudates did not show formation of monohydrate. This visualization technique can aid future tablet design.

Multiphoton microscopy (MPM), with the advantages of improved penetration depth, decreased photo-damage, and optical sectioning capability, has become an indispensable tool for biomedical imaging. The combination of multiphoton fluorescence (MF) and second-harmonic generation (SHG) microscopy is particularly effective in imaging tissue structures of the ocular surface. This work is intended to be a review of advances that MPM has made in ophthalmic imaging. The MPM not only can be used for the label-free imaging of ocular structures, it can also be applied for investigating the morphological alterations in corneal pathologies, such as keratoconus, infected keratitis, and corneal scar. Furthermore, the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty (CK) can also be studied with MPM. Finally, qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation. With additional development, multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.

Skin scar is unique to humans, the major significant negative outcome sustained after thermal injuries, traumatic injuries, and surgical procedures. Hypertrophic scar in human skin is investigated using non-linear spectral imaging microscopy. The high contrast images and spectroscopic intensities of collagen and elastic fibers extracted from the spectral imaging of normal skin tissue, and the normal skin near and far away from the hypertrophic scar tissues in a 10-year-old patient case are obtained. The results show that there are apparent differences in the morphological structure and spectral characteristics of collagen and elastic fibers when comparing the normal skin with the hypertrophic scar tissue. These differences can be good indicators to differentiate the normal skin and hypertrophic scar tissue and demonstrate that non-linear spectral imaging microscopy has potential to noninvasively investigate the pathophysiology of human hypertrophic scar.

As a second messenger in signal transduction, calcium ion plays a very important role in neuronal information processing and integrating. Limited by the imaging technique, it is difficult to simultaneously perform deep tissue imaging and measure intracellular free calcium concentration ([Ca2+]i) in different compartments of neurons in brain slice noncollinearly. By means of random access two-photon microscopy, which provides high optical penetration into tissues and low photo damage, we successfully measured [Ca2+]i of different parts of pyramidal neurons in neocortical layer V in rat brain slices with high spatial and temporal resolution. Combining the patch clamp technique, we stimulated the soma with depolarizing current and explored the dynamics of calcium in pyramidal neurons.

Photodynamic therapy (PDT) has received increased attention since the regulatory approvals of several photosensitizers and light applicators in numerous countries and regions around the world. In recent years, much progress has been seen in basic research as well as clinical application. PDT clinical application has now extended from treating malignant diseases to nonmalignant diseases. This review article will present recent clinical data published in English journals. The data will be organized according to their clinical specialties. The new development and future direction in clinical applications of PDT for the management of both malignant and nonmalignant diseases will be discussed.

Photobiomodulation (PBM) is a modulation of monochromatic light or laser irradiation (LI) on biosystems. It is reviewed from the viewpoint of extraocular phototransduction in this paper. It was found that LI can induce extraocular phototransduction, and there may be an exact correspondence relationship of LI at different wavelengths and in different dose zones, and cellular signal transduction pathways. The signal transduction pathways can be classified into two types so that the Gs protein-mediated pathways belong to pathway 1, and the other pathways such as protein kinase Cs-mediated pathways and mitogen-activated protein kinase-mediated pathways belong to pathway 2. Almost all the present pathways found to mediate PBM belong to pathway 2, but there should be a pathway 1-mediated PBM. The previous studies were rather preliminary, and therefore further work should be done.

We proposed a method to evaluate the material dispersion of the dielectric film in dielectriccoated silver hollow fiber. By taking into consideration the derived material dispersion, the wavelengths of the loss peaks and valleys in the loss spectra of the hollow fiber can be predicted more accurately. Then, we fabricated the dielectric-coated silver hollow fiber according to the parameters obtained by using the improved design method. The measured data showed good agreement with the calculated results. The loss for medical laser of Er:YAG and CO2 was less than 0.3 dB/m. The loss for green or red pilot beams was around 5 dB/m, which is sufficiently low for the purpose of pilot beam transmission. The derived material dispersion plays an important role in the design and fabrication of the hollow fiber for multiwavelength delivery.

Studies of non-invasive glucose measurement with optical coherence tomography (OCT) in tissue-simulating phantoms and biological tissues show that glucose has an effect on the OCT signal slope. Choosing an efficient fitting range to calculate the OCT signal slope is important because it helps to improve the precision of glucose measurement. In this paper, we study the problem in two ways: (1) scattering-induced change of OCT signal slope versus depth in intralipid suspensions with different concentrations based on Monte Carlo (MC) simulations and experiments and (2) efficient fitting range for glucose measurement in 3% and 10% intralipid. The results show that the OCT signal slope expresses a contrary change with scattering coefficient below a certain depth in high intralipid concentrations, so that there is an effective fitting depth. With an efficient fitting range from 100μm to the effective fitting depth, the precision of glucose measurement can be 4.4mM for 10% intralipid and 2.2mM for 3% intralipid.

A swept-source optical coherence tomography (SSOCT) system based on a high-speed scanning laser source at center wavelength of 1320 nm and scanning rate of 20 kHz is developed. The axial resolution is enhanced to 8.3 μm by reshaping the spectrum in frequency domain using a window function and a wave number calibration method based on a Mach-Zender Interferometer (MZI) integrated in the SSOCT system. The imaging speed and depth range are 0.04 s per frame and 3.9 mm, respectively. The peak sensitivity of the SSOCT system is calibrated to be 112 dB. With the developed SSOCT system, optical coherence tomography (OCT) images of human finger tissue are obtained which enable us to view the sweat duct (SD), stratum corneum (SC) and epidermis (ED), demonstrating the feasibility of the SSOCT system for in vivo biomedical imaging.