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The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Skin aging is an important issue in geriatric and cosmetic dermatology. To quantitatively analyze changes in keratinocytes related to intrinsic aging, we exploited a 1230 nm-based in vivo harmonic generation microscopy, combining second- and third-harmonic generation modalities. 52 individuals (21 men and 31 women, age range 19-79) were examined on the sun-protected volar forearm. Through quantitative analysis by the standard algorithm provided, we found that the cellular and nuclear size of basal keratinocytes, but not that of granular cells, was significantly increased with advancing age. The cellular and nuclear areas, which have an increase of 0.51 μm(2) and 0.15 μm(2) per year, respectively, can serve as scoring indices for intrinsic skin aging.

We present an imaging method, dSLIM, that combines a novel deconvolution algorithm with spatial light interference microscopy (SLIM), to achieve 2.3x resolution enhancement with respect to the diffraction limit. By exploiting the sparsity of the phase images, which is prominent in many biological imaging applications, and modeling of the image formation via complex fields, the very fine structures can be recovered which were blurred by the optics. With experiments on SLIM images, we demonstrate that significant improvements in spatial resolution can be obtained by the proposed approach. Moreover, the resolution improvement leads to higher accuracy in monitoring dynamic activity over time. Experiments with primary brain cells, i.e. neurons and glial cells, reveal new subdiffraction structures and motions. This new information can be used for studying vesicle transport in neurons, which may shed light on dynamic cell functioning. Finally, the method is flexible to incorporate a wide range of image models for different applications and can be utilized for all imaging modalities acquiring complex field images.

In July 2011 a new concept of a closed microfluidic system equipped with a fixed micropipette, optical tweezers and a UV-Vis spectrometer was presented [Biomed. Opt. Express 2, 2299 (2011)]. Figure 1 showed falsely oriented mirrors. To clarify the design of the setup, this erratum presents a correct schematic We present a new concept of integrating a micropipette within a closed microfluidic system equipped with optical tweezers and a UV-Vis spectrometer. A single red blood cell (RBC) was optically trapped and steered in three dimensions towards a micropipette that was integrated in the microfluidic system. Different oxygenation states of the RBC, triggered by altering the oxygen content in the microchannels through a pump system, were optically monitored by a UV-Vis spectrometer. The built setup is aimed to act as a multifunctional system where the biochemical content and the electrophysiological reaction of a single cell can be monitored simultaneously. The system can be used for other applications like single cell sorting, in vitro fertilization or electrophysiological experiments with precise environmental control of the gas-, and chemical content. Validerad; 2011; 20110721 (ahmahm)

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.

To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 µm x 1 µm x 1 µm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are bio-compatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and

A new type of freeze-drying microscope based upon time-domain optical coherence tomography is presented here (OCT-FDM). The microscope allows for real-time, in situ 3D imaging of pharmaceutical formulations in vials relevant for manufacturing processes with a lateral resolution of

High-speed high-contrast imaging modalities that enable image acquisition of transparent media without the need for chemical staining are essential tools for a broad range of applications; from semiconductor process monitoring to blood screening. Here we introduce a method for contrast-enhanced imaging of unstained transparent objects that is capable of high-throughput imaging. This method combines the Nomarski phase contrast capability with the ultrahigh frame rate and shutter speed of serial time-encoded amplified microscopy. As a proof of concept, we show imaging of a transparent test structure and white blood cells in flow at a shutter speed of 33 ps and a frame rate of 36.1 MHz using a single-pixel photo-detector. This method is expected to be a valuable tool for high-throughput screening of unstained cells.

Accurate microvascular morphometric information has significant implications in several fields, including the quantification of angiogenesis in cancer research, understanding the immune response for neural prosthetics, and predicting the nature of blood flow as it relates to stroke. We report imaging of the whole mouse brain microvascular system at resolutions sufficient to perform accurate morphometry. Imaging was performed using Knife-Edge Scanning Microscopy (KESM) and is the first example of this technique that can be directly applied to clinical research. We are able to achieve ≈ 0.7μm resolution laterally with 1μm depth resolution using serial sectioning. No alignment was necessary and contrast was sufficient to allow segmentation and measurement of vessels.

We present a novel technique for three-dimensional (3D) image processing of complex fields. It consists in inverting the coherent image formation by filtering the complex spectrum with a realistic 3D coherent transfer function (CTF) of a high-NA digital holographic microscope. By combining scattering theory and signal processing, the method is demonstrated to yield the reconstruction of a scattering object field. Experimental reconstructions in phase and amplitude are presented under non-design imaging conditions. The suggested technique is best suited for an implementation in high-resolution diffraction tomography based on sample or illumination rotation.

The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After the refinements of the microring's working wavelength and in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×10(5) was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29 Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM with high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5 μm and an axial resolution of 8 μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue specimens or thicker tissue sections, which is not now imageable with current optical or acoustic microscopes of comparable resolution.

We describe a versatile, catheter-type two-photon probe, designed for in vivo and ex vivo imaging of the aqueous outflow pathway in the eye. The device consists of a silica double cladding fiber used for laser delivery and fluorescence collection, a spiral fiber scanner driven by a miniature piezoelectric tube, and an assembly of three micro-size doublet achromatic lenses used for focusing the laser and collecting the two-photon excitation signal. All the components have a maximum diameter of 2 mm and are enclosed in a length of 12-gauge stainless steel hypodermic tubing having an outer diameter of 2.8 mm. The lateral and axial resolutions of the probe are measured to be 1.5 μm and 9.2 μm, respectively. Different lens configurations and fibers are evaluated by comparing their spatial resolutions and fluorescence signal collection efficiencies. Doublet achromatic lenses and a double cladding fiber with a high inner cladding numerical aperture are found to produce a high signal collection efficiency, which is essential for imaging live tissues. Simple methods for reducing image distortions are demonstrated. Images of human trabecular meshwork tissue are successfully obtained with this miniature two-photon microscope.

We have developed an adaptive optics photoacoustic microscope (AO-PAM) for high-resolution imaging of biological tissues, especially the retina. To demonstrate the feasibility of AO-PAM we first designed the AO system to correct the wavefront errors of the illuminating light of PAM. The aberrations of the optical system delivering the illuminating light to the sample in PAM was corrected with a close-loop AO system consisting of a 141-element MEMS-based deformable mirror (DM) and a Shack-Hartmann (SH) wavefront sensor operating at 15 Hz. The photoacoustic signal induced by the illuminating laser beam was detected by a custom-built needle ultrasonic transducer. When the wavefront errors were corrected by the AO system, the lateral resolution of PAM was measured to be better than 2.5 µm using a low NA objective lens. We tested the system on imaging ex vivo ocular samples, e.g., the ciliary body and retinal pigment epithelium (RPE) of a pig eye. The AO-PAM images showed significant quality improvement. For the first time we were able to resolve single RPE cells with PAM.

Since the photoacoustic effect relies only on the absorbed optical energy, the back-reflected photons from samples in optical-resolution photoacoustic microscopy are usually discarded. By employing a 2 x 2 single-mode fiber optical coupler in a laser-scanning optical-resolution photoacoustic microscope for delivering the illuminating laser light and collecting the back reflected photons, a fiber-optic confocal microscope is integrated with the photoacoustic microscope. Thus, simultaneous multimodal imaging can be achieved with a single light source and images from the two modalities are intrinsically registered. Such capabilities are demonstrated in imaging both phantoms and small animals in vivo.

Refractive index properties, especially at the nanoscale, have shown great potential in cancer diagnosis and screening. Due to the intrinsic complexity and weak refractive index fluctuation, the reconstruction of internal structures of a biological cell has been challenging. In this paper, we propose a simple and practical approach to derive the statistical properties of internal refractive index fluctuations within a biological cell with a new optical microscopy method – Low-coherence Statistical Amplitude Microscopy (SAM). We validated the capability of SAM to characterize the statistical properties of cell internal structures, which is described by numerical models of one-dimensional Gaussian random field. We demonstrated the potential of SAM in cancer detection with an animal model of intestinal carcinogenesis – multiple intestinal neoplasia mouse model. We showed that SAM-derived statistical properties of cell nuclear structures could detect the subtle changes that are otherwise undetectable by conventional cytopathology.