Fast Multiplane Functional Imaging Combining Acousto-optic Switching and Remote Focusing

Networks of neurons are inherently three-dimensional in nature, whereas conventional imaging methods, such as laser scanning two-photon microscopy, usually provide only fast two-dimensional imaging. Rapid volumetric imaging would however be preferable for imaging neurons. To get a more complete picture of the dynamics of the neuron-to-neuron interactions, we have developed a pseudo-parallelised multi-plane two-photon excitation imaging system through the incorporation of an acousto-optic switching and a remote focusing technique into a resonant scanning microscope. This permits the recording of millisecond scale fluorescence transients of calcium indicators from large populations of neurons upon neural firing events at multiple chosen axial planes in very short time frame. While the remote focusing system offers aberration-free axial scanning over a few hundreds of micrometres of depth, the acousto-optic deflector provides high speed optical switching between different laser beam paths in sub-microsecond timescale which in turn, controls the axial focal plane to be targeted. Here, we report on the development of the high temporal resolution multi-plane targeted microscope and its potential application

Achieving superresolution with illumination-enhanced sparsity.

Recent advances in superresolution fluorescence microscopy have been limited by a belief that surpassing two-fold resolution enhancement of the Rayleigh resolution limit requires stimulated emission or the fluorophore to undergo state transitions. Here we demonstrate a new superresolution method that requires only image acquisitions with a focused illumination spot and computational post-processing. The proposed method utilizes the focused illumination spot to effectively reduce the object size and enhance the object sparsity and consequently increases the resolution and accuracy through nonlinear image post-processing. This method clearly resolves 70nm resolution test objects emitting ~530nm light with a 1.4 numerical aperture (NA) objective, and, when imaging through a 0.5NA objective, exhibits high spatial frequencies comparable to a 1.4NA widefield image, both demonstrating a resolution enhancement above two-fold of the Rayleigh resolution limit. More importantly, we examine how the resolution increases with photon numbers, and show that the more-than-two-fold enhancement is achievable with realistic photon budgets

Algorithms for extracting true phase from rotationally-diverse and phase-shifted DIC images

In this paper, we report on the status of our current algorithms and extensions for improved algorithms for extracting phase from images acquired with differential-interference-contrast (DIC) microscopy. Our algorithms are based on two different approaches for the computation of a specimen\u27s phase function or optical path length (OPL) distribution from DIC images. The first approach uses an iterative phase-estimation method that minimizes the 1-divergence discrepancy measure using the conjugate-gradient technique to estimate the OPL from multiple DIC images acquired at different specimen rotations. The method is based on the assumption that the specimen does not absorb light. The second approach is a non-iterative method that is based on a geometric-optics model and the phase-shifting technique that allows separation of the amplitude and phase gradient information from DIC images thereby allowing computation of the desired phase from its gradient. We show results from both methods and discuss the tradeoff between complexity (with respect to data-acquisitiona and computation) and accuracy. Our long term goal is to develop a new and improved method based on a combination of our two approaches

Quantitative DIC microscopy using a geometric phase shifter

In this paper we investigate the use of a geometric phase- shifting (GPS) technique which allows us to convert conventional transmission or reflection differential interference contrast (DIC) microscopy into a quantitative mode. A phase-shifting algorithm is employed to extract the specimen phase gradient from the mixture of phase and amplitude information which is common in DIC. Fourier techniques are then used to recover the exact phase (i.e. optical path length variations) throughout the biological specimen viewed. In addition to this quantitative 'phase map,' we demonstrate that the GPS process simultaneously yields an 'amplitude-only' representation in which various absorption and transmission properties of the specimen are displayed as intensity variations in the image, similar to brightfield microscopy. These two resulting images can then be analyzed or further processed in a number of ways that are not possible with conventional DIC and which improve the microscopist's ability to correctly identify, interpret and measure features in the specimen

Calibration of a phase-shifting DIC microscope for quantitative phase imaging

Phase-shifting differential interference contrast (DIC) provides images in which the intensity of DIC is transformed into values linearly proportional to differential phase delay. Linear regression analysis of the Fourier space, spiral phase, integration technique shows these values can be integrated and calibrated to provide accurate phase measurements of objects embedded in optically transparent media regardless of symmetry or absorption properties. This approach has the potential to overcome the limitations of profilometery, which cannot access embedded objects, and extend the capabilities of the traditional DIC microscope, which images embedded phase objects, but does not provide quantitative information

Quantitative phase microscopy through differential interference imaging

An extension of Nomarski differential interference contrast microscopy enables isotropic linear phase imaging through the combination of phase shifting, two directions of shear, and Fourier space integration using a modified spiral phase transform. We apply this method to simulated and experimentally acquired images of partially absorptive test objects. A direct comparison of the computationally determined phase to the true object phase demonstrates the capabilities of the method. Simulation results predict and confirm results obtained from experimentally acquired images. © 2008 Society of Photo-Optical Instrumentation Engineers