In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic
scan rate. This means that the focal spot can be scanned at high speed along any direction in the
specimen, without introducing systematic aberrations. We present experimental point spread function measurements
for this system using an Olympus 0.8 NA 40X water dipping objective lens that demonstrates an axial
range of operation greater than 200 μm. We give details of a novel actuator device used to displace the focusing
element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present a bioscience application of this
system to image dendritic processes that follow non-linear paths in three-dimensional space. The focal spot was
scanned along one such process at 400 Hz with an axial range of more than 90 μm.
In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic
scan rate. This means that the focal spot can be scanned at high speed along any direction in the
specimen, without introducing systematic aberrations. We present experimental point spread function measurements
for this system using an Olympus 1.4 NA 60X oil immersion lens that demonstrates an axial range of
operation greater than 70 μm. We give details of a novel actuator device used to displace the focusing element
and demonstrate axial scan responses up to 3.5 kHz. Finally, we present an application of this system in liquid
crystal research to image the dynamic response of a nematic device during switching. Information about the
director field at different levels in the device can be inferred from images acquired with a temporal resolution of
2.5 ms.
In this paper, we present a novel technique that permits sectioning microscopes to refocus and acquire images
from a large range of specimen depths without introducing movements near the specimen. In contrast to other
such remote focusing methods, this technique avoids systematic aberrations that degrade image quality when
imaging planes away from the optimal focal plane. Furthermore, the specific geometry that is employed in this
work enables refocusing to be carried out at high speed and hence permits, for the first time, a number of
dynamical biological processes to be observed. Although this technique can be applied to any optical imaging
system, it is particularly suited to the case of high numerical aperture microscope systems. To demonstrate
this we present results from two prototype systems built in our laboratory based on a slit scanning confocal
fluorescence microscope and a two photon fluorescence microscope.
In this paper we describe a simple method of optical refocusing for high numerical aperture imaging systems.
As the first stage of this new method satisfies both the sine and Herschel conditions of geometric optics, images
obtained are free from spherical aberration over a large scan range.
We demonstrate a simple and light-efficient way of generating non-diffracting Bessel beams for use in confocal microscopy. A number of imaging modalities using such beams is discussed. Preliminary experimental results including brightfield, fluorescence and two-photon images are presented.
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