Fluorescence microscopy is an essential tool in bio-imaging, yet there are no widely adopted standards for the calibration of fluorescent microscopes. Calibration provides a wide range of information relating to microscope performance. Without calibration, images taken on two separate microscopes cannot be directly compared as they may have differing magnifications, illumination intensities or detector sensitivities. As the range of microscopy techniques capturing 3D information continues to increase, the need for standardisation becomes ever greater. Widely used methods for determining microscope performance are currently limited to basic techniques such as fluorescent beads, which don’t form a regularly spaced pattern and reflective etched gratings, which are limited to being two-dimensional and require changes to the microscope filter sets. Using ultrafast laser processing inside plastic substrates, we demonstrate the generation of bright fluorescent patterns in three dimensions offering new possibilities for calibration in fluorescence microscopy. The fabricated calibration slides can be used to quantify a range of parameters that determine microscope performance. For example, spatial distortions within the field of view can be quantified by a regular array of bright fluorescent points. Other patterns can determine factors such as detector linearity, field flatness and changes in the point spread function across the field of view and over depth. The patterns can additionally be used to calibrate spatial length-scales and for colour channel registration.
KEYWORDS: Multiplexing, Two photon excitation microscopy, Microscopy, High speed imaging, 3D scanning, 3D image processing, Objectives, Spatial resolution, Luminescence, Signal detection, Mirrors, Microscopes, Spatial light modulators, Electron multiplying charge coupled devices, Signal to noise ratio, Light scattering
Optical sectioning techniques using two-photon excitation of fluorescent indicators are central to diverse imaging applications. The limitations of the technique are low speed and undesirable specimen agitation. In our design, high-speed axial scanning is carried out by moving a reference objective to axially displace the focal spot without introducing significant spherical aberration and any agitation of the specimen. Further, the system is configured to allow switching between single spot and multiple focal spot remote focusing to allow either high dynamic range or high speed imaging.
In cardiac imaging, the spacing between sub-cellular sarcomere structures is of great importance to physiologists in understanding muscle design and performance. Making accurate measurements of the sarcomere length (SL) presents a significant imaging challenge owing to the size of the SL (~2μm) and its naturally low variability (<6%), requiring a high level of precision to determine subtle changes between heart disease models. Moreover, measurements of SL from traditional two-photon imaging have so far been ambiguous to within a factor of cos(α), where α is the inclination of the tissue with respect to the focal plane. By remotely focussing a customised two-photon microscope, it is possible to image heart cells at two oblique angles within 200ms. The oblique images uniquely resolve the tissue inclination ambiguity and reduce the variance of SL measures by as much as 23%. This improved precision is crucial in discerning between pathological models of chronic hypertension. As well as improving measurement precision, the distribution of α across the field of view provides additional structural information which can be related to disease morphology. To validate this new imaging protocol, the value of α calculated from the oblique planes provided the input to a rigid model cell which was used to predict the appearance of the cell in the conventional focal plane. The comparison of the model to the image data provided a confidence metric for our measurements. Finally, by considering the optical transfer function, the range of cell orientations for which the method is valid could be calculated.
Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberrationcompensated imaging extends over volumes as large as 450 μm x 450 μm x 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire (<200ms) depth information temporally freezes residual tissue motion in the arrested heart.
This paper discusses the progress of remote focussing microscopy from a novel imaging technique to a reliable tool in the life sciences. Specifically, we describe recent efforts to achieve the accurate calibration of both distance and orientation within the imaging volume. Using a laser machined fluorescent specimen it is possible to identify, with high sensitivity, small (<1%) depth-dependent magnification changes which are a linear function of axial misalignment of the imaging objective. The sensitivity of the calibration procedure limits distortion to <1 μm over the entire imaging volume. This work finds direct application in identifying the microscopic effects of chronic disease in the living heart.
In cardiac imaging, the spacing between sub-cellular sarcomere structures is of great importance to physiologists in understanding muscle design and performance. Making accurate measurements of the sarcomere length (SL) presents a significant imaging challenge owing to the size of the SL (~2μm) and its naturally low variability (<6%), requiring a high level of precision to determine subtle changes between heart disease models. Moreover, measurements of SL from traditional two-photon imaging have so far been ambiguous to within a factor of cos(α), where α is the inclination of the tissue with respect to the focal plane.
By remotely focussing a customised two-photon microscope, it is possible to image heart cells at two oblique angles within 200ms. The oblique images uniquely resolve the tissue inclination ambiguity and reduce the variance of SL measures by as much as 23%. This improved precision is crucial in discerning between pathological models of chronic hypertension. As well as improving measurement precision, the distribution of α across the field of view provides additional structural information which can be related to disease morphology. To validate this new imaging protocol, the value ofα calculated from the oblique planes provided the input to a rigid model cell which was used to predict the appearance of the cell in the conventional focal plane. The comparison of the model to the image data provided a confidence metric for our measurements. Finally, by considering the optical transfer function, the range of cell orientations for which the method is valid could be calculated.
Currently, in most adaptive optical systems, the control loop between the wavefront sensor and the deformable mirror involves intense mathematical calculations, both during calibration and operation of the system. Although thorough research has been done to optimise the control loop, some issues like error propagation and system bandwidth will always be ultimately limited by the coupling between the mirror and the wavefront sensor. Closed-loop by direct feedback from the wavefront sensor to the deformable mirror was proposed by F. Roddier in his well-quoted curvature wavefront sensing paper. However, due to the natural properties of the defocused-image, this direct feed-back method is limited to bimorph mirror applications only. Recently, M.A.A Neil et al proposed a new modal wavefront sensor (MWFS), which can detect several Zernike modes by a simple intensity subtraction operation. One drawback of this method is that it can only handle a limited number of modes. However, in this paper, we refine this method to detect the orthogonal modes of a deformable mirror instead of Zernike modes in a to-be corrected wavefront. Since the number of actuators of a deformable mirror limits the number of mirror modes, the drawback is minimised in this application. Considering the mirror modes can be directly transformed to the deformable mirror control command set by a proper gain coefficient, it is reasonable to construct a direct-feed back adaptive optical system with the modal wavefront sensing. We will report our first stage investigation on direct feedback adaptive optical system which is to understand the response of MWFS to mirror modes.
The accurate measurement of the double-pass ocular wave front has been shown to have a broad range of applications from LASIK surgery to adaptively corrected retinal imaging. The ocular wave front can be accurately described by a small number of Zernike circle polynomials. The modal wave front sensor was first proposed by Neil et al. and allows the coefficients of the individual Zernike modes to be measured directly. Typically the aberrations measured with the modal sensor are smaller than those seen in the ocular wave front. In this work, we investigated a technique for adapting a modal phase mask for the sensing of the ocular wave front. This involved extending the dynamic range of the sensor by increasing the pinhole size to 2.4mm and optimising the mask bias to 0.75λ. This was found to decrease the RMS error by up to a factor of three for eye-like aberrations with amplitudes up to 0.2μm. For aberrations taken from a sample of real-eye measurements a 20% decrease in the RMS error was observed.
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