Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Internal structure of an intact Convallaria majalis pollen grain observed with X-ray Fresnel coherent diffractive imaging.

We have applied Fresnel Coherent Diffractive Imaging (FCDI) to image an intact pollen grain from Convallaria majalis. This approach allows us to resolve internal structures without the requirement to chemically treat or slice the sample into thin sections. Coherent X-ray diffraction data from this pollen grain-composed of a hologram and higher resolution scattering information-was collected at a photon energy of 1820 eV and reconstructed using an iterative algorithm. A comparison with images recorded using transmission electron microscopy demonstrates that, while the resolution of these images is limited by the available flux and mechanical stability, we observed structures internal to the pollen grain-the intine/exine separations and pore dimensions-finer than 60 nm. The potential of this technique for further biological imaging applications is discussed.

Influence of confocal scanning laser microscopy specific acquisition parameters on the detection and matching of speeded-up robust features.

The robustness and distinctiveness of local features to various object or scene deformations and to modifications of the acquisition parameters play key roles in the design of many computer vision applications. In this paper we present the results of our experiments on the behavior of a recently developed technique for local feature detection and description, Speeded-Up Robust Features (SURF), regarding image modifications specific to Confocal Scanning Laser Microscopy (CSLM). We analyze the repeatability of detected SURF keypoints and the precision-recall of their matching under modifications of three important CSLM parameters: pinhole aperture, photomultiplier (PMT) gain and laser beam power. During any investigation by CSLM these three parameters have to be modified, individually or together, in order to optimize the contrast and the Signal Noise Ratio (SNR), being also inherently modified when changing the microscope objective. Our experiments show that an important amount of SURF features can be detected at the same physical locations in images collected at different values of the pinhole aperture, PMT gain and laser beam power, and further on can be successfully matched based on their descriptors. In the final part, we exemplify the potential of SURF in CSLM imaging by presenting a SURF-based computer vision application that deals with the mosaicing of images collected by this technique.

On the suitability of SIFT technique to deal with image modifications specific to confocal scanning laser microscopy.

Computer vision tasks such as recognition and classification of objects and structures or image registration and retrieval can provide significant information when applied to microscopy images. Recently developed techniques for the detection and description of local features make the extraction and description of local image features that are invariant to various changes possible. The invariance and robustness of feature detection and description techniques play a key role in the design and implementation of object recognition, image registration, or image mosaicing applications. The scale-invariant feature transform (SIFT) technique is a widely used method for the detection, description, and matching of image features. In this article we present the results of our experiments regarding the repeatability of SIFT features, and to the precision of the SIFT feature matching, under image modifications specific to confocal scanning laser microscopy (CSLM). We have analyzed the behavior of SIFT while changing the pinhole aperture, photomultiplier gain, laser beam power, and electronic zoom. Our experiments, conducted on CSLM images, show that the SIFT technique is able to match detected key points between images acquired at different values of the acquisition parameters with good precision and represents a consistent tool for computer vision applications in CSLM.

Rapid hyperspectral fluorescence lifetime imaging.

We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue.