Here, we describe a method of acquisition of fast fluorescent signals with the help of the laser scanning confocal microscope (LSCM). Our method permits an increase in the temporal resolution of acquired signals. The method is based on LSCM recordings of fast fluorescent signals with the shortest achievable time sweep, which are performed with the help of a proprietary algorithm. A series of recordings is made in multiple steps; at each step, the fluorescent signal is incremented by a time interval smaller than the time sweep of the frame of LSCM. The size of the increment determines the achievable time resolution. The convolution of the recorded images results in a signal with the temporal resolution determined by the chosen time increment. This method was applied to register the change in fluorescence (calcium transient) of calcium dye preloaded into peripheral nerve endings by electrical stimulation of the motor nerve. Calculated parameters of the calcium transient were identical to the parameters obtained earlier with the help of a high-speed camera and photodiode. We conclude that the method described here can be applied for the registration of fast fluorescent signals by LSCM with a high spatial and temporal resolution.

In this article, a new scanning electron microscopy (SEM) image composition technique is described, which can significantly reduce drift related image corruptions. Drift distortion commonly causes blur and distortions in the SEM images. Such corruption ordinarily appears when conventional image-acquisition methods, i.e., “slow scan” and “fast scan,” are applied. The damage is often very significant; it may render images unusable for metrology applications, especially where subnanometer accuracy is required. The described correction technique works with a large number of quickly taken frames, which are properly aligned and then composed into a single image. Such image contains much less noise than the individual frames, while the blur and deformation is minimized. This technique also provides useful information about changes of the sample position in time, which may be applied to investigate the drift properties of the instrument without a need of additional equipment.

An electromechanical video camera coupler was developed to rotate a light microscope field of view (FOV) in real time without the need to physically rotate the stage or specimen. The device, referred to as the Camera Thetarotator, rotated microscopical views 240° to assist microscopists to orient specimens within the FOV prior to image capture. The Camera Thetarotator eliminated the effort and artifacts created when rotating photomicrographs using conventional graphics software. The Camera Thetarotator could also be used to semimanually register a dataset of histological sections for three-dimensional (3D) reconstruction by superimposing the transparent, real-time FOV to the previously captured section in the series. When compared to Fourier-based software registration, alignment of serial sections using the Camera Thetarotator was more exact, resulting in more accurate 3D reconstructions with no computer-generated null space. When software-based registration was performed after prealigning sections with the Camera Thetarotator, registration was further enhanced. The Camera Thetarotator expanded microscopical viewing and digital photomicrography and provided a novel, accurate registration method for 3D reconstruction. The Camera Thetarotator would also be useful for performing automated microscopical functions necessary for telemicroscopy, high-throughput image acquisition and analysis, and other light microscopy applications.