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«Technische Universität München Friedrich-Schiedel Institut für Neurowissenschaften Ultra-fast two-photon microscopy for in vivo brain imaging ...»

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Technische Universität München

Friedrich-Schiedel Institut für Neurowissenschaften

Ultra-fast two-photon microscopy for in vivo

brain imaging

Ulrich Leischner

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München

zur Erlangung des akademischen Grades eines

Doctor of Philosophy (Ph.D.)

genehmigten Dissertation.

Vorsitzender: Univ.- Prof. Dr. Thomas Misgeld

Prüfer der Dissertation:

1. Univ.- Prof. Dr. Arthur Konnerth

2. Univ.- Prof. Dr. Dieter Braun Ludwig-Maximilians-Universität München Die Dissertation wurde am 24.2.2011 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 4.3.2011 angenommen.

Table of Contents Chapter 1: Introduction

The general layout of a two-photon scanning microscope

A simple scanning unit with galvanometric mirrors

The use of acousto-optical deflectors as scanning units

Galvanometric mirrors:

Resonance mirrors:

Acousto-optical deflectors (AODs):

Special properties of AODs

The fly back time

The number of resolvable points of an AOD

The cylindrical lens effect

The chromatic dispersion

Movement artifacts require the acquisition of fast 2d image data

Chapter 2: The current status of acousto-optical scanning systems

The chromatic compensation by Lechleiter:

The chromatic compensation by Roorda:

The systematic analysis of the position of the prism

The compensation by using an AOD instead of a prism

Systems without chromatic compensation

Random access scans

Chapter 3: The work of my PhD thesis: The layout and assembly of a custom built two-photon scanning microscope.

Software development for a fast scanning system.

The design of a fast scanning system with a compensation of the non-homogeneous grating of the AOD

The cylindrical lens effect as origin of image distortions and their compensation

The best position and assembly of the compensation optics for the cylindrical lens effect........ 29 Theoretical prediction of the beam distortions caused by the chirped grating of the AOD......... 34 Measurement of beam distortions, caused by the chirped acoustic grating

The construction of different scanning systems

The first AOD-based scanning system with two AODs for x- and y scanning and a two-prism correction for the chromatic dispersion

Improvement of the two-AOD scanning system with one AOD for compensating the chromatic dispersion

A scanning system using one AOD for fast line-scanning, and a galvanometric mirror for slow yscan, together with an AOD for compensating the chromatic dispersion.

The advantages of the AODs from CTI Inc., compared to the products from AA-Optoeletronics 51 Thee-dimensional scanning

Extension for three-dimensional raster scan with video-rate

Scanning in the depth-direction by using an electrical tunable lens

The new scanning mode theoretically increases the signal-to-noise ratio about one magnitude and additionally reduces photo-damage

Image processing: Correcting the movement artifact

Image processing:

Chapter 4: Measurements

The measurement of the Calcium-signal in spines of Purkinje cells in an acute slice preparation

In-vivo measurements of spine-activity in the auditory cortex

Chapter 5: Discussion:

Chapter 6: Summary

Chapter 7: Publications

H.-U. Dodt, U. Leischner, et al., Nature Methods 2007, Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain

W. Wein, M. Blume, U. Leischner, et al., MICCAI 2007, Quality-Based Registration and Reconstruction of Optical Tomography Volumes

U. Leischner, et al., PLoS ONE 2009, Resolution of Ultramicroscopy and Field of View Analysis..... 76 U. Leischner, et al. PLoS ONE 2010, Formalin-Induced Fluorescence Reveals Cell Shape and Morphology in Biological Tissue Samples


Calculation of the chromatic dispersion of a prism

Abbreviations and technical terms:



Chapter 1: Introduction Optical imaging is a good method for investigation of physiological signals in medical research, such as the changes in calcium concentration in cells, because these signals can be detected simultaneously throughout the whole image. For example, when we acquire images with video-rate of a group of cells, loaded with an indicator dye for monitoring the intracellular calcium concentration, we can measure calcium changes inside the different cells at the same time. Calcium is needed for a lot of biological processes inside the cell, and a sudden rise in the calcium concentration in a cell indicates the activation of a cell. The calcium then triggers a lot of secondary chemical reactions. Changes of the membrane potential of nerve cells are also often accompanied by calcium entry into the cell, and therefore the changes of the calcium-concentration are indicators of a wide range of cell activity. By monitoring such a signal in a group of cells, we can distinguish between more active and passive cells, and observe the patterns of the activity in a group of nerve cells in the brain.

These physiological signals are also of interest on a smaller scale, like a small section of a dendritic branch of a nerve cell in the brain. A nerve cell in the brain is connected to thousands of other cells by synapses on spines. These spines are of a very characteristic shape, mushroom like extrusions from the dendritic branch. It is unknown how strong these connections are, and what kind of activity pattern they form in the intact brain. When we are able to resolve the calcium signal in such a spine, we can deduce the connection and transmission strength between two cells, and compare the response of a cell to different input pattern. Such measurements can investigate the mechanisms of the integration of different inputs to a single cell, and reveal the properties of input from other cells on the scale of a single cell. A lot of details of the network connections and the transmission properties are still unknown, mainly because the cellular networks in the brain are highly complex with thousands of connections, and measurements on an intact brain are very difficult. However, a lot of network malfunctions cause mental diseases like Alzheimer’s dementia or schizophrenia, and details of the underlying network alterations are largely unknown. Optical measurements are an excellent tool for investigation of the normal network functions like the spatial organization of input to a cell, and to compare the overall response of a cell to different stimulation pattern. In another step, optical methods have the potential to detect alterations of the network functions in mental diseases.

Although optical imaging is a good method for investigation of network connections in brain cells, the measurements on an intact brain are very difficult, because brain tissue is highly opaque and is not suited for optical imaging in deeper layers. Without a specialized technique, we can only acquire images from the brain surface. The opaqueness of brain tissue is caused by the highly nonhomogeneous composition of substances in the brain. Most components of brain cells are transparent, like lipids, proteins or water, but the non-homogeneous mixture with many membranes embedded by water generates a lot of light-scattering bounding surfaces. Scattering causes the photons to change their direction, and after one scattering process, we can no longer determine the origin of the photon, and use it for an image projection. Such direct widefield projection imaging is only suited for images from the surface. The resolution of the images rapidly degrades when we want to acquire images in deeper layers of brain tissue. Confocal microscopy (Minsky, 1961) was the first technique to increase the imaging depth. This technique illuminates only one point, and removes all scattered photons not coming from that point. This increases the imaging depth in biological tissue up to the ‘mean free path’ of a photon between two scattering events. In practice this ‘mean free path’ is about 40µm (Ntziachristos, 2010), and this can still be considered as the surface region.

The invention of two-photon (2p) microscopy was a major improvement in this respect (Denk et al., 1990). The 2p excitation technique generates fluorescence just at one point by using a focused highpower pulsed laser. Only highest photon densities allow for a simultaneous excitation of a fluorophore with two photons at the same time, and these high photon densities are only present at the focus spot of a laser beam. As fluorescence excitation can only occur at the focus spot, the fluorescence emission can only originate from this location as well. This technique does not need an optical projection technique for image formation, but gathers all photons coming from the sample and directs them to the photo detector. We can get 2D images by scanning the object. As scattering processes in the detection pathway do not degrade image quality, this technique allows for the image acquisition much deeper than the ‘mean free distance’ between two scattering processes, like it is the limit of confocal microscopy. With the 2p technique, it is possible to image up to a depth of 1mm, which means a 25-fold increase in imaging depth.

This technique needs a movement of the focus spot to scan the sample in 2D or 3D to get the raster image information. Moving a spot of focused light through the image plane is mainly done by revolving mirrors, but revolving a mirror is time consuming, as a change of the orientation angle of a mirror requires an acceleration and deceleration. The mirrors are the speed-limiting component of a scanning microscope, and most microscopes are limited to an image speed of about 10 images per second. Therefore, investigations with 2p microscopy are limited to slow processes, as we can not observe fast processes with such a slow detection technique.

There is an alternative device for beam deflection: acousto-optical deflectors (AOD). These devices can deflect a beam at different angles by changes of the diffraction pattern of a grating. The grating is generated by an acoustic wave in a transparent crystal, and this grating can be changed as fast as we can change the acoustic frequency. These devices are inertia free, and therefore they can perform a line-scan with a 10-fold frequency increase compared to a revolving mirror.

The subject of this PhD thesis is the design and construction of a high-speed 2p imaging microscope by using AODs for beam deflection. The beam deflection in AODs is done by changes of the diffraction pattern of an optical grating. This is a different physical principle than a light reflection on a flat mirror, and hence such a beam deflection is accompanied by different optical artifacts, requiring different compensation mechanisms. I will analyze the different artifacts, how they can be compensated, and will present an apparatus that can acquire images of high quality with more than 1000 images per second. In the end I will present some measurements realized with the newly developed apparatus, and demonstrate the capabilities and the image quality of this new microscope.

As two-photon imaging is a central technique, I will first give a detailed introduction of the 2p imaging technique in the first part of this thesis, and I will describe the current status of the technical developments.

The general layout of a two-photon scanning microscope We can modify a standard commercial microscope and use it as a 2p microscope by simply exchanging some filters, mounting a photomultiplier (PMT) at the right position and attach the scanning optics for the two-photon laser. A normal brightfield microscope needs a homogeneous illumination, and good optics for capturing the photons and projecting them to the camera chip. A 2p-microscope needs such optics in the opposite way: good optics for a precise illumination, and a possibility to capture all photons emitted from the sample. Therefore we can just use the detection pathway for illumination, and the illumination-pathway for detection. This requires mounting the PMT to the place where we previously attached the light fiber for the illumination light (figure 1).

This allows for the detection of all photons coming from the surface of the sample. The other pathway for image detection can be used for a precise illumination. To do this, we need to project a focused spot of the 2p-laser at a location called the ‘image plane’. The ‘image plane’ denotes the location of the CCD chip, where the sharp image from the sample is projected. The camera chip is normally placed at this location. But as we do not detect the image with a camera, we can simply dismount the camera and mount the scanning device to perform the image scan at the image plane.

In this way we use the detection pathway in the other way: When we project a spot of light at the location of the image plane, the light is guided through the optical lenses of the microscope downwards to the sample, and illuminates one sharp point there.

Altogether, to modify a microscope to use it for 2p-imaging, we need some minor optical modifications. We must dismount the camera, and attach a scanning device that projects a spot of light at the position of the camera chip. We then just need to exchange some filters and mount the PMT. I present the general layout of such an apparatus in figure 1. In the later sections I will explain some details of the scanning device, and how to achieve high-speed scanning with good results.

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