Monday, November 14, 2016

Week 2: Optics and microscopy

The use of light in neuroscience extends back to Camillo Golgi and Ramon y Cajal’s disputes on the structure of the nervous system, with Golgi believing it comprised of a large continuous fibrous net, and Cajal arguing it's formed of many non-continuous individual cells. Cajal won this argument, and was likely on the right side of history due to having a superior microscope. The importance of quality microscopes has only increased since, and has undergone a new revolution in the past 20 or so years. In the spirit of Golgi and Cajal, this week in the Experimental Neuroscience course at the SWC we aimed to build a microscope capable of visualising neurons. Microscopy 101 Light can be considered to be both particulate in the form of photons, or wavelike; we focused on the macroscopic wavelike properties of light due to the scale on which we were working. On this scale, light can be described as being an electromagnetic wave with a speed that depends only on the medium through which it is travelling, and a variable wavelength and frequency (which are interrelated). Visible light, which we will be dealing with to build our microscope, has a wavelength in the range of 400-700nm. Microscopy manipulates these features of light to enable visualisation of microscopic structures, such as neurons. Designing a microscope Conventional light microscopy makes use of the fact that the speed of light depends on the medium through which it is travelling. Thus, when light passes from one medium to another, its speed changes. As light can be considered as a wave, if light is shone into the change in medium at an angle, then one ‘side’ of the wave will change speed before the other, causing it to change direction. By analogy, if the right hand wheel of a car hits a puddle in the road, the right hand side of the car slows down, causing the car to turn right. The degree of turning, or ‘refraction’, depends on the relative speed of light between the two mediums, known as the index of refraction. Lenses make use of this ability to manipulate the direction of light. By adjusting the angle of incidence and the refractive index of material used, the direction of light can be controlled. This is achieved by adding a curvature to the surface of glass. A key feature of lenses is their focal length, which determines the distance at which light entering the lens will converge to a single point, and hence the size of an image at a given distance from the lens. As lenses use the curvature of their surface to bend light, the focal distance is largely determined by the curvature of the surface of the lens. This plays a crucial role in deciding where to place lenses relative to each other and the sample being imaged. The first step of the microscope is to shine light through the sample. For this purpose, we used an LED light source. However, some light sources produce collimated light, which prevents an image from forming. To ensure our light was not collimated, we used a diffuser prior to light entering the sample, meaning light hit our sample at multiple angles. To reduce contamination from stray light hitting parts of our sample other than our region of interest, we introduced an aperture after the diffuser, but before the light source. Thus our sample is subjected to non-collimated light illuminating only a small region of interest. Having passed through the sample, the light then passes through an objective lens placed 1 focal length away from the source, with the aim of collecting as much of the light from the sample and collimating it. This light then passes into our second lens, which bends the light such that it converges onto our image collection point. To collect our image, we used a webcam with the lens removed placed after the second lens at its focal point, thus providing a photosensitive chip that could easily be connected to a computer. [caption id="attachment_162" align="alignnone" width="1806"]Figure 1 - Schematic diagram of microscope Figure 1 - Schematic diagram of microscope[/caption] [caption id="attachment_164" align="alignnone" width="1124"]Figure 2 - Opening the components of the microscope Figure 2 - Opening the components of the microscope. Left: Before. Right: After.[/caption]   [caption id="attachment_165" align="alignnone" width="783"]Figure 4 - Microscope setup Figure 3 - Microscope setup[/caption] Results Having set up our system we attempted to produce images from different samples at various magnifications. During testing and alignment, we used a slice of rat brain from Kampff lab. Our first successful production of an image can be seen in figure 4, where it is projected onto the forehead of SWC PhD student Jesse Geerts. [caption id="attachment_163" align="alignnone" width="399"]Figure 3 - Image of a brain slice projected onto Jesse's head Figure 4 - Image of a brain slice projected onto Jesse's head[/caption]   We next increased the magnification and replaced Jesse’s head with our modified webcam. As biological tissue can be hard to image, we tested our system by imagine a cloth, before moving onto imaging biological tissue (figure 5).  We successfully imaged populations of neurons, and were able to identify a single neuron filled with biocytin (figure 6). We therefore succeeded in our aim of building a microscope capable of visualising neurons. [caption id="attachment_166" align="alignnone" width="1016"]Figure 5 - Left - Image of a cloth (left) and Golgi stained brain tissue (right) at high magnification. Figure 5 - Left - Image of a cloth (left) and Nissl  stained brain tissue (right) at high magnification.[/caption] [caption id="attachment_167" align="alignnone" width="571"]Figure 6 - Image of a biocytin filled neuron (denoted by arrow) Figure 6 - Image of a biocytin filled neuron (denoted by arrow)[/caption]   Conclusions By using a very simple configuration of lenses, in a relatively short period of time we made a microscope capable of visualising neurons and produce a digital image. With some minor adjustments we could adapt this microscope to use fluorescence, or with a different configuration of lenses produce a higher magnification.

No comments:

Post a Comment