Week 5 - Fluorescence microscopy

In week 2 we built a simple light microscope and imaged neurons (fig 1). A glance at the image produced shows three key limitations of this approach: we lack contrast between our neuron of interest and the background; we observe a static readout of a dynamic structure; and we view the brain in slices, rather than in its complete form. A combination of fluorescence and genetic engineering have produced techniques to circumvent these limitations of microscopy.

Fluorescence

Fluorescent molecules absorb light of a given wavelength, which excites electrons into a high energy state. When these electrons relax, they emit light at a different, but specified, wavelength. Fluorescence microscopy utilizes these molecules by shining light at their absorption wavelength, and detecting light at their emission wavelength. If these molecules can be confined only to cells of interest, this produces a nearly infinite contrast between the sample and the background. Confining fluorescent molecules to cells of interest can be done in many different ways, but in many cases it relies upon advances in genetic engineering.

Famously, green fluorescent protein (GFP) was first discovered in glowing jellyfish in Friday Harbor. When the molecule absorbs (blue) light with a wavelength of around 488 nm, it undergoes a conformational change and emits (green) light with wavelengths around 509 nm (see figure 1). Its potential for applications in biology became apparent when Douglas Prasher and Martin Chalfie managed to managed to clone its nucleotide sequence and to express it in E. Coli and C. Elegans 1994. In neuroscience, GFP fused with Calmodulin (CaM) has proven to be an excellent marker of neural activity: when calcium binds to this complex, it undergoes a conformational change, enabling the absorption of light at the excitation wavelength. This is extremely useful, as it provides us with a method for monitoring neural activity by observing calcium transients through a microscope.

Figure 1. Emission and excitation spectra of our set-up. Because the emission and excitation spectra of GFP overlap (blue and green lines), we used filters whose transmission bandwidths are shown in transparent blue and green. Lastly, the dichroic mirror (black line) transmitted the emitted (green) light from the sample, while reflecting the blue excitation light from our photo diode. Filter transmission data were retrieved from thorlabs.com and GFP excitation/emission spectral data from chroma.com.

Designing the microscope

In order to exploit these properties of GFP for microscopy, the design of our microscope has to satisfy a few key characteristics that we summed up in figure 2.

Firstly, to avoid contamination, we needed a way to make sure that there was no overlap between the light used for excitation and the light detected by our camera. As the excitation/emission spectra of GFP show some degree of overlap (fig 1), we placed a band-pass excitation filter in front of our LED light source to ensure only blue light (wavelength 469 ± 17.5 nm) would be sent into the sample. A green emission filter (525 ± 19.5 nm) was placed in front of our camera to ensure that all detected light came from fluorescence emitted by the sample, rather than from our LED. The emission filter undeniably discards much of the fluorescent light coming from the sample. However, since there is virtually no light that is coming from other sources than the sample, a very good signal to noise ratio can be maintained.

A second goal was to separate the paths of the excitation and the emission light. To achieve this, we placed a dichroic mirror between the tube lens and the objective. This dichroic mirror transmits light of wavelengths longer than 500 nm and reflects most of the light below that wavelength (figure 1), allowing us to avoid any excitation light hitting the camera.

A third, not entirely insignificant change to our microscope from week 2 entailed replacing the dismantled consumer webcam with a high-performance, highly efficient scientific CMOS camera. This camera has a quantum efficiency of 82%, meaning that 82% of the photons hitting the light-sensitive surface of the camera will produce an electric response.

Figure 2. Schematic representation of our fluorescence microscope 

Figure 3. The fluorescence microscopy set-up. 

Results

At the end of the week, we tested our fluorescence microscope by imaging zebrafish larvae that were provided by Elena Dreosti. These zebrafish expressed GCaMP, so that their neurons showed green fluorescence whenever the calcium concentration in their neurons increased. The fish were fixed in a gel, such that they could not move, and we imaged their neural activity in vivo by using a 16x magnification objective that can be immersed in water.

The results of this imaging can be seen in this video (turn on HD for better resolution):

Neural activity in zebrafish tectum from Jesse Geerts on Vimeo.

Conclusions

By using fluorescent proteins and a combination of different wavelength filters and a dichroic mirror, we were able to build a fluorescence microscope that overcomes many of the limitations we saw in week 2; capturing only green light ensured that all light that hit our camera came from the neurons, solving most of our contrast problem. Moreover, this meant that we were able to look at a zebrafish brain in vivo and in real time, which was a major improvement from the static readout from a brain slice.

However, our microscope has some disadvantages that become apparent when looking at the video; GFP is expressed also in neurons outside the focal plane, and the excitation light will also hit these neurons, and they will fluoresce as well. Our wide field of view captures this light, meaning that part of our signal is polluted with light from outside the focal plane. As we will see in the next blog, this problem can only be circumvented by looking at a single point in the sample at a time.