Background to the investigation

1.1 Scope

Almost all modern biology laboratories employ fluorescence as a tool for studying biological systems. In this activity you will explore how fluorescence imaging can be used to investigate cellular Ca²⁺ signals that affect mitochondrial respiration.

The development of fluorescent Ca²⁺ indicators transformed studies of cellular Ca²⁺ signalling because they allow characterisation of Ca²⁺ signals in single cells with millisecond temporal resolution, and micrometre spatial resolution. In this investigation, you will assess changes in Ca²⁺ concentration and mitochondrial respiration by quantitating the output from fluorescent Ca²⁺ indicators and that of endogenous NADH within individual cells.

There is some background information relating to the investigation, as well as an overview of the theory and principles of the methods employed:

fluorescence and its applications
interpretation of fluorescent images
capturing meaningful data.

This practical consists of a series of three preliminary activities and an onscreen laboratory experiment in two parts. The activities are concerned with technical aspects of fluorescence imaging, and the experiment uses fluorescence imaging data to answer specific biological questions. In each section you will be presented with specific aims and guidance.

1.2 Skills and learning outcomes

As well as helping you understand aspects of cellular Ca²⁺ signalling, this investigation develops a number of key practical skills.

After completing this investigation, you will be able to:

Explain the importance of using settings appropriate for the working range of a fluorescent imaging system in order to capture valid data.
Choose, and apply, appropriate parameters for the purposes of integrating fluorescent signals.
Calculate mean integrated responses using fluorescent signals.
Produce a tabular and graphical summary of experimental data obtained from fluorescence imaging of single cells.
Develop an hypothesis to explain experimental observations.

Although this practical concerns fluorescence imaging, the principles underlying these key skills are applicable to other experimental situations.

1.3 Advice

An understanding of the background material is critical if you are to successfully complete the experiment and correctly interpret the results. To check your understanding, you should undertake the practice activities and complete the quiz questions that are included.

While you should work through the investigation from start to finish, you may well wish to return to some of the background and theory sections at particular points when you are analysing and interpreting the results from your experiments. These sections are structured to help you to readily find the appropriate information.

You should keep a hard-copy or electronic experimental notebook to record what you do and the results that you obtain. The protocols provided can be copied and pasted into your notebook and then modified as required. You should copy and save any results from your experiments (for example, a note of parameters you choose for integrating cellular responses, and the data values you obtain for integrated responses) as you work. You should also record any calculations that you perform and note any observations that you make.

1.4 Principles of fluorescence

When fluorescent molecules absorb energy, the electrons within the molecules change from a resting condition called the ‘ground state’ to an ‘excited state’ for a very brief period of time. Eventually, the electrons return to their ground state, and emit energy as photons of light (Figure 2).

**Figure 2** A single atom within a molecule is shown absorbing energy, which excites an electron from its ground state to an excited state. When the electron returns to its ground state, light is emitted.

In many cases, the energy that is absorbed by fluorescent molecules is in the form of light, and the molecules emit light to return to their ground state. The light emitted by a fluorescent molecule is a different colour to the light that it absorbs, and this colour change can be used to visualise the molecule in biological materials.

The reason why the light emitted by a fluorescent molecule is a different colour to the light it absorbed is because electrons in their excited state lose a tiny amount of energy before they return to the ground state.

You may remember the colours of the visible spectrum from previous studies (Figure 3). Recall that the wavelength of light increases as colour changes from blue to red, and that the energy of light decreases as wavelength increases. Specifically, the light that excites a fluorescent molecule has a shorter wavelength (higher energy) than the light that is emitted.

**Figure 3** The visible spectrum, showing the colours with their corresponding wavelength in nanometres (10^–9 m).

The wavelength of light needed to excite a fluorescent molecule is characteristic of that particular molecule, as is the wavelength of light it emits.

The absorption and emission of specific colours allow biologists to monitor different fluorescent molecules at the same time within a single biological sample. For example, a fluorescent molecule that is excited by ultraviolet (UV) light and emits blue light could be used at the same time as a different fluorescent molecule that is excited by green light and emits red light.

1.5 Protein expression and localisation

Many different fluorescent proteins have been cloned from various organisms (particularly aquatic organisms such as coral), or modified through deliberate changing of gene sequences, to develop a range of fluorescent proteins with specific properties such as colour, environmental sensitivity and stability. Biologists can use simple techniques to transfer the DNA coding for fluorescent proteins, such as the well-known ‘green fluorescent protein’ (GFP) into cells. In the right context, the host cell will benignly express GFP molecules and then start to show fluorescence.

Figure 4 shows a cell expressing GFP and a red fluorescent protein (RFP), taken using a light microscope adapted to record fluorescence.

**Figure 4** A highly magnified portion of a HeLa epithelial cell expressing both GFP targeted to the endoplasmic reticulum (green) and a red fluorescent protein (red) in mitochondria. (Taken from Collins et al. (2001) EMBO J)

The fluorescent proteins were targeted to the endoplasmic reticulum (GFP) or mitochondria (RFP) by appending known targeting sequences that direct cellular proteins to specific locations. Close interaction between the ER and mitochondria is evident; in fact there are some places where the green and red colours have merged to produce yellow indicating where the two organelles are so close together that they cannot be distinguished using light microscopy.

The image in Figure 4 was obtained by introducing two different exogenous DNA molecules into the cell; one encoding GFP and the other encoding an RFP. The cell produces the fluorescent proteins by the same transcription and translation processes that are used for all its other proteins, but using the exogenous DNA as a template. It is actually very easy to introduce exogenous DNA into cells by mixing the DNA with a lipophilic reagent. The lipophilic reagent masks the negative charges of the phosphate groups within DNA, and helps to ‘smuggle’ the exogenous DNA across the cell membrane. This technique is known as transfection.

1.6 Organelle dynamics

Fluorescence can also be used to obtain information about temporal changes in cellular activities. In Video 1, the mitochondria within HeLa cells were labelled with a fluorescent molecule that accumulates within mitochondria, and emits red light when it fluoresces. Images of the cells, and the red fluorescence from mitochondria, were captured at regular intervals for 15 minutes. The movie runs much faster than real time, and you can see that far from being static organelles, some mitochondria are highly motile within a cell.

Video 1 HeLa cells containing a red fluorescent molecule within their mitochondria. (0:06 min)

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1.7 Fluorescent Ca²⁺ indicators

Fluorescent Ca²⁺ indicators allow Ca²⁺ to be measured from many individual cells at the same time. Ca²⁺ binds to, and unbinds from, these indicators very rapidly, so they can be used to measure Ca²⁺ signals that might arise within a few milliseconds (e.g. during muscle contraction) or much slower Ca²⁺ oscillations (e.g. during fertilisation of oocytes by sperm).

There are many different types of fluorescent Ca²⁺ indicator available, including genetically encoded indicators, which can be introduced into cells by transfection. Expression of such indicators can be targeted to specific cell types in transgenic animals, enabling the recording of Ca²⁺ signals in vivo in active animals.

This investigation uses Ca²⁺ indicators that were initially developed by Roger Tsien and colleagues. Tsien and colleagues first synthesised Ca²⁺ indicators by conjugating a fluorescent molecule with another that was known to bind Ca²⁺. By doing so, they obtained a hybrid molecule that would bind Ca²⁺, and where the binding of a Ca²⁺ ion caused electron displacement in the molecule that changed its fluorescence characteristics.

The initial Ca²⁺ indicator developed by Tsien when he was a post-doctoral researcher in Cambridge was called Quin-2. Despite having a modest change in fluorescence emission between its Ca²⁺-free and Ca²⁺-bound states, Quin-2 proved useful in a number of published studies and showed how Ca²⁺ indicators could be used for studying Ca²⁺ signals in living cells. Tsien, and others, progressively refined the properties of fluorescent Ca²⁺ indicators, and development of the indicator Fluo-4, which has a much larger change in fluorescence emission between its Ca²⁺-free and Ca²⁺-bound states, represented a significant advance.

1.8 Fluo-4 – a widely used Ca²⁺ indicator

The structure of the fluorescent Ca²⁺ indicator Fluo-4 is shown in Figure 5.

Question

How many carboxylate groups does Fluo-4 have?

Answer

There are four carboxylate groups (COO⁻). It is to these negatively charged groups that Ca²⁺ binds.

When Ca²⁺ binds to Fluo-4 it causes an increase in Fluo-4’s fluorescence emission, as shown in Figure 6.

**Figure 6** The fluorescence emitted by Fluo-4 increases as the concentration of Ca²⁺ in the medium in which Fluo-4 is dissolved increases.

You can see from Figure 6 that the light emitted by Fluo-4 has a broad spectrum, ranging from 500nm (green) to 600 nm (red). It is quite typical for fluorescent molecules to have a broad spectrum for both the light they absorb and the light they emit. It is also evident from Figure 6 that the position of the emission spectrum does not vary with concentration of Ca²⁺. Rather, the emission increases over the whole spectrum as the Ca²⁺ concentration increases.

Note that fluorescence is commonly expressed in arbitrary units, i.e. as a relative unit of measurement.

1.9 Monitoring cytosolic Ca²⁺ concentrations

A simple scheme showing how a Ca²⁺ indicator such as Fluo-4 can be used to monitor the cytosolic Ca²⁺ concentration is shown in Figure 7.

**Figure 7** A scheme showing the change in fluorescence emission from a Ca²⁺ indicator as it binds, or releases, Ca²⁺.

If cells are loaded with Fluo-4, it is possible to measure changes in fluorescence emission and calibrate the actual change in cytosolic Ca²⁺ concentration. An example of Ca²⁺ signals that were recorded from a cell loaded with Fluo-4 can be seen in Video 2.

Video 2 A cell loaded with Fluo-4. The cell is showing spontaneous Ca²⁺ signals that are evident as increases in the brightness of the image as the movie plays. (0:22 min)

1.10 Sensitivity and affinity

Quantitation of how cytosolic Ca²⁺ concentration changes over time is readily done using modern software, such as a freely available programme called ImageJ. Essentially, a cell (or subcellular region of interest) can be outlined, and the intensity of the fluorescence emission in the outlined area is measured for each image of the movie sequence (Figure 8).

**Figure 8** (a) Fluo-4-loaded cell with an outline that defines the region over which the fluorescence intensity will be measured.
(b) Plot of Fluo-4 fluorescence against time. Recall from Figure 6 that Fluo-4 fluorescence emission is proportional to cytosolic Ca²⁺concentration.

Ca²⁺ indicators allow the precise Ca²⁺ concentration to be calculated because their affinities for Ca²⁺ are known. In fact there are numerous fluorescent Ca²⁺ indicators, which differ in their affinities for Ca²⁺, and therefore in their suitability for measuring large or small Ca²⁺ signals. Plots illustrating the relationship between fluorescence emission and Ca²⁺ concentration for well known Ca²⁺ indicators are shown in Figure 9.

The affinity with which one molecule binds to another is often denoted by a value called a dissociation constant (written as K_d in shorthand). The smaller the K_d value, the higher the affinity for binding.

**Figure 9** The relationship between fluorescence emission and Ca²⁺ concentration for three well known Ca²⁺ indicators, showing their K_d values.

1.11 Questions

**Figure 9 (repeat)** Relationship between fluorescence emission and [Ca²⁺]_cyt for three indicators, showing their K_d values.

The useful working range for an indicator is limited to the Ca²⁺ concentrations over which its fluorescence emission changes in an approximately linear manner. You can see that the working ranges for the Ca²⁺ indicators in Figure 9 do not overlap. Magfluo-4, for example, does not show a change in fluorescence signal over the working range of Indo-1 and Fluo-8. These Ca²⁺ indicators all have different uses because of their differing affinities.

Question 1

Rank the three indicators in Figure 9 in order of increasing affinity for Ca²⁺. Remember that a lower K_d indicates higher affinity.

Answer

In terms of affinity for Ca²⁺, the order is Magfluo-4 < Fluo-8 < Indo-1.

Question 2

Rank the three indicators in Figure 9 in order of increasing sensitivity to Ca²⁺.

Answer

In terms of sensitivity for Ca²⁺, the order is Magfluo-4 < Fluo-8 < Indo-1.

Question 3

Which of the three indicators in Figure 9 would be sensitive to small changes in cytosolic Ca²⁺ concentration?

Indo-1
Fluo-8
Magfluo-4

Question 4

Which of the three indicators in Figure 9 would be suitable for detecting substantial pathological Ca²⁺ changes?

Indo-1
Fluo-8
Magfluo-4

1.12 Calibrating Ca²⁺ signals

The fluorescence signal from a Ca²⁺ indicator can be converted into units of Ca²⁺ concentration by performing a simple calibration experiment that involves the determination of the minimal fluorescence from a Ca²⁺ indicator when it is not bound with any Ca²⁺ at all (F_min) and the maximal fluorescence when the Ca²⁺ indicator is saturated with Ca²⁺ (F_max).

The values for F_min and F_max are usually obtained by making cells permeable to Ca²⁺ by addition of a Ca²⁺ ionophore, such as ionomycin, and then superfusing the cells with solutions containing either no Ca²⁺ (to get F_min) or saturating Ca²⁺ (to get F_max). A typical calibration experiment would look something like the trace in Figure 10.

**Figure 10** Results from calibration experiment.

Once F_min and F_max have been determined, the actual Ca²⁺ concentration during an experiment recording can be calculated from the equation:

where F is the fluorescence emission from the Ca²⁺ indicator at any time during the experiment, and K_d is the published affinity of the Ca²⁺ indicator for Ca²⁺.

This equation might look a little daunting, but it is actually quite straightforward. It relates the fluorescence emission (which is dependent on how many of the indicator molecules have bound Ca²⁺) to the concentration of Ca²⁺. The equation generates the kind of concentration-response curves shown in Figure 9. You are not expected to remember the equation, or how it is used, but simply to remember that fluorescence can sometimes be calibrated and expressed as an actual number of ions or molecules.

1.13 Mitochondrial sequestration of Ca²⁺

It has been known for more than half a decade that mitochondria within cells have the capacity to sequester Ca²⁺ from the cytosol. However, it took many years to discover why, and how, mitochondria sequester Ca²⁺. In fact, a long sought-after protein called the ‘mitochondrial Ca²⁺ uniporter’ that allows the passage of Ca²⁺ from the cytosol into the mitochondrial matrix has only recently been identified.

Video 3 shows a group of cells loaded with a Ca²⁺ indicator that preferentially loads into the mitochondrial matrix.

Video 3 Four HeLa cells with a fluorescent Ca²⁺ indicator within their mitochondria. The indicator is only located within the mitochondrial matrix, so the edges of the cells are not defined and the dark parts are the cytosol and nuclei. (0:12 min)

The presence of a Ca²⁺ indicator within the matrix provides a way of monitoring mitochondrial Ca²⁺uptake in living cells. For the first 10 seconds of the movie, the cells are unstimulated and mitochondrial matrix Ca²⁺ concentration is low. At 10 seconds, the cells were stimulated with an IP₃-generating hormone, and the concentration of Ca²⁺ in the mitochondrial matrix rapidly rises. The rise of matrix Ca²⁺ concentration is evident from the sudden increase in brightness of the Ca²⁺ indicator.

Only the change of mitochondrial Ca²⁺ concentration is evident in the video because that is where the Ca²⁺ indicator is located. However, mitochondria sequester Ca²⁺ from the cytosol, so there would have been cytosolic Ca²⁺ signal before the mitochondria responded. The cytosolic Ca²⁺ increase is not visible in the movie because there was no Ca²⁺ indicator in the cytosol.

The electron transport chain on the inner mitochondrial membrane provides the driving force for Ca²⁺ sequestration into the mitochondrial matrix. The transport of protons from across the inner mitochondrial membrane by the electron transport chain during respiration means that the mitochondrial matrix is negatively charged with respect to the outside of mitochondria. So, during a Ca²⁺ oscillation, when the cytosolic Ca²⁺ concentration increases, Ca²⁺ ions can flow down an electrochemical gradient from the cytosol and into the mitochondrial matrix, via the mitochondrial Ca²⁺ uniporter protein described above. Interestingly, the affinity of the mitochondrial Ca²⁺ uniporter for Ca²⁺ is relatively low. Mitochondria are therefore ideally suited to sequester Ca²⁺ ions when there are large, rapid increases in cytosolic Ca²⁺ concentration, such as during Ca²⁺ oscillations.

1.14 Working range of recording systems

All biological experiments rely on the accurate sampling and recording of data. It is vitally important to consider how this will be done before experiments are attempted. For example, if the sampling of data for an experiment requires the use of particular equipment it is essential to know that the equipment has sufficient sensitivity and working range (sometimes called dynamic range) to detect the results.

The term working range is often used to describe the difference between the dimmest and brightest signals that a system can record. Ideally, a system would be sensitive to dim fluorescent samples, but also be able to accurately record very bright samples too.

Question

By way of illustration, consider an experiment in which there are temperature changes that an experimenter wishes to measure. How would working range be a consideration in choosing a thermometer to make these measurements?

Answer

As you will be aware, there are lots of different types of thermometer; they operate over different temperature ranges, and they have different scales. It would be essential to choose a thermometer that works over the range that covers the temperatures expected in the experiment. The same principles apply to experiments involving fluorescence, and other types of experiment too.

The microscopes that are commonly used to record images of fluorescent cells have controls that alter the brightness of the acquired images. These controls have to be correctly adjusted to ensure that meaningful fluorescent images are acquired.

1.15 Using correct settings – 1

To illustrate the importance of using correct settings in fluorescence imaging, Figure 11 shows images of the same fluorescent sample, but acquired with different instrument settings. Blue fluorescence indicates nuclei; red fluorescence indicates ER-associated protein abundant around the nuclei; green fluorescence indicates microtubules.

Use the buttons to show the outcome of increasing or decreasing red or green signals.

**Figure 11** Images of cells with green, red and blue fluorescent labels. Note alternative images are referred to as 11a, 11b and 11c in the text.

Although the same structures are evident in all three images in Figure 11, the images look rather different. Figure 11a was acquired first, and then the microscope was adjusted as follows:

for (b), the brightness of the red fluorescence was increased
for (c), the brightness of the green and red fluorescence were substantially diminished.

Figure 11a represents the most accurate depiction of the fluorescence emitted by the cells. This can be briefly explained by stating that the red, green and blue fluorescence levels were all correctly adjusted before the image in (a) was captured. The activities you will conduct later in this practical explain this more fully, and illustrate how we can tell that Figure 11a is the most accurate depiction of the biological sample.

For Figure 11b, the red fluorescence was increased so much that many parts of the image now look yellow because the red and green colours overlap. For Figure 11c, the green fluorescence is far too dim. The images in Figure 11 are an illustration that it is sometimes possible to record different observations from the same sample.

Question

Given that the image in Figure 11a provides the most accurate depiction of the distribution of proteins in the cells shown, what erroneous conclusions could the images in Figures 11b and c lead to?

Answer

Overlap of the green and red fluorescence in Figure 11b produces lots of yellow pixels, which could give the impression that the ER-associated protein and the microtubules are always closely associated. The weak green fluorescence in Figure 11c could suggest that microtubules are only around the nucleus.

1.16 Using correct settings – 2

Images of the same fluorescent sample can look very different if the settings used to capture the image are changed; so how can you know when capture settings are correct, and that the image is a valid representation of the light emitted by a fluorescent molecule?

An experienced researcher is likely to be able to judge whether the settings used to capture a fluorescent image are appropriate or not on visual inspection, but such a subjective method is not sufficiently rigorous, for example, where data are to be published. There is a better guide than simple visual inspection, based on making sure that the data obtained fit the working range of the system used to capture it.

To explain what working range means for a fluorescence image, the range of intensities of all the pixels (also known as picture elements) that make up an image must be examined. You may have come across the term pixels in relation to a camera or a television. In essence, pixels are small regions that sit side by side, and together they comprise an image. The more pixels there are in an image the sharper it will look.

To illustrate this, consider just the green fluorescence from the image shown in Figure 11a, which is reproduced in Figure 12. You can see that in the image with 512 x 512 pixels (that is 512 pixels in both the vertical and horizontal directions), the green microtubules look quite clear and detailed. However the quality of the image deteriorates as the number of pixels is reduced.

**Figure 12** Images of the same cells, but with different numbers of pixels. Note that when the number of pixels is reduced, images get smaller, but to show the poor quality of images with fewer pixels the images in panels have been stretched to the same size as the 512 x 512 pixel image.

1.17 Using correct settings – 3

How can we ensure that the recorded fluorescence signal is within the working range of the recording system?

Consider the same green fluorescence image with 512 x 512 pixels, reproduced again in Figure 13. Recall that a 512 x 512 pixel image will have 262 144 pixels in it (i.e. 512² = 262 144). The intensity of all of the 262 144 pixels that make up the image, or the pixels within a defined area of the image, can be plotted in the form of a histogram with the number of pixels plotted on the y-axis against intensity on the x-axis. This is shown in Figure 13b. You can see that there is a wide spread of pixel intensities ranging from ^~10 to ^~250. This is ideal because the system that was used to capture the image has a working range of pixel intensities from 0 to 255. It appears that the pixels that make up the green fluorescence image in Figure 13a were appropriately distributed within the working range of the recording system.

It is important to understand what pixel intensity values tell us. An intensity value of 0 means that a pixel will be entirely black, whereas an intensity value of 255 means that a pixel will be as bright as it can be. At a pixel intensity of 255, a pixel is said to be ‘saturated’ as it cannot be any brighter. To obtain an accurate fluorescence image, we want to use as much of the 0–255 working range as possible. It is important that all the pixel intensities fit inside this range, if not it is likely that information will be lost. Having many black pixels (pixel value of 0) or many saturated pixels (pixel value of 255) usually indicates that the wrong settings have been used, and that the fluorescence data are not likely to be accurate.

**Figure 13** Distribution of pixel intensities in a fluorescence image. (a) A green fluorescence image. (b) The intensities of the pixels that make up the image in (a). Note that pixel intensity has arbitrary units.

1.18 Using correct settings – 4

The reason for the particular range of 0–255 is that the system used to capture the data could discriminate 256 intensity levels. This type of system is sometimes referred to as an 8-bit system (2⁸ = 256 intensity levels, ranging from black to saturation in a captured image). Sometimes systems have more, or fewer, levels of discrimination. For example, an image could be 10-bit (2¹⁰ = 1024 intensity levels). However many levels of discrimination there are, the data must fit between the minimum and maximum values.

It is usually possible to obtain a histogram of pixel intensities when an imaging system is running and the biological sample is being viewed, so that the correct settings can be applied before an image is captured. When this is not possible, and an image is taken without examining pixel intensities, it is necessary to check the distribution of pixel intensities in the captured image. If the image is found to have an inappropriate pixel intensity distribution then a new image should be taken with appropriate changes to the settings.

You have now completed ‘Unit 1: Background to the investigation’. Return to the Fluorescence imaging landing page to begin ‘Unit 2: Activities – Capturing and quantitating fluorescence signals’.

1.1 Scope

1.2 Skills and learning outcomes

1.3 Advice

1.4 Principles of fluorescence

1.5 Protein expression and localisation

1.6 Organelle dynamics

1.7 Fluorescent Ca2+ indicators

1.8 Fluo-4 – a widely used Ca2+ indicator

Question

Answer

1.9 Monitoring cytosolic Ca2+ concentrations

1.10 Sensitivity and affinity

1.11 Questions

Question 1

Answer

Question 2

Answer

Question 3

Question 4

1.12 Calibrating Ca2+ signals

1.13 Mitochondrial sequestration of Ca2+

1.14 Working range of recording systems

Question

Answer

1.15 Using correct settings – 1

Question

Answer

1.16 Using correct settings – 2

1.17 Using correct settings – 3

1.18 Using correct settings – 4

1.7 Fluorescent Ca²⁺ indicators

1.8 Fluo-4 – a widely used Ca²⁺ indicator

1.9 Monitoring cytosolic Ca²⁺ concentrations

1.12 Calibrating Ca²⁺ signals

1.13 Mitochondrial sequestration of Ca²⁺