Experiments on optical recording techniques for culturing single neurons

Summary

Techniques for optically recording the course of activity of individual viable neurons must be considered in the light of two fundamental questions, namely, what to record and how to record it. In particular, in determining what is to be recorded, meaningful parameters should be selected (e.g., membrane potential or ion concentration), the nature of the information required (e.g., qualitative or quantitative) should be determined, and the optical indicator best suited for these assays should be selected.

Modern Neuroscience Research Techniques

Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun

Operation method

Experiments on optical recording techniques for culturing single neurons

Principle

The important steps in performing single neuron optical recording can be divided into three parts: design and installation of the apparatus; design and implementation of the experiment; and analysis and display of the signal. The following section details the design and implementation of the experiment, and the analysis and display of the signal.

Materials and Instruments

Cells Solution
Optical indicators
Optical recording systems Auxiliary electrophysiologic devices Data acquisition systems

Move

This section focuses on a number of methodological issues or experimental techniques that are important to the successful recording of optics, but does not present a single methodology, since the methods used to carry out the recording of optics in experiments are practically varied, i.e., only factors that are common to all experiments will be considered here. Many of these factors are quite common, but they are often what makes the difference between experimental success and unnecessary failure. These factors include:

Preparation and storage of a light indicator

-Loading and staining programs

I Experimental design

Calibration procedure

-Signal processing method

I. Preparation and storage of light indicators 1. Dissolution and storage of VSD

There is no standardized method for dissolving and storing these indicators, and in most cases the optimum conditions for the preparation of the various dyes are empirical. Most VSDs are not naturally water-soluble due to the nature of biphilic molecules, and sometimes surfactants are required to dissolve them. It is also sometimes necessary to help these dyes penetrate into the cell membrane with other reagents, including various solvents or mixtures thereof, as well as surfactants such as ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), polyether (Pluronic) F-127, bile salts (e.g., sodium bile acids), and staining vesicles.

Examples of dissolution and storage methods for VSD commonly used in single-cell studies are shown below:

2. Dissolution and storage of CaSD

Ion sensitive indicators are generally divided into two groups: free salts and methyl acetate (AM) esters. These two types of CaSD are different in terms of dissolution and storage. Free salts: Most CaSD free salts are water soluble and are stable at -20T for long periods of time, both in the dissolved and solid state. Usually these salts are used for microinjection or dialysis, so concentrated storage solutions can be prepared in pure water (without calcium). There are no special requirements for the preparation of these solutions, but it is better to prepare concentrated master batches (50?IOOX) for storage. These fractions should be stored at dry, -20X.

Note: Some researchers have mixed these dyes with the membrane clamp electrode internal fluid and stored them frozen, but our experience suggests that the dyes stored in this way degrade more rapidly.

II. Loading/staining schemes

There are a number of possible methods for loading/staining of photoindicators, and these fall into two categories: batch loading and single-cell loading.

In batch loading studies, all cells present are loaded, or stained without difference. Batch loading methods include:

-Bath incubation

I AM ester loading

Electroporation

-Cationic liposome transduction

-Low-osmotic oscillations

The most commonly used method for mediating the entry of calcium stains into cells is to utilize AM ester.AM ester shields the strongly negatively charged portion of the dye molecule (Table 4-2), so it can be retained in the cell membrane.Once AM ester enters the cell, a nonspecific esterase removes the ester from the calcium-sensitive dye, and the dye is intracellular.AM ester has been used for a variety of purposes, including the removal of calcium stains from cells and the removal of calcium stains. Loading is usually performed in single-cell studies by microinjection or membrane clamp electrode dialysis, and localized electroporation is also used.

The following are the three most commonly used methods for optical recording: (i) incubation; (ii) microinjection; and (iii) dialysis (via membrane-clamp electrodes).

In each case, the optimal staining/loading conditions depend on the indicator used. The determination of optimal conditions is usually empirical, but a few representative examples listed below can serve as a guide.

III. Experimental Design

The design and execution of experiments using optical indicators requires careful consideration of many factors, some of which are prerequisites for obtaining useful data and avoiding artifactual results. Consideration of these factors can be divided into two categories, general factors that are applicable to all experiments, and factors that are applicable specifically to experiments with optical indicators.

1. Aspects to be considered for general design

Several basic criteria need to be met in order to make a reliability judgment about the experimental manipulation or administration of a drug that produces an effect:

A baseline of measurement: is there a stable baseline prior to the experimental manipulation or application of the drug?

I Repeatability: Are the observed experimental results reproducible?

Reversibility: Can the experimental effect be restored after withdrawal of the experimental manipulation or drug?

A hierarchical response: does the response vary hierarchically with the intensity of the stimulus?

-Pharmacological characterization: can the response be blocked or augmented by the corresponding drug?

2. Aspects to be considered for special design

Experimental designs specifically for use with optical indicators usually take into account: the use of dyes, signal optimization, and the combination of additional electrophysiological techniques.

1. Dye selection: special criteria need to be established to judge whether the concentration and/or sensitivity of the dye is stable throughout the experiment. The problem of non-uniformity of dye concentration and sensitivity can be caused by incomplete dialysis of membrane clamp electrodes or internalization of voltage-sensitive dyes. Stability of reactivity is usually confirmed with responses elicited by standard or control stimuli.

2. Signal optimization: sometimes the overall signal contains both specific and non-specific signals, so it is important to find ways to distinguish between these components. An example of non-specific fluorescence is the production of autofluorescence by cells. This intrinsically emitted incandescent light is not dependent on other extrinsically fluorescent molecules, and autofluorescence becomes a problem when irradiating biological samples with light sources close to UV wavelengths. The solution to this problem is to measure the fluorescence of the cell in the absence of an optical indicator and then subtract that value from the resting fluorescence value in the presence of a dye. This value is generally measured before staining, or in the same area of the experiment where the dye is not stained. Another important experimental question is whether the signal needs to be averaged or digitally oversampled to detect the signal we want to observe. In cases where the signal is small and needs to be averaged, enough of the same recordings must be taken to perform the averaging process. Finally, if the light source is intense, consideration should be given to the need for a bleach correction, which is usually performed by making control recordings, which are taken under experimental conditions without a stimulus or during experimental manipulation.

3. Synthesis: The combination of optical recordings with electrophysiological techniques often requires specific procedural changes. For example, VSD dissolved in solvents, especially DMSO/F-127, inhibits the sealing of the membrane clamp electrode to the cell membrane, so it is sometimes necessary to form this seal before the cells are stained.

   IV. Methods of Calibration

   Optical signal calibration< is necessary if the purpose of the experiment is to require a quantitative result or to measure the absolute change in the value of a desired parameter; similarly, if comparisons of signals are required across experiments, or signals at different points in the same experiment, these signals have to be calibrated. The quantitative method demonstrates its advantages if it is used to record all signals, although this method is not always available for other reasons, such as the recording bandwidths often involved.

   Calibration of optical signals is often chosen from the following methods: single wavelength determination, ratio determination, and hybrid determination.

   Undoubtedly, the ratio method gives the most reliable results. This method can be used with some indicators that have spectral offsets to either excitation or emission light, depending on the variable to be observed. These spectral shifts allow comparison of two wavelengths with fluorescence intensity changes in opposite directions, or comparison of a single wavelength with a spectral isosbestic point (i.e., a point that is insensitive to the parameter being measured). In addition to providing quantitative results, the ratio assay reduces or eliminates systematic errors in fluorescence caused by: indicator concentration; excitation path length; excitation intensity; and detector effectiveness.

   More importantly, the ratio assay also eliminates many artifacts and non-systematic factors, including: photobleaching; overtime leakage of the indicator; uneven distribution of the indicator; and varying cell thickness.

   In some cases, the ratio assay is also more sensitive because the change in fluorescence at each wavelength is usually an inverse change in signal, so that the change in signal ratio is greater than the change at any single wavelength.

   However, under some experimental conditions, it is impractical to apply the ratio method, which can be applied to the hybrid method (Lev-Rametal.1992). Hybrid determination is the combination of quantitative determination and qualitative estimation at different transients, e.g., the initial baseline can be quantitatively determined by ratio determination. Subsequently, rapid changes in the same parameter at a single wavelength can be measured qualitatively at a much higher measurement frequency. It is important to note, however, that this method assumes that all other variables (especially the indicator concentration) are held constant during the recording of the single wavelength measurement.

   Both the ratio-measurement method and the non-ratio-measurement method have been used for calcium-sensitive dyes and voltage-sensitive dyes. Examples of each type of calibration method are discussed below. Table 4-6 graphically summarizes how the measurements are made in each case. In addition, general guidelines that are commonly used for both types of indicators are summarized below:

   1. VSD Calibration

   Voltage-sensitive fluorescent dyes are often referred to as 'scale-free linear voltmeters'. However, they can only provide information about voltage changes, while the absolute amplitude of the electrical signal varies depending on the dye staining and local variability in sensitivity. Therefore, these dyes are most commonly used for non-calibrated absolute comparisons between points, and inter-sample comparisons are not feasible. However, calibrated measurements are possible in some cases, and the success of their measurements is easily verified by synchronized electrical measurements. Measurements of this type include the following assays: single wavelength method; double excitation wavelength method based on an excitation spectrum offset; and double emission wavelength method based on an emission spectrum offset.
   
   
   
   The <img alt="" calibration method (Zhang etal. 1998). The disadvantage of this excitation ratio composition method is that it requires alternating two images and/or switching excitation light flashes, which is not only time-consuming but also limits the overall time bandwidth, which cannot meet the requirements of acquiring fast events such as action potentials.9 The formula for converting standardized ratio data to absolute membrane potential (mV) is V m = C F X R ' where CF is the conversion factor between the ratio data and the membrane potential value. where CF is the conversion factor between the ratio data and the membrane potential value; and J?' is the standardized ratio value (usually standardized to - R for OmV). 3 . Ratio measurements for detecting absolute changes in membrane potential based on dual-emission wavelengths with an offset in the emission spectrum (Bullen and Saggau 1999; BeachetaL 1996): This is another ratio measurement for measuring changes in membrane potential with TTC agents, i.e., using a single excitation wavelength along with a dual-emission wavelength, which relies on a voltage-sensitive dye, and is based on a single excitation wavelength, but with a dual-emission wavelength. This method relies on a voltage-dependent spectral shift in the emission spectrum of the voltage-sensitive dye di-8-ANEPPS. Typically, this is measured using a follow-on bidirectional beam spectrometer (e.g., DCLP5 70 ) or a prism and dual photodetector (e.g., DCLP5 70 ).

   2. Calibration of CaSD

   There are three possible calibration methods for these indicators: the single wavelength method, the ratio method based on an excitation offset or an emission offset, and the hybrid method.
   
   

   3. General guidelines for calibration of photoindicators

   An important step in the conversion of optical measurements to observed physiological parameters is post-experimental calibration. Although conversion factors for voltage and calcium ion indicator calibration can be measured in solution or in various simplified specimens (e.g., vesicles), these conditions generally do not correspond to the true values of the intracellular environment. Factors that do not reproduce well under these conditions are temperature, pH, ion concentration, and interaction of the dye with proteins or membranes.

   Also, some CaSD and intracellular protein interactions have been found to alter the apparent 2^○^^-bayashietal.1993). In short, in situ calibration is superior to the equivalent ex vivo process, so the ex vivo method should be used whenever possible.

   These calibration methods can often be accomplished in a cytochemical clamp fashion with pore-breaking ion carriers_^ For example, membrane potential ratios can be calibrated with valinomycin, i.e., a series of valinomycin-mediated K+ diffusion potentials are specifically applied to establish a gradient over the range of membrane potentials desired to be measured and the fluorescence ratios are simultaneously determined. Details of this procedure are given in Loew (1994). Similarly, the calcium ion ratio assay can be calibrated in situ with ion carriers such as ionomycin or casithromycin (or their analogues 4-brom 〇-A23187)? However, care should be taken when using these compounds, which have a rather strong autofluorescence, especially visible under UV light. For a detailed description of these methods see the relevant chapter by Kao (Nucdtelli 1994).

   This chapter will also discuss some general assumptions and practical limitations of the calibration process.

   Note: Generally subtract any autofluorescence values or other deviations before calculating the ratio or AF/F.

   V. Signal Processing Methods

   Even when the best indicators and the best instruments are used, some optical signals are still weak, accompanied or only by noise.

   On the other hand, the sensitivity of the detector used may be poor, or the physiological indicator measured may be quantum in nature. In any case, extra care must be taken to extract the desired signal from the background noise. Noise may include: systematic noise and random noise.

   In some cases, systematic noise can be detected and its effect can then be canceled out by subtraction or division. In contrast, random noise becomes difficult to separate from a valid signal. Signal averaging is one of the methods of eliminating the effects of truly random noise, however, signal averaging is sometimes not feasible (e.g. for non-stationary events). In a single-scan record, only those components of random noise that are spectrally separable from the signal can be removed (e.g., by filtering).

   In order to overcome the noise problem in optical recording experiments, a number of signal processing and noise reduction techniques can be used; they include: source noise ratio methods, digital filtering, and signal averaging.

   1. Source voice ratio method

   System noise present in experimental recordings can be divided into two categories: additive or multiplicative. Usually, phase force curtain noise can be removed by subtraction, while multiplicative noise can be best calibrated by the ratio method. Multiplicative noise, generated by the variation in the intensity of the signal source, is the most common source of noise in optical recording experiments, and is particularly noticeable when the relative change in fluorescence is equal to or less than the fluctuations produced by the light source. In such cases, it is difficult to isolate a valid signal from the noise, and this is a common problem for laser light sources, which are capable of variations in source intensity of up to 5% peak-to-peak. However, these variations can be measured and eliminated from the signal using the ratio method. In fact, calculating the ratio of a signal measurement to a reference measurement is the most effective way to remove source noise. This method is superior to subtraction in that it does not require amplitude matching between the signal and the reference, and the effectiveness of this method is described by Bullen et al. (1997) and illustrated by an example in Figure 4-5.
   

   Another method of eliminating the source noise variance is to calculate the ratio between the two emitted wavelengths, in which case the source noise variance exists as a common mode signal at both wavelengths and can be effectively eliminated by the ratio calculation process.

   2. Digital Filtering Method

   Digital filtering is an important signal processing tool that is often used to reduce the effects of random noise or harmful signals in recorded signals (see Chapter 45), which is useful for signals that are not stationary and cannot be averaged. The principle of the digital filtering method is that useful frequencies are separated out according to whether they are signal or noise. The following are the four main types of digital filtering: low-pass, high-pass, band-pass, and band-stop.

   Low-pass filters are important in controlling the bandwidth of an experimental recording to retain useful signal components while removing high-frequency components. High-pass filtering, also known as A/C coupling, facilitates the removal of the DC component of the signal, revealing only the changing parts. Band-stop (or tangent) filters block a particular bandwidth and are particularly useful in removing AC noise from experimental recordings.

   There are also special filters that retain the high-frequency components while functioning as low-pass filters; one example is the Savitzky-Golay filter method, which essentially performs a multinomial regression over a localized region, which in turn determines a smoothed value for each numerical point. What makes this method superior to other filtering methods is that it preserves data features such as peak heights and wave widths that would normally be "washed out" by averaging neighboring data and low-pass filtering.

   Various applications of these digital filters are usually found in scientific mapping software packages (e.g. Origin or SigmaPlot), and also in specialized mathematical software (e.g. Matlab or Mathematica).

   Note: It is important to avoid filtering frequencies that contain significant signal components, and it must be recognized that some filtering methods can result in small phase shifts in the data, although nowadays FIR (Finite Impulse Response) digital filters can be inverted to solve this problem. Finally, care should be taken not to violate the principles of sampling (see Chapter 45, "Acquisition and Digitization of Data").

   Note: It is certainly wise to apply analog filters (active or passive) at all stages of data acquisition or processing (starting with the detector), as this significantly reduces the build-up of harmful noise at each stage and reduces the extent to which subsequent digital filtering is required.

   3. Signal Averaging

   Signal averaging reduces the effects of random noise by grouping tests temporally or spatially by region or repetition (see Chapter 45). If the noise is truly random, then signal averaging reduces the noise by a factor Jvl, N being the number of test repetitions. This averaging method requires that the events be fixed and include all occurring frequency components, i.e., the signals used for averaging are strictly time-locked events. If signal averaging is used, care should be taken not to introduce any temporal chatter (e.g., biological or instrumental), which could cause low-pass filtering effects. In cases where this is a problem, events like action potentials can be classified by their peaks and averaged to improve the quality of the overall signal.

   An example of the effect of signal averaging is shown in Figures 4-6. In this example, experiments are given to detect problems with the linearity of the data from the VSD ratio method, and the averaged signal method results in a proportional reduction in relative noise as the number of experiments is increased.

   A disadvantage of the real-time averaging method, is that the overall test frequency is usually reduced because of the time it takes to collect a sufficient number of recordings for averaging. This limits the number of data points that can be captured in a given time window, which in turn can be a problem in those experiments where the response timescale of the drug or experimental manipulation is important.
   

   VI. Results.

   This section describes important principles in the presentation and display of experimental results. They include.

   -Data presentation

   -Important characteristics of experimental records

   Types of experiments and typical records

   1. Data expression

   Optical recording results obtained from single neurons or partial neurons can be expressed in a variety of ways, including the following.

   -Dimensional recordings: signals recorded from a single locus, or data extracted from a single point or region of interest (ROI) in an image, are displayed as a one-dimensional line of recordings plotted against time.

   Artifactual color imaging: a series of color images to distinguish changes in activity levels or ion concentrations. Live video: a sometimes speed-adjustable reproduction of images obtained directly from an experiment. Although video and artifactual color imaging provide a high-quality, qualitative display of data, it is often difficult to show the time course and/or quantitative variations in these methods. (e.g., currents, voltages) to synthesize and analyze.

   2. Important Characteristics of Experimental Records

   There is an unfortunate tendency in current articles to provide over-simplified data, and in many cases the original data are omitted altogether. This situation does exist in optical recording studies, i.e., many records are often reduced to single pseudo-color images. However, it is still important to provide some raw records in order for others to be able to make judgments about the quality of valid data. In examining this type of data, it is important to consider various issues such as whether the records provided show ...

   . the sensitivity of the test: whether the method of recording and the indicators used are sufficiently sensitive for the measurements made.

   -Signal/noise ratio: is the given signal/noise ratio sufficient to obtain useful experimental conclusions? Can it be further optimized?

   Spatio-temporal resolution: whether the chosen method has sufficient spatio-temporal resolution to answer the questions posed by the experiment

   Fidelity: Are the records provided an accurate reflection of valid physiological events? Are there any interferences and variations caused by the recording method itself?

   3. Types of experiments and their representative records

   A number of experimental recordings are provided in this chapter to illustrate the various types of experimental recordings that may be seen in single neuron experiments. For example, the ratio signal provided earlier was taken from an experiment examining the VSDdi-8-ANEPPS linear relationship. This recording was obtained at a scanning site immediately adjacent to the diaphragm clamp electrode, demonstrating the existence of good agreement in response time and amplitude between the optical signal and the voltage clamp command waveform. Such calibrations can subsequently be quantified for similar optical detection data done under more connected ia physiological conditions.

   
   In a different example using the same VSD, postsynaptic potentials in dendrites of cultured hippocampal neurons were examined for integration in the hippocampus and conduction patterns (Figs. 4-7). This practical experiment further illustrates the utility of this optical recording method in a number of ways. In particular, these recordings were obtained from very small cells using non-invasive methods. Moreover, several assays can be performed simultaneously at different recording sites (2 ym in diameter), whic

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