Once raw data files are organized following requirements in Input file format, analysis can get started. A first step is to follow the guidelines in 10 minute tutorial. Here we go into more detail about:

• how to parse your data,
• how to define the observable to look at, and
• how to define conditions.

## Experiment and filters¶

To start the analysis, you need to tell tunacell which experiment to analyse, and whether to apply filters.

To set up the experiment, you have to give the path to the experiment folder on your computer. We will denote this path as <path-to-exp>, then use:

from tunacell import Experiment
exp = Experiment(<path-to-exp>)


By default, no filter is applied. But it is possible to associate a set of filters to an experiment, giving instructions to how data structures will be parsed.

### Defining the statistical ensemble¶

The statistical ensemble is the set of cells, lineages, colonies, and containers that are parsed to compute statistics. In some cases, you might be likely to remove outliers, such as cells carrying anomalous values.

To do so a FilterSet instance must be defined and associated it with the Experiment object. Detailed description about how to define filters and filter sets is in Filters. Here we give a simple, concrete example. Suppose you’d like to filter out cells that didn’t divide symmetrically. To do so, you first instantiate the FilterSymmetricDivision class:

from tunacell.filters.cells import FilterSymmetricDivision
myfilter = FilterSymmetricDivision(raw='length', lower_bound=0.4, upper_bound=0.6)


length is used as the raw quantifier (assuming you have a column length in your data files). Such filter requires that the daughter cell length at birth must be bound within 40 and 60 percent of the mother cell’s length at division. Then:

from tunacell import FilterSet
myfset = FilterSet(filtercells=myfilter)


In the last line, the keyword argument specifies filtercells since our filter myfilter acts on Cell instances. You can define one filter for each type of structures: Cell, Colony, Lineage, and Container.

Once that a FilterSet instance is defined, load it with:

exp.set_filter(fset)


Note

Filtering cell outliers may affect the tree structure, decomposing original tree in multiple subtrees where outlier node has been removed. Hence the number of trees generated from one container file depends on the filter applied to cells.

### Defining particular samples¶

All samples from an experiment are used for statistics, under the filtering assumption discussed above. However, visualization of trajectories is performed over a subset of reasonable size: this is what we’ll be calling small samples.

Small samples can be chosen specifically by user (“I am intrigued by this cell, let’s have a look on its trajectory”), or randomly. To do so:

from tunacell import Parser
parser = Parser(exp)


Note that a sample is identified by a couple of labels: the container label, and the cell identifier. For example:

parser.add_sample({'container_label': 'FOV_001', 'cellID': 12})


or synonymously:

parser.add_sample(('FOV_001', 12))


This information is stored under the samples, and you can get a print of the registered samples with:

print(parser.info_samples())


You can also add randomly chosen samples:

parser.add_sample(10)


### Iterating through samples¶

The exp object provides a set of iterators to parse data at each level, with the appropriate applied filters:

• Container level with the method iter_containers(), filtered at the container level,
• Colony level with the method iter_colonies(), filtered at the container, cell, and colony levels,
• Lineage level with the method iter_lineages(), filtered at the container, cell, colony, and lineages levels
• Cell level with the method iter_cells(), filtered at the container, cell, colony, and lineages levels.

The idea behind tunacell is to decompose colonies into sets of lineages, i.e. into sets of sequences of parentally linked cells. This way, it is possible to extract time-series that span time ranges larger than single cell cycles.

Note

Decomposition in lineages is performed randomly: at cell division, one daughter cell is chosen randomly to be the next step in the lineage. This way, lineages are independent: a given cell belongs to one, and only one independent lineage.

#### Iterating over listed samples¶

Use above-mentioned methods on the Parser instance.

See Parser for more details.

#### Iterating over all samples¶

Use above-mentioned methods on the Experiment instance.

See Experiment for more details.

## Defining the observable¶

To define an observable, i.e. a measurable quantity that evolves through time, use the Observable class:

from tunacell import Observable


and instantiate it with parameters to define a particular observable.

First parameter is the name to give to the observable (to find it back in the analysis process).

Second, mandatory parameter is the column to use as raw data (e.g. ‘length’, ‘size’, ‘fluo’, …).

Then, it is possible to use time-lapse data (as stored in data files, or processed using a time-derivative estimate) or to determine the value of said raw observable at a particular cell cycle stage, for example length at birth.

### Indicating raw data¶

First, one needs to indicate which column to be used in the raw data file, by specifying raw='<column-name>'.

When raw data is expected to be steady, or to be a linear function of time within cell cycle, then use scale='linear' (default setting). When it is expected to be an exponential function of time within cell cycle, use scale='log'. We will mention below how this parameter affects some procedures.

Raw data can be used as is, or further processed to provide user-defined observable. Two main modes are used to process raw data:

• The dynamics mode is used when one wants to analyze observables for all time points; examples are: length, growth rate, …
• The cell-cycle mode indicates observables that are defined as a single value per cell cycle; examples are: length at birth, average growth rate, …

### Dynamic mode¶

It corresponds to the parameter mode='dynamics'. It sets automatically the timing parameter as timing='t' where t stands for time-lapse timing. It is meant to study observables for all time points (time-lapse, dynamic analysis).

### Cell-cycle modes¶

Cell-cycle modes are used when one observable need to be quantified at the cell-cycle level, i.e. quantified once per cell cycle.There are few cell cycle modes:

• mode='birth': extrapolates values to estimate observable at cell birth;
• mode='division': extrapolates values to estimate observable at cell division;
• mode='net-increase-additive': returns the difference between division and birth values of observable;
• mode='net-increase-multiplicative': returns the ratio between division and birth values of observable;
• mode='average': returns the average value of observable along cell cycle;
• mode='rate': proceeds to a linear/exponential fit of observable depending on the chosen scale parameter. In fact, the procedure always performs linear fits, when scale='log' the log of raw data is used, thereby performing an exponential fit on raw data.

### Choosing the timing¶

For dynamic mode, the only associated timing is t (stands for “time-lapse”). The parameter tref may be used to align time points. When provided as a number, it will be substracted to acquisition time. A string code can be given, 'root' that aligns data with the colony’s root cell division time (caution: when filtering happens, some cells that were acquired at the middle of your experiment can become root cells if their parent cell is an outlier; this may affect dangerously the alignement of your time-series).

For cell-cycle modes it associates to the estimated observable a time-point to be chosen between:

• b: time at birth, when known;
• d: time at division, when known;
• m: time at mid-point trhough cell-cycle;
• g: generation index, which can be used in conjunction with the parameter tref. When the later is set to a floating number, generation index will be offset to the generation index of the cell’s ancestor that lived at this time of reference if it exists, otherwise, data from this lineage is discarded in analysis. When tref=None, then the generation index is relative to the colony to which belongs current cell.

End-point values are estimated by extrapolation. This is because cell divisions are recorded halfway between parent cell last frame and daughter cell first frame. The extrapolation uses local fits over join_points points.

Warning

generation index may be used with care in statistical estimates over the dynamics of dividing cells, since generation 0 for a given colony does not necessarily correspond to generation 0 of another colony.

### Differentiation¶

In dynamics mode, differentiation is obtained either by default using finite differences with two consecutive points, either by a sliding window fit. For an observable $$x(t)$$, depending on the chosen scale, linear or log, it returns the estimate of $$\frac{dx}{dt}$$ or $$\frac{d}{dt} \log x(t)$$ respectively.

### Local fit estimates¶

As finite difference estimates of derivatives are very sensitive to measurement precision, the user can opt for a local fitting procedure.

This procedure can be applied to estimate derivatives, or values of the observables by performing local linear fit of the scaled observable over a given time window. To use said option, user needs to provide the time window extent, e.g. time_window=15, will proceed to a local fit over a time window of 15 units of time (usually minutes).

Such a local fit procedure restricted to scanning cell-cycle time segments would lead to a loss of exploitable times, as large as the time window, for each cell. To deal with that, the procedure provide a way to use daughter cell information to “fill data estimates” towards the end of cell-cycle. The parameter join_points=3 indicates that end-point values are estimated using 3 first frames, or 3 last frames.

Warning

Using local fitting procedure is likely to artificially correlate time points over the time window time range. Such option can help with data visualization since it smoothens measurement errors, but extreme caution is adviced when this feature is used in statistical analysis.

### Examples¶

Let’s assume that raw data column names include 'length' and 'fluo'.

#### Example 1: length vs. time¶

This is the trivial example. We stay in dynamic mode, and we do not associate any further processing to collected data:

>>> length = Observable(name='length', raw='length')


#### Example 2: length at birth¶

We go to the corresponding cell-cycle mode with the appropriate timing:

>>> length_at_birth = Observable(name='birth-length', raw='length', mode='birth', timing='b')


Note

one could associate the observable length at birth with another timing, e.g. time at mid cell cycle.

#### Example 3: Fluorescence production rate (finite differences)¶

>>> k = Observable(name='prod-rate', raw='fluo', differentiate=True)


#### Example 4: Fluorescence production rate (local fit)¶

We found that the later led to really noisy timeseries, so we choose to produce local estimates over 3 points, in an experiment where acquisition period is 4 minutes, it means to have a 12 minutes time-window:

>>> kw = Observable(name='window-prod-rate', raw='fluo', differentiate=True, scale='linear',
local_fit=True, time_window=12.)


It computes

$\frac{d}{dt} \mathrm{fluo}(t)$

using 12 minutes time windows.

#### Example 5: Fluorescence production rate (using local fits) at birth¶

And we want to have it as a function of generation index, setting 0 for cells that live at time 300 minutes:

>>> kw = Observable(name='window-prod-rate-at-birth'raw='fluo', differentiate=True, scale='linear',
local_fit=True, time_window=12.,
mode='birth', timing='g', tref=300.)


## Conditional analysis¶

We saw in Defining the statistical ensemble that one can define filters that act on cells, or colonies, and to group them in a FilterSet instance that essentially sets the statistical ensemble over which analysis is performed.

There is another utility of these FilterSet objects: they may define sub-ensembles over which analysis is performed in order to compare results over chosen sub-populations. One example is to “gate” cell-cycle quantifiers and observe the statistics of the different sub-populations. Here we extend the gating procedure to analyse any dynamic observable.

To do os, a list of FilterSet instances, one per condition, can be provided to our analysis functions. We refer to the following users pages for further reading on how to use filters, see Filters, and how to run statistical analysis Statistics of the dynamics.